Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
COMPOSITIONS AND METHODS FOR INCREASING MUSCLE MASS AND MUSCLE STRENGTH BY SPECIFICALLY ANTAGONIZING GDF8 AND/OR ACTIVIN A
Document Type and Number:
WIPO Patent Application WO/2013/074557
Kind Code:
A1
Abstract:
The present invention provides compositions and methods which involve specifically antagonizing GDFS and Activir) A. Sn certain embodftTtents, compositions are provided which comprise a GDF8-specifie binding protein and an Activin A-specific binding protein. For example, the invention includes compositions comprising an anti-GDF8 antibody and an anti- Activin A antibody, in other embodiments, antigen-binding molecules are provided which comprise a GDFB-specific binding domain and an Activin A-specific binding domain. For example, the invention includes bispecific antibodies that bind GDF 8 and Activin A, The compositions of the present invention are useful for the treatment of diseases and conditions characterized by reduced muscle mass or strength, as well as other conditions which are treatable by antagonizing GDF8 and/or Activin A activity.

Inventors:
STITT TREVOR (US)
LATRES ESTHER (US)
Application Number:
PCT/US2012/064911
Publication Date:
May 23, 2013
Filing Date:
November 14, 2012
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
REGENERON PHARMA (US)
International Classes:
C07K16/22; A61K39/395; A61P21/06
Domestic Patent References:
WO2008031061A22008-03-13
WO2007047112A22007-04-26
WO2004037861A22004-05-06
WO2011150008A12011-12-01
WO2010070094A12010-06-24
Foreign References:
US6096506A2000-08-01
US7320789B22008-01-22
US7261893B22007-08-28
US7807159B22010-10-05
US7888486B22011-02-15
US7635760B22009-12-22
US7632499B22009-12-15
US20070178095A12007-08-02
US20100166764A12010-07-01
US20090148436A12009-06-11
US201113115170A2011-05-25
US20090234106A12009-09-17
Other References:
TAYLOR ET AL., NUCL, ACIDS RES., vol. 20, 1992, pages 6287 - 6295
POWELL ET AL.: "Compendium of excipients for parenteral formulations", J PHARM SCI TECHNOL, vol. 52, 1998, pages 238 - 311, XP009119027
WU ET AL., J. BIOL. CHEM., vol. 262, 1987, pages 4429 - 4432
SEFTON, CRC CRIT. REF. BIOMED. ENG., vol. 14, 1987, pages 201
LANGER AND WISE: "Medical ApplicatiOns of Controlled Release.", 1974, CRC PRES.
GOODSON: "Medical Applications of Controlled Release", vol. 2, 1984, pages: 115 - 138
LANGER, SCIENCE, vol. 249, 1990, pages 1527 - 1533
Attorney, Agent or Firm:
WESTBERG, Christopher (Inc.777 Old Saw Mill River Roa, Tarrytown NY, US)
Download PDF:
Claims:
What is claimed is:

1. A composition comprising a GDFS-specific binding protein and an Activin A- specific binding protein.

2. The composition of claim 1 , wherein the GDFS-specific binding protein is an anti- GDF-8 antibody or antigen-binding fragment thereof.

3. The composition of claim 1 , wherein the Activin A-specific binding protein is an anti-Activin A antibody or antigen-binding fragment thereof.

4. The composition of claim 1 , wherein the GDFS-specific binding: protein: is an anti- GDF-8 antibody or antigen-binding fragment thereof, and wherein the Activin A-specific binding protein is an anti-Activin A antibody or antigen-binding fragment thereof.

5. The composition of claim 2, wherein the anti-GDFS antibody or antigen-binding fragment thereof comprises the heavy chain compiementarity determining regions (HCDRs) of a heavy chain variable region (HCVR) comprising SEQ ID NQ:9S and the Sight chain

compiementarity determining regions (LCDRs) of a light chain variable region (LCVR) comprising SEQ !D NO;13.

6. The composition of claim 5, wherein the anti-GDFS antibody or antigen-binding fragment thereof comprises three HCDRs comprising SEQ ID NO:10, SEQ ID O:11 , and SEQ !D NO:12, and three LCDRs comprising SEQ !D NO:14, SEQ ID NO:15, and SEQ ID ΝΟ:1β.

7. An antigen-binding molecule comprising a GDFS-specific binding domain and an Activin A-specific binding domain.

8. The antigen-binding moiecuie of claim 7, wherein the GDF8-specific binding domain comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR).

9. The antigen-binding moiecuie of claim 7, wherein the Activin A-speeific binding domain comprises a heavy chain variabie region (HCVR) and a light chai variable region (LCVR).

10. The antigen-binding moiecuie of claim 8, wherein the HCVR comprises three heavy chain compiementarity determining regions (HCDRs) comprising SEQ ID NO:10, SEQ !D NO:1 and SEQ ID ΝΟ.Ί2, and wherein the LCVR comprises three light chain compiementarity determining regions (LCDRs) comprising SEQ ID NO: 14, SEQ ID NO:1S, and SEQ ID NO:16.

11. The antigen-binding mo!ecuie of claim 7, wherein the GDF8-specifie binding domain comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), and wherein the Activin A-specific binding domain comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR),

12. The antigen-binding molecule of claim 11. wherein the antigen-binding molecule is a bispecific antibody.

13 A method for increasing muscle mass or strength in a subject, the method comprising administering to the subject an Activin A-specific binding protein.

14. The method of claim 13, wherein the Activin A-specific binding protein is an anfi- Activin A antibody or antigen-binding fragment thereof.

15. A method for increasing muscle mass or strength in a subject, the method comprising administering to the subject a GDFS-specific binding protein and an Activin A- specifie binding protein.

16. The method of claim 15, wherein the GDFS-specific binding protein is an anti- GDFS antibody or antigen-binding fragment thereof.

17. The method of claim 15, wherein the Activin A-specific binding protein is an anti- Activin A antibody or antigen-binding fragment thereof.

18. The method of claim 15, wherein the GDFS-specific binding protein is an anti- GDFS antibody or antigen-binding fragment thereof, and wherein the Activin-A-specific binding protein is an anti-Activin A antibody or antigen-binding fragment thereof,

19. The method of ciaim 16, wherein the anti-GDFS antibody or antigen-binding fragment thereof comprises the heavy chain complementarity determining regions (HCDRs) of a heavy chain variable region (HCVR) comprising SEQ !D NO:9, and the Sight chain

complementarity determining regions (LCDRs) of a light chain variable region (LCVR) comprising SEQ ID ΝΟ.Ί 3.

20. The method of ciaim 19, wherein the anti-GDFS antibody or antigen-binding fragment thereof comprises three HCDRs comprising SEQ ID NQ;10« SEQ ID NO;11, and SEQ ID NO:12, and three LCDRs comprising SEQ ID NO;14, SEQ !D NO:15, and SEQ ID NO:16.

21. A method for increasing muscle mass or strength in a subject, the method comprising administering to the subject an antigen-binding molecule comprising a GDFS-specific binding domain and an Activin A-specific binding domain.

22. The method of claim 21 , wherein the GDFB-specific binding domain comprises a heavy chain variable region {HCVR} and a Sight chain variable region (LCVR).

23. The method of claim 21 , wherein the Activin A-specific binding domain comprises a heavy chain variable region {HCVR) and a light chain variable region (LCVR).

24. The method of claim 22, wherein the HCVR comprises three heavy chain complementarity determining regions (HCDRs) comprising SEQ !D NO;10, SEQ ID NO; 11 , and SEQ !D NO: 12, and wherein the LCVR comprises three light chain complementarity determining regions (LCDRs) comprising SEQ ID NO: 14, SEQ ID NO: I S, and SEQ !D NO: 16.

25. The method of claim 21 , wherein the GDFS-specific binding domain comprises a heavy chain variable region (HCVR) and a light chain variabie region (LCVR), and wherein the Activin A-specific binding domain comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR).

26. The method of claim 25, wherein the antigen-binding molecule is a bispedfic antibody,

27. A method for treating a disease or disorder characterized by decreased muscle mass or strength, the method compfising administering to a subject in need thereof an Activin A- specific binding protein.

28. A method for treating a disease or disorder characterized by decreased muscle mass or strength, the method comprising administering to a subject in need thereof a GDFS- specific binding protein and an Activin A-specific binding protein.

29. A method for treating a disease or disorder characterized by decreased muscle mass or strength, the method comprising administering to a subject in need thereof an antigen- binding molecule comprising a GDFS-specific binding domain and an Activin A-specific binding domain,

30. Use of a GDFS-specific binding protein and an Activin A-specific binding protein in the manufacture of a medicament for increasing muscle mass or strength in a subject, the method comprising administering to the subject a GDFS-specific binding protein and an Activin A-specific binding protein.

31. The use of claim 30, wherein the GDFS-specific binding protein is an anti-GDF8 antibody or antigen-binding fragment thereof.

32. The use of claim 30, wherein the Activin A-specific binding protein is an anti- Activin A antibody or antigen-binding fragment thereof.

33. The use of claim 30, wherein the GDFS-specifie binding protein is an anti-GDF8 antibody or antigen-binding fragment thereof, and wherein the Acttvin-A-specific binding protein is an anti-Activln A antibody or antigen-binding fragment thereof.

34. The use of claim 31 , wherein the anti-GDF8 antibody or antigen-binding fragment thereof comprises the heav chain complementarity determining regions (HCDRs) of a heavy chain variable region (HCVR) comprising SEQ !D O:9, and the light chain complementarity determining regions (LCDRs) of a light chain variable region (LCVR) comprising SEQ iD NO: 13.

35. The use of claim 34, wherein the anti-GDFS antibody or antigen-binding fragment thereof comprises three HCDRs comprising SEQ iD NO: 10, SEQ ID NO:11 , and SEQ !D NQ.12, and three LCDRs comprising SEQ iD NO:14, SEQ ID NO; 15, and SEQ !D NO:16.

36. Use of an antigen-binding molecule comprising a GDF8-specific binding domain and an Activin A-specific binding domain in the manufacture of a medicament for increasing muscle mass or strengt in a subject.

37. The use of claim 38, wherein the GDFS-specifie binding domain comprises a heavy chain variabSe region (HCVR) and a Sight chain variable regio (LCVR).

38. The use of claim 36, wherein the Activin A-specific binding domain comprises a heavy chain variable region {HCVR} and a Sight chain variable region (LCVR).

39. The use of claim 37, wherein the HCVR comprises three heavy chain

complementarity determining regions (HCDRs) comprising SEQ SD NO: 10, SEQ iD NO:11. and SEQ SD NO; 12, and wherein the LCVR comprises three Sight chain complementarity determining regions (LCDRs) comprising SEQ !D O:14, SEQ ID NO: 1 S, and SEQ SD NO:16.

40. The use of claim 36, wherein the GDF8-speciftc binding domain comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), and wherein the Activin A-specific binding domain comprises a heavy chain variable region (HCVR) and a light chain variabSe region (LCVR).

41 . The use of claim 40, wherein the antigen-binding molecule is a bispecffic antibody,

42. Use of an Activin A-specific binding protein in the manufacture of a medicament for treating a disease or disorder characterized by decreased muscle mass or strength.

43. Use of a GDFS-specif ic binding protein and an Activin A-speeific binding protein in the manufacture of a medicament for treating a disease or disorder characterized by decreased muscle mass or strength.

44. Use of an antigen-binding molecule comprising a GDFS-specific binding domain and an Activin A-specific binding domain in the manufacture of a medicament for ireaitng a disease or disorder characterized by decreased muscle mass or strength.

Description:
COMPOSITIONS AND METHODS FOR INCREASING MUSCLE MASS AND MUSCLE STRENGTH BY SPECIFICALLY ANTAGONIZING GDP 8 AND/OR ACTJVJN A

FIELD OF THE INVENTION

[OOOiJ The present invention relates to compositions and methods for increasing muscle mass and muscle strength in a subject. More specifically, the invention relates to compositions thai specifically bind GDF8 and Activin A and the use of such compositions to treat diseases and disorders characterized by decreased muscle mass or strength.

BACKGROUND

[0002} Growth and differentiation factor-8 (GDF8, also known as myostatin), is a secreted ligand belonging to the transforming growth faetor-p (TGF-p) superfamily of growth factors, GDF8 plays a central role in the development and maintenance of skeletal muscle, acting as a negative regulator of muscle mass. While the myostatin null mouse phenotype demonstrates the importance of GDF8 in the control of muscle size during development, muscle hypertrophy can also be elicited in adult muscle through inhibition of GDF8 with neutralizing antibodies, decoy receptors, or other antagonists. Administration of GDF8 neutralizing antibodies has been reported to result in muscle mass increases of between 10 and 30%. The increased muscle mass seen is due to increased fiber diameter as opposed to myofiber hyperplasia (fiber number). A number of studies have also reported increases in muscle strength or performance commensurate with increased size including twitch and tetanic force. Use of a cleavage resistant version of the GDF8 propeptide also leads to increased muscle size.

[0003} Other GDF8 antagonists have been used in adult mice with significant effects on skeletal muscle mass. These include the extracellular portion of the Type I! GDF8 receptor, ActR!iB, stabilized by fusion to an IgG Fc domain ("ActRliB-Fc"). The clinical molecule "ACE- 031" is an exampl of an AetRI!B-Fc molecule.

[00041 Although ActRIIB-Fc has been shown to increase muscle mass in experimental animals, in human clinical trials this molecule was shown to cause various adverse side effects. For example, administration of ACE-031 to postmenopausal women in a Phase lb ascending dose study was shown to cause undesired increases in hemoglobin and decreases in FSH levels, in addition, a Phase !! study of ACE-031 in pediatric patients with muscular dystrophy was discontinued due to adverse effects including nose and gum bleeding. Dilated blood vessels are also observed in patients treated with ActR!IB-Fc.

0005J Experiments have shown that the muscle growth-inducing effects of ActRI!B-Fc are attenuated but not eliminated in myostatin null mice, suggesting that ActRi!B-Fc exerts its muscle mass-inducing effects by antagonizing other ActRI IB iigand(s) in addition to GDF8. Other iigands that bind AciRi!B include Aciivin A, Activin B, Activin AB, !nhibin A, inhibin B ; GDF3, GDF11 , Nodal, BMP2, BMP4, and 8MP7.

BRIEF SUMMARY OF THE INVENTION

[00061 The present inventors hypothesized that the enhanced muscle growth effects of ActRliB-Fe, as we!i as its unwanted side effects, are due to the binding of this molecule to additional iigands beside GDF8. Thus, the in ventors sought to determine if it was possible to specifically antagonize only certain AetR!IB Iigands but not others in order to produce the enhanced muscle growth effects of AciRilB-Fc while at the same time avoiding the unwanted adverse side effects associated with this molecule. Through the experimentation set out in the Examples herein, it was surprisingly discovered that significant muscle growth enhancement could be achieved by specifically antagonizing Activin A. importantly, it was also determined that the desired therapeutic effects of ActRiSB-Fc (e.g., enhanced skeletal muscle growth) couid be achieved without unwanted side effects by specifically antagonizing GDF8 and Activin A but not antagonizing other ActRiiB iigands (e.g., GDF11 , BMP9, BMP 10, etc.).

[0007J Thus, according to one aspect of the present invention, a composition is provided comprising a GDF8-specific binding protein and an Activin A-specific binding protein. In certain embodiments, the GDF8-specific binding protein is an anti-GDF6 antibody and/or the Activin A- specific binding protein is an anti-Acfivin A antibody. According to a related aspect of the invention, an antigen-binding molecule is provided comprising a GDFB-specific btndtng domain and an Activin A-speeific binding domain. In one embodiment of this aspect of the invention, the antigen-binding molecule is a bispecifse antibody comprising a first variable domain that specifically binds GDF8 and a second variable domain that specifically binds Activin A,

0008} The present invention provides methods for increasing muscle mass or strength in a subject by administering to the subject an Activin A-specific binding protein. The present invention also provides methods for increasing muscle mass or strength in a subject by administering to the subject a GDFS-spectfic binding protein and an Activin A-specific binding protein, or by administering to the subject an antigen-binding molecule comprising a GDF8- specific binding domain and an Activin A-specific binding domain. The methods according to this aspect of the invention are useful for treating diseases or disorders associated with decreased muscle mass, strength or power, including, e.g. , cachexia, sarcopenia and other muscle-wasting conditions.

[0009] Other embodiments of the present invention will become apparent from a review of the ensuing detailed description. DETAILED DESCRIPTION

[OO G] Before the present invention is described, it is to be understood that this invention is not limited to particular methods and experimenta! conditions described, as such methods and conditions may vary, it is also to be understood that the terminology used herei is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

{0011! Unless defined otherwise, al! technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1 %. For example, as used herein, the expression "about 100" includes 99 and 101 and ali values in between (e.g., 99.1 , 99.2, 9,3, 99,4, etc.).

Antigen-Specific Binding Proteins

[0012! The present invention relates to compositions comprising antigen-specific binding proteins. More specifically, the present invention provides a composition comprising a GDF8- speeific binding protein and an Activin A-specific binding protein.

[00131 As used herein, the expression "antigen-specific binding protein" means a protein comprising at !east one domain which specifically binds a particular antigen. Exemplary categories of antigen-specific binding proteins include antibodies, antigen-binding portions of antibodies, peptides that specifically interact with a particular antigen (e.g., pepfibodies), receptor molecules that specifically interact with a particular antigen, and proteins comprising a iigand-binding portion of a receptor that specifically binds a particular antigen.

[001 1 The present invention includes antigen-specific binding proteins that specifically bind GDF8, i.e., "GDFS-speeific binding proteins". The term "GDFB" {also referred to as "growth and differentiation factor-β" and "myostatin") means the protein having the amino acid sequenc of SEQ iD NO:25 {mature protein). According to the present invention. GDFS-specific binding proteins specifically bind GDF8 but do not bind other ActRi!B ligands such as GDF3, BMP2, 8MP4, B P7, BMP8, BMP1G, GDF11 , Activi A, Activin 8, Activin AS, Nodal, etc.

[0015] The present invention also includes antigen-specific binding proteins that specifically bind Activin A, i.e., "Activin A-specific binding proteins", Activins are homo- and heter-dimeric molecules comprising pA and/or j3B subunits. The βΑ subunit has the amino acid sequence of SEQ iD NQ:26 and the βΒ subunit has the amino acid sequence of SEQ iD NO;28, Activin A is a homodimer of two βΑ subunits; Activin B is a homodimer of two βΒ subunits; and Activin AB is a heferodimer of one j3A subunit and one βΒ subunit. An Activin A-specific binding protein may be an antigen-specific binding protein that specifically binds the βΑ subunit. Since the pA subunit is found in both Activin A and Activin AB molecules, an "Activin A-specific binding protein" can be an antigen-specific binding protein that specifically binds Activin A as we!! as Activin AB (by virtue of its interaction with the βΑ su unit). Therefore, according to the present invention, an Activin A-specific binding protein specifically binds Activin A, or Activin A and Activin AB, but does not bind other ActRI IB Iigands such as Activin B, GDF3, GDF8, BMP2, BMP4, BMP7, BMP9, SMP10, GDF11 , Nodal, etc.

[0016J in the context of the present invention, moiecuies such as ActR!iB-Fc (e.g. , "ACE- 031"), which comprise the Sigand-binding portion of the ActRlSB receptor, are not considered "GDF8- specific binding proteins" or "Activin A-specific binding proteins" because such moiecuies bind multiple Iigands besides GDFS, Activin A and Activin AB.

Antigen-Binding Molecules with Two Different Antigen-Specific Binding Domains

[0017J The present invention also includes antigen-binding moiecuies comprising two different antigen-specific binding domains. In particular, the present invention includes antigen-binding moiecuies comprising a GDFS-specific binding domain and an Activin A-specific binding domain. The term "antigen-specific binding domain," as used herein, includes poiypeptides comprising or consisting of: (i) an antigen-binding fragment of an antibody molecule, (ii) a peptide that specifically interacts with a particular antigen (e.g., a peptibody). and/or (I) a ligand-binding portion of a receptor that specifically binds a particular antigen. For example, the present invention includes bispecific antibodies with one arm comprising a first heavy chain variable region/light chain variable region (HCVR/LCVR) pair that specifically binds GDFS and another arm comprising a second HCVR/LCVR pair that specifically binds Activin A.

Specific Binding

[00181 The term "specifically hinds" or the like, as used herein, means that an antigen-specific binding protein, or an antigen-specific binding domain, forms a complex with a particular antigen characterized by a dissociation constant (Ks) of 500 pfvl or less, and does not bind other unrelated antigens under ordinary test conditions. "Unrelated antigens" are proteins, peptides or polypeptides that have less than 95% amino acid identity to one another. Methods for

determining whether two moiecuies specifically bind one another are well known in the art and include, for example, equilibrium dialysis, surface p!asmon resonance, and the like. For example, an antigen-specific binding protein or an antigen-specific binding domain, as used in the context of the present invention, includes molecules that bind a particular antigen (e.g. , GDFS, or Activin A and/or AB) or a portion thereof with a , of less than about 500 pM, less than about 400 pM, less than about 300 M, iess than about 200 pM, less than about 100 pM, iess than about 90 pM. less than about 80 pM, iess than about 70 pM, less than about 80 pM, iess than about 50 pM, less than about 40 pM, iess than about 30 pM, iess than about 20 pM, iess than about 0 pM, iess than about 5 pM, less than about 4 pM, less than about 2 pM, iess than about 1 , less than about 0.5 pM, less than about 0.2 pM, less than about 0.1 pfvL or less than about 0.05 pM, as measured in a surface p!asmon resonance assay.

[0019} As used herein, an antigen-specific binding protein or antigen-specific binding domain "does not bind" to a specified molecule (e.g., "does not bind GDF1 V, "does not bind BMP9", "does not bind BMP 10", etc.) it the protein or binding domain, when tested for binding to the molecule at 2S°C in a surface piasmon resonance assay, exhibits a K<- ; of greater than 1000 pM, or fails to exhibit any binding in such an assay or equivalent thereof.

[00201 The term "surface piasmon resonance", as used herein, refers to an optical phenomenon that allows for the analysis of real-time interactions by detection: of alterations in protein concentrations within a biosensor matrix, for example using the SIAcore™ system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).

[00211 The term "Ko ". as used herein, means the equilibrium dissociation constant of a particular protein-protein interaction (e.g., antibody-antigen interaction). Unless indicated otherwise, the K D values disclosed herein refer to K 0 values determined by surface piasmon resonance assay at 25°C.

Antibodies and Antigen-Binding Fragments of Antibodies

[00221 As indicated above, an antigen-specific binding protein can comprise or consist of an antibody or antigen-binding fragment of an antibody. Furthermore, in the case of antigen- binding molecules comprising two different antigen-specific binding domains, one or both of the antigen-specific binding domains may comprise or consist of an antigen-binding fragment of an antibody.

[0023} The term "antibody", as used herein, is intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as mu!timers thereof (e.g., !givl). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCV or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, (¾1 , CM2 and C H 3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V'J and a light chain constant region. The light chain constant region comprises one domain The VH and Vs . regions can be further subdivided into regions of hypervariabi!ity, termed

complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each V M and V L is composed of three CDRs and four FRs, arranged from amsno-terminus to carboxy-terminus in the following order; FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the antibodies of the invention (or antigen-binding portion thereof) may be identical to the human germiine sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. [0024J The term "antibody/' as used herein, also includes antigen-binding fragments of full antibody mo!ecuies. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein:, include any naturally occurring, enzymaticaily obtainabie, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a comp!ex. Antigen-binding fragments of an antibody may be derived, e.g... from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries}, or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, fo example, to arrange one or more variabie and/or constant domains into a suitable configuration, or to introduce eodons, create cysteine residues, modify, add or delete amino acids, etc.

[0O2SJ Non-limiting examples of antigen-binding fragments include; (i) Fab fragments; (is) F{ab')2 fragments; {$) Fd fragments; <iv) Fv fragments; (v) single-chain Fv (scFv) moiecuies; (vt) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariab!e region of an antibody (e.g., an isolated complementarity determining regio (CDR) such as a CDR3 peptide), or a constrained FR3-CD 3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain- deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies {e.g. monovalent nanobodses, bivalent nanobodses, etc.), smal! modular immunQpharrnaceuiica!s (SM!Ps), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.

[0026} An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain ma be of any size or amino acid composition and wi!l generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences, in antigen-binding fragments having a V H domain associated with a V L domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VR-V l or V L -VY dimers.

Alternatively, the antigen-binding fragment of an antibody may contain a monomelic V H or V L domain.

[0027J In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covaient!y linked to at least one constant domain. Non-limiting, exemplary configurations of variabie and constant domains that may be found within an antigen- binding fragment of an antibody of the present invention include: (j) VH-CK1 ; (ii) VH-C H 2; (i) V H - C H 3; (iv) VH-C H 1-CH2; (V) VH-C H 1 -C H 2-C H 3; (vi) V h -¾2-CH3; (vii) V H -C,.; (viii) VL-C H 1 ; (ix) VL-CB2; (x) V L -CH3: (xi) V L -CH1 -C H 2; (xii) V L -CH1 -C H 2-CH3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C L . In any configuration of variable and constant domains, including any of th exemplary configurations listed above, the variable and constant domains may be either direetiy linked to one another or may be linked by a full or partiai hinge or linker region. A hinge region may consist of at ieast 2 {e.g., 5, 10, 15, 20, 40, 80 or more) amino acids which resu!t in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a horno-dimer or hetero-dimef (or other mutii er) of any of the variable and consiani domain configurations listed above in non-covaient association with one another and/or with one or more monomeric V'H or Vs. domain {e.g., by disulfide bondfs)),

[0028I The molecules of the present invention may comprise or consist of human antibodies and/or recombinant human antibodies, or fragments thereof. The term "human antibody", as used herein, includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may nonetheless include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation n vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended fo include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0029] The molecules of the present invention may comprise or consist of recombinant human antibodies or antigen-binding fragments thereof. The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector Iran sfected into a host eel (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes {see e.g. , Taylor et al, (1992} Nuc!, Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V¾ and V ; . regions of the recombinant antibodies are sequences that, while derived from and related to human germline V H and V L sequences, may not naturally exist within the human antibody germline repertoire in vivo,

Anti-GDPS Antibodies and Antigen-Binding Fragments Thereof

00301 In certain specific embodiments of the present invention, the GDF8-specific binding protein, or the GDFS-specific binding domain, comprises or consists of an anti-GDFS aritibody or antigen-binding fragment thereof . Anti-GDFS antibodies are mentioned in, e.g., US Patent Nos. 6,098,506; 7,320,789; 7,261 ,893; 7,807,159; 7,888,488; 7,635,760; 7,632,499; in US Patent AppL Pubi. Nos. 2007/0178095; 2010/0166764; 2009/0148436; and international Patent Appi. Pub!. No. WO 20 0/070094. Anti-GDFS antibodies are also described in US Patent Appi, No. 13/115,170, fled on May 25, 2011 , and published as US 2011/xxxxxxx, including the antibodies designated 8D12, H4H1657N2, and H4H1669P. Any of the anti-GDFS antibodies mentioned and/or described in any of the foregoing patents or publications, or antigen-binding fragments thereof, can be used in the context of the present invention, so long as such antibodies and/or antigen-binding fragments "specifically bind" GDF8, as that expression is defined herein.

[00311 Table 1 sets fort the sequence identifiers of the HCVRs, LCVRs, and CDRs of certain non-limiting, exemplary anti-GDFS antibodies that can be used in the context of the present invention.

Table 1

Anti-Acttvin A Antibodies and Antigen-Binding Fragments Thereof

[0032} In certain specific embodiments of the present invention, the Activin A-specific binding protein, or the Activin A-specific binding domain, comprises or consists of an antibody or antigen-binding fragment thereof that specifically hinds Activin A. In certain embodiments, the Activin A-specific binding protein specifically binds the 8A subunit. An antigen-specific binding protein that specifically binds the [¾A subunit may recognize both Activin A (βΑ/βΑ homodimer) and Activin AB (βΑ/ββ heterodimer). Thus, according to the present invention, an Activin A~ specific binding protein may bind both Activin A and Activin AB (but not Activin B), Anti-Activin A antibodies are mentioned in, e.g., US Patent Appi. Publ. No 2009/0234106. A particular anti- Activin A antibody is designated "MAB3381 ," and is a ailable commercially from R&D Systems, inc. Minneapolis, MM MAB3381 specifically binds Activin A {homodimer} as weil as Activin AB (heterodimer). Any of the aforementioned anfi-Activin A antibodies, or antigen-binding fragments thereof, can be used in the context of the present invention, so long as such antibodies and/or antigen-binding fragments "specifically bind" Activin A and/or Activin AB, as defined herein.

Pharmaceutical Compositions and Methods of Administration

[£3033} The present invention includes pharmaceutical compositions comprising a GDFS- specific binding protein and an Activin A-specific binding protein. The present invention also includes pharmaceutical compositions comprising an antigen-binding molecule comprising a GDFS-specifie binding domain and an Aetivin A-speeific inding domain. The pharmaceutical compositions of the invention are formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in, e.g.. Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, PA, Suitable formulations include, for example, powders, pastes, ointments, jeies, waxes, oils, lipids, lipid {caiionic or anionic) containing vesicles (such as LIPOFECT!N™}, DMA conjugates, anhydrous absorption pastes, oii-irt-water and water-in-oil emulsions, emulsions carbovvax (polyethylene glycols of various molecula weights), semi-solid gels, and semi-solid mixtures containing carbowax. Additional suitabl formulations are also describee! in Powell et a!. "Compendium of excipients for parenteral formulations" PDA (1998) J Pbarm Sci Techno! 52:238-311.

[0034J Various deiivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, e.g. , encapsulation in liposomes, micropartic!es, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 282:4429-4432). Methods of

administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by an convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.

[0035} A pharmaceutical composition of the present invention can be delivered

subcutaneous!y or intravenously with a standard needle and syringe, in addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a

pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Onc ail of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen deiivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[Q03SJ Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but are not limited to AUTOPEN™ (Owen umford, Inc., Woodstock, UK),

DiSETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland}, BUMAtOG Mix 75/25™ pen, HUMALGG™ pen, HUMALIN 70/30 w pen {Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ !, If and Hi (Novo Nordisk, Copenhagen., Denmark}, OVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark). SD™ pen (Becton Dickinson, Franklin Lakes, NJ),

OPT! PEN™, OPTiPEN PRO™, OPTiPEN STARLET™, and OPT!CLSK™ isanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present Invention include, but are not limited to the SOLOSTAR™ pen {sanofi-aveniis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eil Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPE (Dey, LP.), and the HUMIRA™ Pen (Abbott Labs, Abbott Park it), to name only a few.

[00371 In certain situations, the pharmaceutical compositions of the present invention can be delivered in a controlled release system. In one embodiment, a pump may be used (see

Langer, supra; Sefton, 1987, CRC Crit. Ref, Biomed, Eng. 14:201 ). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds,), 1974, CRC Pres., Boca Raton, Florida, i yet another embodiment, a control !ed release system can be placed In proximity of the composition's target, thus requiring oniy a fraction of the systemic dose (see, e.g., Goodson, 19S4, in Medical Applications of Controlled Release, supra, vol 2, p, 115-138), Other controlled re!ease systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[00381 T e Injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations ma be prepared by known methods. For example, the injectable preparations may be prepared, e.g... by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium convention-ally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc.. which may be used in combination with an appropriate solubiiizing agent such as an alcohol (e.g., ethanoi), a poiyalcoho! (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., poiysorbate 80, HCO-50

(poSyoxyethylene (50 mo!) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubiiizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.

[0039J Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms In a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

Dosage [0040J The amount of active ingredient (e.g., anti-GDF8 antibodies and anti-Activin A antibodies) that can be administered to a subject is, generally, a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount' 1 means a ciose of antigen-specific binding proteins and/or antigen-binding molecules that results in a detectable increase in one or more of the following parameters: body weight, muscle mass {e.g. , tibialis anterior [TAJ muscle mass, gastrocnemius [GAJ muscle mass, quadriceps [Quad] muscle mass, etc.), muscle strength/power, and/or muscle function. For example, a "therapeutically effective amount" of a GDF8-specific binding protein and/or an Activin: A-specific binding protein includes, e. g., an amount of GDFS-specific binding protein and/or Activin A-specific binding protein that, when administered to a test subject, causes an increase in TA or GA muscle mass of at feast 2%. 5%, 10%, 15%. 20%, 25%, 30%, 40%, 50%. 60% or more, compared to control treated subjects, e.g., as illustrated in Example 1 , herein.

[0Q4iJ In the case of antibodies of the present invention (e.g. , anti-GDFS antibodies, anti- Activin A antibodies, or bispecific antibodies that specifically bind GDF8 and Activi A), a therapeutically effective amount can be from about 0.05 mg to about 600 mg; e.g., about 0.05 mg, about 0.1 mg, about 1 .0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 80 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 1 10 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 580 mg, about 570 mg, about 580 mg, about 590 mg, or about 800 mg, of the respective antibody.

[0042J The amount of antibody of the present invention (e.g., anti~GDF8 antibodies, anti- Activin A antibodies, or bispecific antibodies that specifically bind GDF8 and Activin A) contained within the individual doses may be expressed in terms of milligrams of antibody per kilogram of patient body weight {i.e., mg/kg). For example, the anti~GDF8, anti-Activin A and/or anti- GDF8/anfi-Activin A bispecific antibodies of the present invention may be administered to a patient at a dose of about 0,0001 to about 50 mg kg of patient body weight (e.g. 0,5 mg/kg, 1 ,0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, 5.0 mg/kg, 5,5 mg/kg, 6,0 mg/kg, 6,5 mg/kg, 7,0 mg/kg, 7,5 mg/kg, 8,0 mg/kg, 8.5 mg/kg, 9.0 mg/kg, 9.5 mg/kg, 10.0 mg/kg, 10.5 mg/kg, 1 1.0 mg/kg, 11 .5 mg/kg, etc.).

f.0043] The compositions of the present invention may comprise equal amounts of GDFS- specific binding protein and Activin A-specific binding protein. Alternatively, the amount of GDFS-specific binding protein in the composition may be less than or greater than the amount of Activin A-specific binding protein. A person of ordinary skill in the art, using routine

experimentation, vvi!l be able to determine the appropriate amounts of the individua! components in the compositions of the present invention necessary to produce a desired therapeutic effect

Therapeutic Methods

[0044] The present invention includes methods of treating conditions or afflictions which can be cured, alleviated or improved by increasing muscle strength/power and/or muscle mass and/or muscle function in an individual, or by favorably altering metaboiism (carbohydrate, iipid and protein processing) by specifically binding GDF8, and/or Activin A, and/or Activin AS, and not binding other ActRIIB ligands. For examp!e, the present invention includes methods for increasing muscle strength/power and/or muscle mass and/or museie function in a subject, or for treating a disease or disorder characterized by decreased museie mass or strength in a subject, by administering to the subject an Activin A-specific binding protein. The present invention also includes methods for increasing muscle strength/power and/or muscle mass and/or muscle function in a subject, or for treating a disease or disorder characterized by decreased muscle mass or strength in a subject, by administering to the subject a GDFS- specific binding protein and an Activin A-specific binding protein. Any of the GDFS-specific binding proteins and/or Activin A-specific binding proteins disclosed or referred to herein can be used in the context of these aspects of the invention , For example, the therapeutic methods of the present invention include administering to a subject an anti-GDFS antibody and/or an antt- Activin A antibody.

[0045} in methods which comprise administering a GDFS-specific binding protein and an Activin A-specific binding protein to a subject, the GDFS-specific binding protein and the Activin A-specific binding protein may be administered to the subject at the same or substantially the same time, e.g., in a single therapeutic dosage, or i two separate dosages which are administered simultaneously or within less than about 5 minutes of one another. Alternatively, the GDFS-specific binding protein and the Activin A-specific binding protein may be

administered to the subject sequentially, e.g., in separate therapeutic dosages separated in time from one another by more than about 5 minutes.

[0046] The present invention also includes methods for increasing muscle strength/power and/or muscle mass and/or muscle function in a subject, or for treating a disease or disorder characterized by decreased museie mass or strength in a subject, b administering to the subject an antigen-binding molecule comprising a GDF8-specific binding domain and an Activin A-specific binding domain. Any of the antigen-binding molecules disclosed or referred to herein can be used in the context of this aspect of the invention. For example, the therapeutic methods of the present invention include administering to a subject a bispecific antibody comprising a first variable domain comprising a HCVR/LCVR pair that specifically binds GDF8 and a second variable domain comprising a HCVR/LCVR pair that specifically binds Activin A.

[0047} The compositions of the present invention may be administered to a subject along with one or more additional therapeutic agents, including, e.g., growth factor inhibitors,

immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and cytotoxic/cytostatic agents. The additional therapeutic agent(s) may be administered prior to, concurrent with, or after the administration of the GDF8- and Activin A-speeific binding proteins of the present invention.

00 SJ Exemplary diseases, disorders and conditions that can be treated with the

compositions of the present invention include, but are not limited to, sa copenia, cachexia {either idiopathic or secondary to other conditions : e.g., cancer, chronic reoai failure, or chronic obstructive pulmonary disease), muscie injury, muscie wasting and muscle atrophy, e.g., muscie atrophy or wasting caused by or associated with disuse, immobilization, bed rest, injury, medical treatment or surgical intervention {e.g. , hip fracture, hip replacement, knee replacement, etc.) or by necessit of mechanical ventilation. The compositions of the invention may also be used to treat, prevent or ameliorate diseases such as cancer, obesity, diabetes, arthritis, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis, Parkinson's disease, osteoporosis, osteoarthritis, osteopenia, metabolic syndromes (including, but not limited to diabetes, obesity, nutritional disorders, organ atrophy, chronic obstructive pulmonary disease, and anorexia).

Avoidance of Side Effects

[0049} The present invention includes methods for increasing muscie strength/power and/or muscie mass and/or muscle function in a subject, or for treating a disease or disorder characterized by decreased muscle mass or strength in a subject, without causing adverse side effects associated with the administration of mo!ecuies which bind multiple {e.g., 3 or more) ActRi!B ligands. For example, the clinical molecule referred to as AC £-031 (Aceeleron Pharma, inc., Cambridge, MA) is a mu!timer consisting of the extracellular portion of ActR!iB fused to an igG Fc domain {this molecule is also referred to herein as "ActRiiB-Fc"). ActRliB-Fc binds GDF8 as well as other Act HB ligands such as, e.g., Activin A, Activin B, GDF11 , 8 P9,

Β ΊΡΙΟ, and TGFp, and is known to cause various adverse side effects when administered to human patients. Significantly, the present inventors have unexpectedly discovered that specificaiiy inhibiting GDF8 and Activin A {e.g., by administering an anti-GDFB antibody and an anti-Activin A antibody), while not inhibiting other ActRi!B ligands such as Activin 8, GDF11 , BMP9, BMP10, and TGFp, results in an increase in muscie mass that is at least equivalent to that observed by administration of AcfRHS-Fc, without causing the adverse side effects associated with non-specific Activin binding agents such as ActR!SB-Fc. Administration Regimens

[0050J According to certain embodiments of the present invention, multiple doses of the compositions of the present invention (e.g., compositions comprising GDF8- and/or Activin A- specific binding proteins or antigen-binding molecules comprising a GDFS-specific binding domain and an Activin A-specific binding domain), may be administered to a subject over a defined time course. The methods according to this aspect of the invention comprise sequentially administering to a subject muitip!e doses of the compositions of the present invention . As used herein, "sequentially administering" means thai each dose of the compositions of the present invention are administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval {e.g. , hours, days, weeks or months). The present invention includes methods which comprise sequentially administering to the patient an initial dose of a composition of the present invention, followed by one or more secondary doses of the composition, and optionally followed by one or more tertiary doses of the composition.

[0051} The terms "initial dose," "secondary doses," and "tertiary doses," refer to the temporal sequence of administration of the compositions of the present invention. Thus, the "initial dose" is the dose which is administered at the beginning of the treatment regimen {also referred to as the "baseline dose"); the "secondary doses" are the doses which are administered after the initial dose; and the "tertiary doses' 1 are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of active ingredient(s), but will generally differ from one another in terms of frequency of administration, in certain embodiments, however, the amount of active ingredients) contained in the initial, secondary and/or tertiary doses will vary from one another {e.g., adjusted up o down as appropriate) during the course of treatment.

[00521 In one exemplary embodiment of the present invention, each secondary and/or tertiary dose is administered 1 to 30 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, I S, 18, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 28, 27, 28, 29, 30, or more) days after the immediately preceding dose. The phrase "the immediately preceding dose," as used herein, means, in a sequence of multiple administrations, the dose(s) of the compositions of the present invention which are administered to a subject prior to the administration of the very next dose in the sequence with no intervening doses.

[0053} The methods according to this aspect of the invention may comprise administering to a patient any number of secondary and/or tertiary doses of the compositions of the present invention. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 8, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient, in other embodiments, two or more (e.g., 2, 3, 4, 5, 6 ; 7, 8, or more) tertiary doses are administered to the patient.

[0054} in embodiments involving multiple secondary doses, eac secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may he administered to the patient 1 to 29 days after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 1 to 60 days after the immediately preceding dose.

Alternatively, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination,

E AMPLES

fOOSS] The following examples are put fort so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average mo!ecutar weight, temperatur is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1. Specific inhibition of GDFS and Activin A Causes Synergistic increases in Skeletal Muscle IVIass

Introduction

[00561 ActRSIB-Fc is a GDFS antagonist consisting of the extracellular portion of the ActRiSB receptor, stabilized by fusion to an SgG Fc domain. ActRi!B-Fc has been shown to increase muscle mass in mice to a greater extent than anti-GDP8 antibodies. The present inventors hypothesized that the enhanced activity of AetRUB-Fc could potentially be due to its ability to bind additional AetRiiB !igands besides GDF8, in particular, it was proposed that antagonism of Activin A, in addition to antagonism of GDFS, might cause greater increases in skeletal muscle mass than what has been observed in animals treated with anfi~GDF8 antibodies alone. Thus, the present Example was designed to determine whether specific inhibition of GDFS and Activin A can increase skeletal muscle mass to an extent that is at least equivalent to the increase observed using ActRIIB-Fc. Results and Discussion

[0057} The extent of skeletal muscle hypertrophy induced by administration of AetR!lB-Fc was compared to the effect of administration of a GDFS-specific antibody, an Activin A specific antibody, or a combination of an anti-GDF8 + anti-Activin A antibody. The ActRilB-Fc construct used in this Example has the amino acid sequence of SEQ ID NO:27, The anti-GDFS antibody used in this Example is the antibody designated H4H1857N2 (see Tabie 1 ). The artii-Activin A antibody used in this Example is the antibody designated MA83381 (available from R&D Systems, inc., Minneapolis, N). An isotype-matched ChigG4) antibody was used as negative control

(00581 Briefly, 25 male CB1 SOD mice at approximately 10 weeks of age, were divided evenly according to body weight into 5 groups based on treatment (isotype Control rrsAb, ActRllB-Fc, H4H1657N2, MAB3381 , or H4H1657N2 + MAB3381), Reagents were administered subcutaneous!y at a dose of 10 mg/kg twice for the first week (on day 0 and day 3} and once a week for the following three weeks {on day 7, day 14 and day 21 }. On day 28, mice were euthanized and weighed, and the tibialis anterior (TA) muscles, and the gastrocnemius (GA) muscles, were dissected and weighed. Tissues were normalized to starting bod weight, and percent change in weight over the isotype-matched (higG4) control antibody was calculated. Results are summarized in Table 2 and are expressed as percent increase over negative control ± standard error of the mean.

Table 2A

[0059} In order to confirm that muscle hypertrophy was the resu!t of an increase in muscle fiber size, the tibialis anterior (TA) musc!e was embedded in OCT and isopentane-frozen for histological examination and immunohistochemical labeling. Cross-sections of the TA muscle were stained with anti-iaminin antibody to outline the muscle fiber, and the average cross- sectienai-area (CSA) was determined by using an imaging analysis system. Results of two independent experiments (£xp#1 and Exp#2) are summarized in Table 28. All data are expressed as means ± the standard error of the mean. Table 2B

[0060J As shown in Table 2A, Act liB-Fc induced significant hypertrophy in aii muscles examined, with increases of 44.88 + 5.35% in TA muscle mass, and 34,25 + 6,97% in GA muscle mass. Treatment with H4H1657N2 (anti-GDFS), or ΜΑΒ33β1 (anti-Activin A) alone also induced significant hypertrophy in TA muscle mass (22.42 + 1 .65% and 19.09 + 2.04%, respectively) and GA muscie mass (24,17 + 1 ,84 and 14,02 + 0,91%, respectively) but not as pronounced as AetR!iB-Fc. However, the combination of H4H1657N2 and fvlAB3381 induced increases in TA {55.13 + 5.16%) and GA {41.72 + 3.63%), that were even greater than what was observed in Acf ilB-h c-ireaied animals. Furthermore, it was confirmed that the muscie hypertrophy observed was the result of a increase in muscie fiber size {see Table 2B).

0061} Importantly, the extent of increases in body weight, TA muscle, and GA muscle for the anti-GDFS + anti-Activin A combination were substantially greater than the sums of the increases in these parameters observed in the anii-GDFS plus anti-Activin A monotherapy subjects. Thus, combined inhibition of GDF8 and Activin A produces synergistic increases in body weight and skeletal muscle mass, and these increases are more pronounced than what is observed in ActRIIB-Fc-treated animals. Moreover, as demonstrated in the following Example, the increases in body weight and skeletal muscle mass in animals that are treated with GDF8- and Activin A-specific binding agents, can be achieved without causing the adverse side effects observed with molecules such as ActRilS-Fc.

Example 2: Specific Antagonism of GDF8 and Activin A Does Mot Cause Adverse Side Effects Associated with Non-Specific Activin Ligand Binding Agents

Background

[£3062} ActRSIB~Fc binds multiple ActRiiS Sigands and causes significant side effects. The present Example demonstrates that the adverse side effects associated with ActRISB-Fc can be avoided by selectively antagonizing only certain ActRilB Sigands, narneiy GDF8 and/or Activin A. in particular, biornarker, protein expression studies, and in vivo red blood ceil characteristics (i.e . elevated endoglin levels and increased red cell distribution width), which appear to be linked to ActRliB-Fc side effects in humans, were only seen in animals treated with ActR!IB-Fc, but not in animals treated with anti-GDFS antibody, anti-Activin A antibody or a combination of anti-GDF8 and anti-Activin A antibodies. Thus, taken together, the results below show that specific antagonism of GPF8 and/or Activin A, without antagonizing other ActRI!B !igands such as Acfivtn 8, GDF1 1 , BMP9, BMP10, and/or TGFp, does not cause the undesired phenotypes associated with ActRf lB-Fc.

Results and Discussion

[00631 Hematology studies were conducted using mice treated with ActRiiB-Fc (SEQ ID NO:27), H4H1657N2 {anti-GDFS}, A83361 (anti-Activin A), or a combination of Ή4Η1657Ν2 + MAB3381 according to the dosing schedule described in Example 1 (i.e.. 10 mg/kg twice for the first week [on day 0 and day 3] and once a week for the following three weeks [on day 7, day 14 and day 21]). Specifically, hemoglobin !eveis and red b!ood eel! distribution width (ROW) (an indicator of certain blood disorders such as anemia) were measured from blood samples taken from mice treated with the various agents. RDVV results {normalized to Isotype Control values) are summarized in Table 3,

Table 3

[0064J After 28 days of treatment, none of the groups had a significant increase in Hb levels. However, as shown in Table 3, mice treated with AetRlSB-Fc showed a significant increase in red blood ceil distribution width (RDVV), which reflects the extent of size variation of red blood ceils in a sample. Surprisingly, mice treated with anti-GDFS antibody, anti-Activin A antibody, or the combination of anti-GDFS + anti-Acttvin A antibodies, did not exhibit an appreciable degree of increase in % RDW as compared to isotype control-treated mice. These experiments therefore demonstrate that antagonism of GDFS or Activin A alone, or the combination of anti- GDFS antibody + anti-Activin A antibodies, do not cause the hematological phenotypes that are observed with ActRiiB-Fc treatment.

[0065} To further investigate the differences in side effects between ActRliB-Fc-treated subjects and anti-GDFS + anti-Activin A combination-treated subjects, mtcroarray and protein expression studies were conducted.

[0066] Microarray analysis was conducted on skeletal muscle samples from mice treated with isotype control, ActRi!B-Fe, and H4H1657N2 (anti-GDF8). From these experiments, a set of genes was identified that is uniquely affected by AcfRi!B-Fc. Of particular interest was the up- reguiation of Endoglin rnRNA levels in skeletal muscle in samples from ActRliB-Fc-treated subjects. Endoglin is a transmembrane protein expressed primarily in endothelial ceils and interacts and promotes signaling through receptors of the TGFp family (ALK > in response to TGFp, BMP , or BMP10. Signaling mediated by Alk1 and Endoglin in endothelial cells is required for maintaining normal vascutar structures. Mutations in the A!kl and Endogiin genes in humans causes Hereditary Haemorrhagic Telangiectasia (HHT). Patients suffering HHT display a vascular phenotype including diiated blood vessels, and bleeding in the nasal, oral, and gastraintenfinaS mucosa. Thus, elevated Endogiin levels caused by ActRISB-Fc potentially reflect at least some of the adverse side effects observed in patients treated with this therapeutic agent.

[0067} Next, expenmenis were conducted to confirm that the changes observed in Endogiin mRNA levels were also reflected at the protein expression level using muscle samples from the previous experiment. Quantitative Western blot analysis of Endogiin protein levels was conducted on samples from mice treated with isotype control, Act SIB-Fc, H4H1857N2 (anti- GDF8), SVSA83381 (anti-Activin A), and the H4H1657N2 + MAB3381 combination. Expression of Endogiin was normalized by the endothelial cell marker CD31 to confirm that AefRil8-hPc treatment does not increase the endotheiiai compartment. Results (normalized to isotype Control values) are summarized in Table 4.

Table 4

[0068} As shown in Table 4, levels of Endogiin protein were significantiy elevated in the ActRI!B-hFe group, but not in the anti-GDFS or anti-Activin A-treated groups, interestingly, Endogiin levels were also not elevated in the anti-GOF8 + anti-Activin A combination-treated group.

Summary and Conclusions

[00691 " Π"»© results presentee! in the prior Example {Example 1 } show that the combination of anti-GDFS + anti-Activin A treatment can produce muscle hypertrophy effects that are at least equivalent to those observed with Act ilB-Fc treatment. The present Example 2 shows that indicators of the adverse side effects of ActRliB-Fc treatment, such as increased RDW and eievated Endogiin expression, are not observed with anti-GDFS, anti-Activin A, or anti-GDFB + anti-Activin A combination treatment. Thus, the present inventors have surprisingly discovered that treatment with a GDF8-specifte binding protein, or an Activin A-specific binding protein, or a combination of a GDFS-specific binding protein and an Activin A-specific binding protein provide highly efficaoious methods for increasing muscl mass and strength that avoid the adverse side effects caused by ActRf lB-Fc.

Example 3: Effects of GDF8 and Activin A Antagonists on Wound Healing

[00701 Pharmaceutical agents which function to increase muscle mass and strength, such as GDF8 antagonists and Activin A antagonists, have utility in settings in which patients have undergone surgery (or will undergo surgery), e.g. , for joint replacement or repair, etc. As such, agents that are administered to promote the rescue of muscle mass would ideally not interfere with other aspects of surgical recovery such as wound healing.

[0071] Accordingly, experiments were conducted to determine the effects of GDF8 blockade, Activin A blockade, and combinations thereof, on wound healing, as compared to the effects of AcfRIIB-Fc treatment. These studies were carried out in SCID mice, in particular, the effects of H4H1657N2 (anti-GDFS) and MAB3381 {anii-Acfsvin A) administration on wound healing, as single treatments or in combination with one another, were compared to the wound healing effects of the more broadly-acting decoy receptor ActRliS-hFc {SEQ ID NO:27). Briefly, circular skin excisionai wounds were made on the left abdominaS flank of 30 male SCID mice approximately 7-8 weeks. The animals were divided into five treatment groups (n=6 per group) each receiving fiv subcutaneous injections of an isotype control antibody, Act IIB-hFc,

H4H1657N2, AB3381 , or H4H1657N2 + MAB3381. Aii reagents were administered at 10 mg/kg every 3-4 days. The first dose was given the day before wounding the animals, and the last one was given two days before terminating the study on day 14. Digital images of the wound were taken on day 0 (day of wounding), 8, 9, 12, and 14, and the excision wound size change was measured and compared to the isotype control group. Results are summarized in Table 5. All data are expressed as mean total wound size ± the standard error of the mean.

Table S

[0072J An analysis of wound size at the end of the experiment, as shown in Tabie 5, revea!ed that treatment with H4H 1657N2, MAB338 , or H4H1657N2 + MAB3381 resulted in no significant difference in wound size at any time after the initial excision as compared to the isotype controi group. By contrast, ActR!iB-hFc significantly deiayed wound ciosure as indicated by the larger wound size at days 6, 9, 12, and 14 as compared to the wound size in mice treated with H4H1657N2, MAB3381 , H4H1657 2 + MAB3381 , or the isotype control.

[0O73J This experiment demonstrates that therapeutic treatments involving GDF6 antagonism, Activin A antagonism, or GDF8 + Activin A dual antagonism, do not significantly impair wound heaiing, whereas the iess specific antagonist ActRHB-hFc does significantly impair wound heaitng. Accordingly, the present Example provides further support for the notion thai specific antagonism of GOF8 and Activin A can produce enhanced muscie mass and function, similar to what is seen with ActRllb-hFc treatment, but without the adverse side effects associated with ActRlib-hFc treatment.

10074} The present invention is not to be limited in scope by the specific embodiments described herein, indeed, various modifications of the invention in addition to those described herein wii! become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fail within the scope of the appended claims.