WO2015195094A1

WO2015195094A1 - Anti-activin and nati-myostatin antibodies and methods of using the same

Info

Publication number: WO2015195094A1
Application number: PCT/US2014/042729
Authority: WO
Inventors: Jasbir S. Seehra
Original assignee: Ember Therapeutics, Inc.
Priority date: 2014-06-17
Filing date: 2014-06-17
Publication date: 2015-12-23

Abstract

The disclosure features a method of treating a subject having a disorder or condition comprising administering to said subject, an effective amount of a bispecific antibody molecule that binds both activin and myostatin, or a combination of an anti-activin antibody molecule and an anti-myostatin antibody molecule, to thereby treat the disorder or condition. The disclosure also features a bispecific antibody molecule that binds both activin and myostatin and optionally also binds one or more (e.g., 1, 2, 3, 4, 5, or all) of GDF-5, GDF-6, GDF-7, BMP-2, BMP-4, and BMP-7.

Description

ANTI-ACTIVIN AND ANTI-MYOSTATIN ANTIBODIES AND METHODS OF USING

THE SAME BACKGROUND

Thermogenesis is the process of heat production in organisms. As a component of the metabolic rate, stimulation of thermogenesis can increase energy expenditure and fat oxidation. Brown adipose tissue (BAT) is specialized for the efficient dissipation of chemical energy in the form of heat. Chronic energy imbalance, for example, excessive energy intake, moderate energy expenditure, or both, can cause various metabolic disorders such as obesity and diabetes. The epidemic of obesity and diabetes has increased the interest in searching for effective treatments.

SUMMARY

The disclosure relates, inter alia, to antibody molecules that bind to activin and/or myostatin. For example, the disclosure provides methods of treating a subject having a disorder or condition comprising administering to said subject, an effective amount of a bispecific antibody molecule that binds both activin and myostatin, or a combination of an anti-activin antibody molecule and an anti-myostatin antibody molecule. The antibody molecules described herein can be used to treat a variety of disorders and conditions, including, but not limited to metabolic disorders {e.g., diabetes, obesity, and non-alcoholic fatty liver diseases (NAFLD)), frailty, loss of muscle mass or function {e.g., muscular dystrophy), and amyotrophic lateral sclerosis (ALS). Also disclosed herein are, for example, compositions, use, kits, and articles of manufacture related to the antibody molecules and methods described herein. In one aspect, the disclosure features a method of treating a subject having a disorder or condition comprising administering to said subject, an effective amount of a bispecific antibody molecule that binds both activin and myostatin, or a combination of an anti-activin antibody molecule and an anti-myostatin antibody molecule, to thereby treat the disorder or condition.

In some embodiments, the method comprises administering an effective amount of a bispecific antibody molecule that binds activin and myostatin. In some embodiments, said antibody molecule comprises a first variable region {e.g., a first antigen-binding site) that binds activin and a second variable region {e.g., a second antigen-binding site) that binds myostatin. The first variable region {e.g., the first antigen-binding site) binds activin specifically, e.g., does not bind or substantially bind myostatin. The second variable region {e.g., the second antigen- binding site) binds myostatin specifically, e.g., does not bind or substantially bind activin. In some embodiments, said antibody molecule does not bind or substantially bind one or more (e.g., 1, 2, 3, 4, 5, or all) of growth differentiation factor (GDF)-5, GDF-6, GDF-7, bone morphogenetic factor (BMP)-2, BMP-4, and BMP-7. In some embodiments, said antibody molecule does not bind or substantially bind one, two, or all of GDF-5, GDF-6, GDF-7. In some embodiments, said antibody molecule does not bind or substantially bind one, two, or all of BMP-2, BMP-4, and BMP-7. In one embodiment, said antibody molecule does not bind or substantially bind one or more of: GDF-9 and GDF- 11.

In some embodiments, said antibody molecule binds one or more (e.g., 1, 2, 3, 4, 5, or all) of GDF-5, GDF-6, GDF-7, BMP-2, BMP-4, and BMP-7. In some embodiments, said antibody molecule binds one, two, or all of GDF-5, GDF-6, GDF-7. In some embodiments, said antibody molecule binds one, two, or all of BMP-2, BMP-4, and BMP-7. In one embodiment, said antibody molecule binds GDF-9 and/or GDF-11.

In some embodiments, said antibody molecule does not modulate, e.g., increase activity of, one or more (e.g., 1, 2, 3, 4, 5, or all) of GDF-5, GDF-6, GDF-7, BMP-2, BMP-4, and BMP-7. In some embodiments, said antibody molecule does not modulate, e.g., increase activity of, one, two, or all of GDF-5, GDF-6, GDF-7. In some embodiments, said antibody molecule does not modulate, e.g., increase activity of, one, two, or all of BMP-2, BMP-4, and BMP-7. In one embodiment, said antibody molecule does not modulate, e.g., inhibit activity of, one or more of GDF-9 and GDF- 11.

In some embodiments, said antibody molecule modulates, e.g., increases activity of, one or more (e.g., 1, 2, 3, 4, 5, or all) of GDF-5, GDF-6, GDF-7, bone morphogenetic factor BMP-2, BMP-4, and BMP-7. In some embodiments, said antibody molecule modulates, e.g., increases activity of, one, two, or all of GDF-5, GDF-6, GDF-7. In some embodiments, said antibody molecule modulates, e.g., increases activity of, one, two, or all of BMP-2, BMP-4, and BMP-7. In one embodiment, said antibody molecule modulates, e.g., inhibits activity of, one or more of GDF-9 and GDF- 11.

In some embodiments, said antibody molecule binds to activin and/or myostatin with a binding affinity of at least about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ and 10¹¹ M^"1. In some embodiments, said antibody molecule binds to activin and/or myostatin with a K_0ff slower than about 1 x 10^" , 5 X 10^"4, or l x l0^"4 s^_1. In some embodiments, said antibody molecule binds to activin and/or

2 3 3 -1 -1

myostatin with a K_on faster than about 1 x 10 , 1 x 10 , or 5 x 10 M s^" . In some embodiments, said antibody molecule inhibits activin activity and/or myostatin activity, e.g., with a Ki of less than about 10^"5, 10^"6, 10^"7, 10^"8, 5xl0^"9, 10^"9, 5xl0^"10 and 10^"10 M. In some embodiments, said antibody molecule has an IC50 for activin and/or myostatin of less than about 100, 10, or 1 nM. In some embodiments, said antibody molecule has an affinity for activin and/or myostatin characterized by a KD of less than about 100 or 10 nM, or about 3, 5, 7, 9, or 10 nM.

In some embodiments, said antibody molecule can be, e.g., an IgGl, IgG2, IgG3, IgG4, or Fab2'. In some embodiments, said antibody molecule comprises a human CDR or human framework region.

In some embodiments, said antibody molecule binds an epitope bound by an antibody molecule described herein, or an epitope that overlaps with such epitope.

In some embodiments, said antibody molecule is administered subcutaneously, intramuscularly, or intravenously. In some embodiments, said antibody molecule is

administered twice weekly, weekly, bi-weekly, or monthly.

In some embodiments, the method comprises administering a combination of an anti- activin antibody molecule and an anti-myostatin antibody molecule.

In some embodiments, said anti-activin antibody molecule and an anti-myostatin antibody molecule are administered in the same dosage form. In some embodiments, said anti- activin antibody molecule and an anti-myostatin antibody molecule are administered as separate dosage forms.

In some embodiments, said anti-activin antibody molecule and said anti-myostatin antibody molecule are administered to said subject at the same time or within about 240, 180, 120, 90, 60, 30, 15, 10, 5, or 1 minute of one another, e.g., such that there is overlap of an effect of each antibody molecule on said subject. In some embodiments, said anti-activin antibody molecule and said anti-myostatin antibody molecule are administered sufficiently close together such that a combinatorial (e.g., synergistic) effect is achieved, e.g., greater than an additive effect, e.g., at least 25, 50, 75, 100, 200, 300, 400, or 500% greater than an additive effect.

In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule does not bind or substantially bind one or more (e.g., 1, 2, 3, 4, 5, or all) of GDF-5, GDF-6, GDF-7, BMP-2, BMP-4, and BMP-7. In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule does not bind or substantially bind one, two, or all of GDF-5, GDF-6, GDF-7. In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule does not bind or substantially bind one, two, or all of BMP-2, BMP-4, and BMP-7. In one embodiment, said antibody molecule does not bind or substantially bind one or more of: GDF-9 and GDF- 11.

In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule binds one or more (e.g., 1, 2, 3, 4, 5, or all) of GDF-5, GDF-6, GDF-7, BMP- 2, BMP-4, and BMP-7. In some embodiments, said anti-activin antibody molecule and/or anti- myostatin antibody molecule binds one, two, or all of GDF-5, GDF-6, GDF-7. In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule binds one, two, or all of BMP-2, BMP-4, and BMP-7. In one embodiment, said antibody molecule binds one or more of: GDF-9 and GDF- 11.

In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule does not modulate, e.g., increase activity of, one or more (e.g., 1, 2, 3, 4, 5, or all) of GDF-5, GDF-6, GDF-7, BMP-2, BMP-4, and BMP-7. In some embodiments, said anti- activin antibody molecule and/or anti-myostatin antibody molecule does not modulate, e.g., increase activity of, one, two, or all of GDF-5, GDF-6, GDF-7. In some embodiments, said anti- activin antibody molecule and/or anti-myostatin antibody molecule does not modulate, e.g., increase activity of, one, two, or all of BMP-2, BMP-4, and BMP-7. In one embodiment, said antibody molecule does not modulate, e.g., inhibit activity of, one or more of: GDF-9 and GDF- 11.

In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule modulates, e.g., increases activity of, one or more (e.g., 1 , 2, 3, 4, 5, or all) of GDF-5, GDF-6, GDF-7, BMP-2, BMP-4, and BMP-7. In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule modulates, e.g., increases activity of, one, two, or all of GDF-5, GDF-6, GDF-7. In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule modulates, e.g., increases activity of, one, two, or all of BMP-2, BMP-4, and BMP-7. In one embodiment, said antibody molecule modulates, e.g., inhibits activity of, one or more of: GDF-9 and GDF- 11.

In some embodiments, said anti-activin antibody molecule binds to activin with a binding affinity of at least about 10⁵, 10⁶, 10⁷,10⁸, 10⁹, 10¹⁰ and 10¹¹ M . In some embodiments, said anti-myostatin antibody molecule binds to myostatin with a binding affinity of at least about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ and 10¹¹ M .

In some embodiments, said anti-activin antibody molecule binds to activin with a K_Qff slower than about 1 x 10^~3, 5 x 10^~4, or 1 x 10^~4 s^"1. In some embodiments, said anti-myostatin antibody molecule binds to myostatin with a K_Qff slower than about 1 x 10^~3, 5 x 10^~4, or 1 x 10^~4 s^"

1

In some embodiments, said anti-activin antibody molecule binds to activin with a K_on

2 3 3 -1 -1

faster than about 1 x 10 , 1 x 10 , or 5 x 10 M s^" . In some embodiments, said anti-myostatin

2 3 3 antibody molecule binds to myostatin with a K_on faster than about 1 x 10 , 1 x 10 , or 5 x 10

M-V¹.

In some embodiments, said anti-activin antibody molecule inhibits activin activity, e.g., with a Ki of less than about 10^"5, 10^"6, 10^"7, 10^~8, 5xl0^"9, 10^"9, 5xl0^"10 and 10^"10 M. In some embodiments, said anti-myostatin antibody molecule inhibits myostatin activity, e.g., with a Ki of less than about 10^"5, 10^"6, 10^"7, 10^"8, 5xl0^~9, 10^"9, 5xl0^~10 and 10^"10 M.

In some embodiments, said anti-activin antibody molecule has an IC50 of less than about 100, 10, or 1 nM. In some embodiments, said anti-myostatin antibody molecule has an IC50 of less than about 100, 10, or 1 nM.

In some embodiments, said anti-activin antibody molecule has an affinity characterized by a K_D of less than about 100 or 10 nM, or about 3, 5, 7, 9, or 10 nM. In some embodiments, said anti-myostatin antibody molecule has an affinity characterized by a K_D of less than about 100 or 10 nM, or about 3, 5, 7, 9, or 10 nM.

In some embodiments, said anti-activin antibody molecule has a t of at least about 10,

20, 30, 40, 50, 60, 120, 240, or 360 minutes. In some embodiments, said anti-myostatin antibody molecule has a t_1/2 of at least about 10, 20, 30, 40, 50, 60, 120, 240, or 360 minutes.

In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule can be, e.g., an IgGl, IgG2, IgG3, IgG4, Fab, Fab2', scFv, minibody, or scFv- Fc fusion. In some embodiments, wherein said anti-activin antibody molecule and/or anti- myostatin antibody molecule comprises a human CDR or human framework region.

In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule binds an epitope bound by an antibody molecule described herein, or an epitope that overlaps with such epitope.

In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule is administered subcutaneously, intramuscularly, or intravenously. In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule is administered twice weekly, weekly, bi-weekly, or monthly.

In some embodiments, said disorder or condition is a metabolic disorder or a symptom thereof, e.g., a metabolic disorder or a symptom described herein. In some embodiments, said metabolic disorder is diabetes, e.g., Type II diabetes. In some embodiments, said metabolic disorder is obesity. In some embodiments, the subject has one or more symptoms of: high blood sugar, insulin resistance, glucose intolerance, abnormal lipid levels (e.g., decreased high-density lipoprotein (HDL) level, increased levels of triglycerides and low-density lipoprotein (LDL)), and high blood pressure. In some embodiments, said disorder or condition is non-alcoholic fatty liver disease (NAFLD), e.g., non-alcoholic steatohepatitis (NASH).

In some embodiments, said disorder or condition is characterized by a need for increased fat browning, increased brown adipose tissue (BAT), or increased thermogenesis, e.g., as described herein. In some embodiments, said disorder or condition is characterized by a need for increased muscle mass, e.g., as described herein. In some embodiments, said disorder or condition comprises frailty, e.g., frailty associated with or arising from decreased muscle mass or strength, e.g., as described herein.

In some embodiments, said disorder or condition is characterized by loss of muscle mass or function, e.g., arising from insufficient use of muscle, e.g., associated with or arising from bed rest or other inactivity arising from or associated with age, disability, or medical condition, or a medical or surgical procedure, e.g., as described herein.

In some embodiments, said disorder or condition comprises an acquired or inherited disorder or condition of the muscles, e.g., a muscular dystrophy, e.g., Duchenne muscular dystrophy, spinal muscular atrophy, or amyotrophic lateral sclerosis (ALS), e.g., as described herein.

In some embodiments, the subject is a human. In some embodiments, the subject is an animal, e.g., a domesticated animal.

In some embodiments, the activin is activin A. In some embodiments, the activin is activin B.

In another aspect, the disclosure features a bispecific antibody molecule that binds activin and myostatin, e.g., an antibody molecule described herein.

In some embodiments, said antibody molecule comprises a first variable region (e.g., a first antigen-binding site) that binds activin and a second variable region (e.g., a second antigen- binding site) that binds myostatin. The first variable region (e.g., the first antigen-binding site) binds activin specifically, e.g., does not bind or substantially bind myostatin. The second variable region (e.g., the second antigen-binding site) binds myostatin specifically, e.g., does not bind or substantially bind activin.

In some embodiments, said antibody molecule does not bind or substantially bind one or more (e.g., 1, 2, 3, 4, 5, or all) of GDF-5, GDF-6, GDF-7, BMP-2, BMP-4, and BMP-7. In some embodiments, said antibody molecule does not bind or substantially bind one, two, or all of GDF-5, GDF-6, GDF-7. In some embodiments, said antibody molecule does not bind or substantially bind one, two, or all of BMP-2, BMP-4, and BMP-7. In one embodiment, said antibody molecule does not bind or substantially bind one or more of: GDF-9 and GDF-11.

In some embodiments, said antibody molecule binds one or more (e.g., 1, 2, 3, 4, 5, or all) of GDF-5, GDF-6, GDF-7, BMP-2, BMP-4, and BMP-7. In some embodiments, said antibody molecule binds one, two, or all of GDF-5, GDF-6, GDF-7. In some embodiments, said antibody molecule binds one, two, or all of BMP-2, BMP-4, and BMP-7. In one embodiment, said antibody molecule binds GDF-9 and/or GDF-11. In some embodiments, said antibody molecule does not modulate, e.g., increase activity of, one or more (e.g., 1, 2, 3, 4, 5, or all) of GDF-5, GDF-6, GDF-7, BMP-2, BMP-4, and BMP-7 In some embodiments, said antibody molecule does not modulate, e.g., increase activity of, one, two, or all of GDF-5, GDF-6, GDF-7. In some embodiments, said antibody molecule does not modulate, e.g., increase activity of, one, two, or all of BMP-2, BMP-4, and BMP-7. In one embodiment, said antibody molecule does not modulate, e.g., inhibit activity of, one or more of GDF-9 and GDF- 11.

In some embodiments, said antibody molecule modulates, e.g., increases activity of, one or more (e.g., 1, 2, 3, 4, 5, or all) of GDF-5, GDF-6, GDF-7, BMP-2, BMP-4, and BMP-7. In some embodiments, said antibody molecule modulates, e.g., increases activity of, one, two, or all of GDF-5, GDF-6, GDF-7. In some embodiments, said antibody molecule modulates, e.g., increases activity of, one, two, or all of BMP-2, BMP-4, and BMP-7. In one embodiment, said antibody molecule modulates, e.g., inhibits activity of, one or more of GDF-9 and GDF- 11.

2 3 3 -1 -1

In some embodiments, said antibody molecule can be, e.g., an IgGl, IgG2, IgG3, IgG4, or Fab2',. In some embodiments, said antibody molecule comprises a human CDR or human framework region.

In yet another aspect, the disclosure features an isolated preparation comprising an anti- activin antibody molecule and an anti-myo statin antibody molecule, e.g., an anti-activin antibody molecule described herein and an anti-myostatin antibody molecule described herein. In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule does not bind or substantially bind one or more (e.g., 1, 2, 3, 4, 5, or all) of GDF-5, GDF-6, GDF-7, BMP-2, BMP-4, and BMP-7. In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule does not bind or substantially bind one, two, or all of GDF-5, GDF-6, GDF-7. In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule does not bind or substantially bind one, two, or all of BMP-2, BMP-4, and BMP-7. In one embodiment, said antibody molecule does not bind or substantially bind one or more of: GDF-9 and GDF- 11.

In some embodiments, said anti-activin antibody molecule binds to activin with a binding affinity of at least about 10⁵, 10⁶, 10⁷,10⁸, 10⁹, 10¹⁰ or 10¹¹ M^"1. In some embodiments, said anti- myostatin antibody molecule binds to myostatin with a binding affinity of at least about 10⁵, 10⁶, 10⁷,10⁸, 10⁹, 10¹⁰ and 10¹¹ M^"1.

In some embodiments, said anti-activin antibody molecule binds to activin with a K_Qff slower than about 1 x 10^~3, 5 x 10^"4, or 1 x 10^~4 s^"1. In some embodiments, said anti-myostatin antibody molecule binds to myostatin with a K_Qff slower than about 1 x 10^~3, 5 x 10^"4, or 1 x 10^"4 s^"

1

In some embodiments, said anti-activin antibody molecule binds to activin with a K_on faster than about 1 2 3 3 1 1

x 10", 1 x 10^J, or 5 x 10^J M^~V\ In some embodiments, said anti-myostatin antibody molecule binds to myostatin with a K_on faster than about 1 x 10 2 , 1 x 103 , or 5 x 103 M^'V¹.

In some embodiments, said anti-activin antibody molecule inhibits activin activity, e.g., with a Ki of less than about 10^"5, 10^"6, 10^"7, 10^~8, 5xl0^"9, 10^"9, 5xl0^"10 or 10^"10 M. In some embodiments, said anti-myostatin antibody molecule inhibits myostatin activity, e.g., with a Ki of less than about 10^"5, 10^"6, 10^"7, 10^~8, 5xl0^"9, 10^"9, 5xl0^"10 and 10^"10 M.

In some embodiments, said anti-activin antibody molecule has an IC50 of less than about

100, 10, or 1 nM. In some embodiments, said anti-myostatin antibody molecule has an IC50 of less than about 100, 10, or 1 nM.

In some embodiments, said anti-activin antibody molecule has a t of at least about 10, 20, 30, 40, 50, 60, 120, 240, or 360 minutes. In some embodiments, said anti-myostatin antibody molecule has a t_1/2 of at least about 10, 20, 30, 40, 50, 60, 120, 240, or 360 minutes.

In some embodiments, the activin is activin A. In some embodiments, the activin is activin B. In one aspect, the disclosure features a composition, e.g., a pharmaceutical composition, comprising a bispecific antibody molecule that binds activin and myostatin described herein, e.g., for treating a disorder or condition described herein, in a subject.

In some embodiments, said antibody molecule binds one or more (e.g., 1, 2, 3, 4, 5, or all) of GDF-5, GDF-6, GDF-BMP-2, BMP-4, and BMP-7. In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule binds one, two, or all of GDF-5, GDF-6, GDF-7. In some embodiments, said anti-activin antibody molecule and/or anti- myostatin antibody molecule binds one, two, or all of BMP-2, BMP-4, and BMP-7. In one embodiment, said antibody molecule binds one or more of: GDF-9 and GDF-11.

In some embodiments, said antibody molecule does not modulate, e.g., increase activity of, one or more (e.g., 1, 2, 3, 4, 5, or all) of GDF-5, GDF-6, GDF-7, BMP-2, BMP-4, and BMP-7. In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule does not modulate, e.g., increase activity of, one, two, or all of GDF-5, GDF-6, GDF-7. In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule does not modulate, e.g., increase activity of, one, two, or all of BMP-2, BMP-4, and BMP-7. In one embodiment, said antibody molecule does not modulate, e.g., inhibit activity of, one or more of: GDF-9 and GDF- 11.

In some embodiments, said antibody molecule modulates, e.g., increases activity of, one or more (e.g., 1, 2, 3, 4, 5, or all) of GDF-5, GDF-6, GDF-7, BMP-2, BMP-4, and BMP-7. In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule modulates, e.g., increases activity of, one, two, or all of GDF-5, GDF-6, GDF-7. In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule modulates, e.g., increases activity of, one, two, or all of BMP-2, BMP-4, and BMP-7. In one embodiment, said antibody molecule modulates, e.g., inhibits activity of, one or more of: GDF-9 and GDF- 11.

In some embodiments, said antibody molecule binds to activin and/or myostatin with a binding affinity of at least about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, or 10¹¹ M . In some embodiments, said antibody molecule binds to activin and/or myostatin with a K_Qff slower than about 1 x 10^" , 5 X 10^"4, or l x l0^"4 s^_1. In some embodiments, said antibody molecule binds to activin and/or

2 3 3 -1 -1

myostatin with a K_on faster than about 1 x 10 , 1 x 10 , or 5 x 10 M s^" . In some embodiments, said antibody molecule inhibits activin activity and/or myostatin activity, e.g., with a Ki of less than about 10^"5, 10^"6, 10^"7, 10^"8, 5xl0^"9, 10^"9, 5xl0^"10, or 10^"10 M. In some embodiments, said antibody molecule has an IC50 for activin and/or myostatin of less than about 100, 10, or 1 nM. In some embodiments, said antibody molecule has an affinity for activin and/or myostatin characterized by a K_D of less than about 100 or 10 nM, or about 3, 5, 7, 9, or 10 nM.

In some embodiments, said antibody molecule is formulated for subcutaneous, intramuscular, or intravenous administration. In some embodiments, said antibody molecule is formulated for administeration twice weekly, weekly, bi-weekly, or monthly.

In some embodiments, said anti-activin antibody molecule and/or anti-myo statin antibody molecule is for use, e.g., in treating a disorder or condition described herein.

In another aspect, the disclosure features a composition, e.g., a pharmaceutical composition, comprising an anti-activin antibody molecule and an anti-myostatin antibody molecule, e.g., an anti-activin antibody molecule described herein and an anti-myostatin antibody molecule described herein, e.g., for treating a disorder or condition described herein, in a subject.

In some embodiments, the composition comprises an effective amount of a combination of an anti-activin antibody molecule and an anti-myostatin antibody molecule.

In some embodiments, said anti-activin antibody molecule and an anti-myostatin antibody molecule are administered in the same dosage form. In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule does not bind or substantially bind one or more (e.g., 1, 2, 3, 4, 5, or all) of GDF-5, GDF-6, GDF-7, BMP-2, BMP-4, and BMP-7. In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule does not bind or substantially bind one, two, or all of GDF-5, GDF-6, GDF-7. In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule does not bind or substantially bind one, two, or all of BMP-2, BMP-4, and BMP-7. In one embodiment, said antibody molecule does not bind or substantially bind one or more of: GDF-9 and GDF- 11.

In some embodiments, said anti-activin antibody molecule binds to activin with a binding affinity of at least about 10⁵, 10⁶, 10⁷,10⁸, 10⁹, 10¹⁰, or 10¹¹ M . In some embodiments, said anti-myostatin antibody molecule binds to myostatin with a binding affinity of at least about 10⁵, 10⁶, 10⁷,10⁸, 10⁹, 10¹⁰, or 10¹¹ M^"1.

1

In some embodiments, said anti-activin antibody molecule binds to activin with a K_on faster than about 1 2 3 or 5 3 1 1

x 10", 1 x 10^J, x 10^J M^~V\ In some embodiments, said anti-myostatin antibody molecule binds to myostatin with a K_on faster than about 1 x 10 2 , 1 x 103 , or 5 x 103 M^'V¹.

In some embodiments, said anti-activin antibody molecule inhibits activin activity, e.g., with a Ki of less than about 10^"5, 10^"6, 10^"7, 10^~8, 5xl0^"9, 10^"9, 5xl0^"10, or 10^"10 M. In some embodiments, said anti-myostatin antibody molecule inhibits myostatin activity, e.g., with a Ki of less than about 10^"5, 10^"6, 10^"7, 10^~8, 5xl0^"9, 10^"9, 5xl0^"10, or 10^"10 M.

In some embodiments, said antibody molecule is formulated for subcutaneous, intramuscular, or intravenous administration. In some embodiments, said anti-activin antibody molecule and/or anti-myostatin antibody molecule is formulated for administeration twice weekly, weekly, bi-weekly, or monthly. In some embodiments, said composition is for use in treating a subject having a disorder or condition described herein.

In one aspect, the disclosure features a composition comprising an antibody molecule that binds activin, e.g., an anti-activin antibody molecule described herein, for use in combination with an anti-myostatin antibody molecule, e.g., an anti-myostatin antibody molecule described herein, for the treatment of a disorder or condition described herein, in a subject.

In some embodiments, the composition comprises an effective amount of an antibody molecule that binds activin.

In some embodiments, said anti-activin antibody molecule and said anti-myostatin antibody molecule are administered to said subject at the same time or within about 240, 180, 120, 90, 60, 30, 15, 10, 5, or 1 minute of one another, e.g., such that there is overlap of an effect of each antibody molecule on said subject. In some embodiments, said anti-activin antibody molecule and said anti-myostatin antibody molecule are administered sufficiently close together such that a combinatorial (e.g., synergistic) effect is achieved, e.g., greater than an additive effect, e.g., at least 25, 50, 75, 100, 200, 300, 400, or 500% greater than an additive effect. In another aspect, the disclosure features a composition comprising an anti-myostatin antibody molecule, e.g., an anti-myostatin antibody molecule described herein, for use in combination with an anti-activin antibody molecule, e.g., an anti-activin antibody molecule described herein, for the treatment of a disorder or condition described herein, in a subject.

In some embodiments, the composition comprises an effective amount of an anti- myostatin antibody molecule.

In one aspect, the disclosure features a kit comprising a bispecific antibody molecule that binds activin and myostatin described herein. In some embodiments, the kit comprises an instruction for using the antibody molecule in a method described herein, e.g., to treat a disorder or condition described herein, in a subject.

In some embodiments, said the kit comprises instructions for administering the antibody molecule, e.g., by subcutaneous, intramuscular, or intravenous administration. In some embodiments, the kit comprises instructions for administering the antibody molecule twice weekly, weekly, bi-weekly, or monthly.

In some embodiments, said disorder or condition is a metabolic disorder or a symptom thereof. In some embodiments, said metabolic disorder is diabetes, e.g., Type II diabetes. In some embodiments, said metabolic disorder is obesity. In some embodiments, the subject has one or more symptoms of: high blood sugar, insulin resistance, glucose intolerance, abnormal lipid levels (e.g., decreased high-density lipoprotein (HDL) level, increased levels of

triglycerides and low-density lipoprotein (LDL)), and high blood pressure. In some

embodiments, said disorder or condition is non-alcoholic fatty liver disease (NAFLD), e.g., nonalcoholic steatohepatitis (NASH).

In some embodiments, said disorder or condition is characterized by a need for increased fat browning, increased brown adipose tissue (BAT), or increased thermogenesis. In some embodiments, said disorder or condition is characterized by a need for increased muscle mass.

In some embodiments, said disorder or condition comprises frailty, e.g., frailty associated with or arising from decreased muscle mass or strength.

In some embodiments, said disorder or condition is characterized by loss of muscle mass or function, e.g., arising from insufficient use of muscle, e.g., associated with or arising from bed rest or other inactivity arising from or associated with age, disability, or medical condition, or a medical or surgical procedure.

In some embodiments, said disorder or condition comprises an acquired or inherited disorder or condition of the muscles, e.g., a muscular dystrophy, e.g., Duchenne muscular dystrophy, spinal muscular atrophy, or amyotrophic lateral sclerosis (ALS).

In another aspect, the disclosure features a kit comprising an anti-activin antibody molecule and an anti-myostatin antibody molecule, e.g., an anti-activin antibody molecule described herein and an anti-myostatin antibody molecule described herein.

In some embodiments, the composition comprises an effective amount of a combination of an anti-activin antibody molecule and an anti-myostatin antibody molecule. In some embodiments, the kit comprises an anti-activin antibody molecule and an anti-myostatin antibody molecule in the same dosage form.

In some embodiments, the kit comprises an anti-activin antibody molecule and an anti- myostatin antibody molecule as separate dosage forms. In some embodiments, the kit comprises instructions for administration of said anti- activin antibody molecule and said anti-myostatin antibody molecule, e.g., administration to said subject at the same time or within about 240, 180, 120, 90, 60, 30, 15, 10, 5, or 1 minute of one another, e.g., such that there is overlap of an effect of each antibody molecule on said subject. In some embodiments, said anti-activin antibody molecule and said anti-myostatin antibody molecule are administered sufficiently close together such that a combinatorial (e.g., synergistic) effect is achieved, e.g., greater than an additive effect, e.g., at least 25, 50, 75, 100, 200, 300, 400, or 500% greater than an additive effect.

In some embodiments, the kit comprises an instruction for using the anti-activin antibody molecule and anti-myostatin antibody molecule in a method described herein, e.g., to treat a disorder or condition described herein, in a subject.

In some embodiments, said disorder or condition comprises an acquired or inherited disorder or condition of the muscles, e.g., a muscular dystrophy, e.g., Duchenne muscular dystrophy, spinal muscular atrophy, or amyotrophic lateral sclerosis (ALS). In one aspect, the disclosure features an article of manufacture comprising a bispecific antibody molecule described herein.

In one embodiment, the antibody molecule described herein, is formulated for subcutaneous or intramuscular administration. In one embodiment, the subcutaneous or intramuscular formulation is a sterile, preservative-free solution that includes the antibody molecule that binds activin and myostatin. In one embodiment, the article of manufacture, e.g., a device described herein (e.g., a syringe or injector pen for subcutaneous administration) contains a subcutaneous formulation comprising an antibody molecule that binds activin and myostatin described herein. In one embodiment, the article of manufacture is a single-use, prefilled pen or as a single-use, prefilled glass syringe (e.g., a pen or syringe described herein. In one embodiment, the article of manufacture is filled with 1 mL of a subcutaneous formulation comprising the antibody molecule that binds activin and myostatin.

In another aspect, the disclosure features an article of manufacture comprising an anti- activin antibody molecule and an anti-myo statin antibody molecule, e.g., an anti-activin antibody molecule described herein and an anti-myostatin antibody molecule described herein.

In one embodiment, the antibody molecule that binds activin and the antibody molecule that binds myostatin, are formulated for subcutaneous or intramuscular administration. In one embodiment, the subcutaneous or intramuscular formulation is a sterile, preservative-free solution that includes the antibody molecule that binds activin and antibody molecule that binds myostatin. In one embodiment, the article of manufacture, e.g., a device described herein (e.g., a syringe or injector pen for subcutaneous administration) contains a subcutaneous formulation comprising an antibody molecule that binds activin and an antibody molecule that binds myostatin. In one embodiment, the article of manufacture is a single-use, prefilled pen or as a single-use, prefilled glass syringe (e.g., a pen or syringe described herein. In one embodiment, the article of manufacture is filled with 1 mL of a subcutaneous formulation comprising the antibody molecule that binds activin and the antibody molecule that binds myostatin.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts the body weight of mice treated with neutralizing antibodies to activin A, myostatin, or a combination of neutralizing antibodies to activin A and myostatin.

FIG. 2 depicts the percentage body weight change of mice treated with neutralizing antibodies to activin A, myostatin, or a combination of neutralizing antibodies to activin A and myostatin. FIG. 3 depicts the lean mass of mice treated with neutralizing antibodies to activin A, myostatin, or a combination of neutralizing antibodies to activin A and myostatin.

FIG. 4 depicts the wet weight of different muscles from mice treated with neutralizing antibodies to activin A, myostatin, or a combination of neutralizing antibodies to activin A and myostatin.

FIG. 5 depicts the weight of fat mass of mice treated with neutralizing antibodies to activin A, myostatin, or a combination of neutralizing antibodies to activin A and myostatin.

FIG. 6 depicts the weight of inguinal fat pad from mice treated with neutralizing antibodies to activin A, myostatin, or a combination of neutralizing antibodies to activin A and myostatin. FIG. 7 depicts the weight of epididymal fat pad from mice treated with neutralizing antibodies to activin A, myostatin, or a combination of neutralizing antibodies to activin A and myostatin. FIG. 8 depicts the induction in brown fat program in the inguinal fat of mice treated with neutralizing antibodies to activin A, myostatin, or a combination of neutralizing antibodies to activin A and myostatin.

FIG. 9 depicts the induction in brown fat program in the epididymal fat of mice treated with neutralizing antibodies to activin A, myostatin, or a combination of neutralizing antibodies to activin A and myostatin.

FIG. 10 depicts the induction in brown fat program in the intra-scapular brown fat of mice treated with neutralizing antibodies to activin A, myostatin, or a combination of neutralizing antibodies to activin A and myostatin.

DETAILED DESCRIPTION

The present disclosure provides antibody molecules that bind to activin, e.g., activin A and/or activin B, and/or myostatin and, in some instances, inhibit activin, e.g., activin A and/or activin B, and/or myostatin activity. The antibody molecules described herein can be used to treat a disorder or condition, e.g., a disorder or condition described herein.

The term "antibody molecule" refers to an antibody or antigen binding fragment thereof. An "antibody molecule that binds activin and/or myostatin" refers to an antibody molecule that can interact with activin (e.g., activin A and/or activin B) and/or myostatin.

The term "antibody" refers to a protein that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')₂, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments (de Wildt et al, Eur J Immunol. 1996; 26(3):629-639)) as well as complete antibodies. An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). Antibodies may be from any source, but primate (human and non-human primate) and primatized are preferred.

The VH and VL regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" ("FR"). The extent of the framework region and CDRs has been precisely defined (see, Kabat, E.A., et al. (1991) Sequences of Proteins of

Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). Kabat definitions are used herein. Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

As used herein, an "immunoglobulin variable domain sequence" refers to an amino acid sequence which can form the structure of an immunoglobulin variable domain such that one or more CDR regions are positioned in a conformation suitable for an antigen binding site. For example, the sequence may include all or part of the amino acid sequence of a naturally- occurring variable domain. For example, the sequence may omit one, two or more N- or C- terminal amino acids, internal amino acids, may include one or more insertions or additional terminal amino acids, or may include other alterations. In one embodiment, a polypeptide that includes immunoglobulin variable domain sequence can associate with another immunoglobulin variable domain sequence to form an antigen binding site, e.g., a structure that preferentially interacts with an activin protein, e.g., a domain of activin described herein, and/or a structure that preferentially interacts with a myostatin protein, e.g., a domain of myostatin described herein.

The VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. In IgGs, the heavy chain constant region includes three

immunoglobulin domains, CHI, CH2 and CH3. The light chain constant region includes a CL domain. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system {e.g., effector cells) and the first component (Clq) of the classical complement system. The light chains of the immunoglobulin may be of types kappa or lambda. In one embodiment, the antibody is glycosylated. An antibody can be functional for antibody-dependent cytotoxicity and/or complement-mediated cytotoxicity.

One or more regions of an antibody can be human or effectively human. For example, one or more of the variable regions can be human or effectively human. For example, one or more of the CDRs can be human, e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3. Each of the light chain CDRs can be human. HC CDR3 can be human. One or more of the framework regions can be human, e.g., FR1, FR2, FR3, and FR4 of the HC or LC. For example, the Fc region can be human. In one embodiment, all the framework regions are human, e.g., derived from a human somatic cell, e.g., a hematopoietic cell that produces immunoglobulins or a non-hematopoietic cell. In one embodiment, the human sequences are germline sequences, e.g., encoded by a germline nucleic acid. In one embodiment, the framework (FR) residues of a selected Fab can be converted to the amino-acid type of the corresponding residue in the most similar primate germline gene, especially the human germline gene. One or more of the constant regions can be human or effectively human. For example, at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of an immunoglobulin variable domain, the constant region, the constant domains (CHI, CH2, CH3, CL1), or the entire antibody can be human or effectively human.

All or part of an antibody can be encoded by an immunoglobulin gene or a segment thereof. Exemplary human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the many immunoglobulin variable region genes. Full-length immunoglobulin "light chains" (about 25 KDa or about 214 amino acids) are encoded by a variable region gene at the NH2- terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH— terminus. Full-length immunoglobulin "heavy chains" (about 50 KDa or about 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids). The length of human HC varies considerably because HC CDR3 varies from about 3 amino-acid residues to over 35 amino-acid residues.

The term "antigen-binding fragment" of a full length antibody refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to a target of interest. Examples of binding fragments encompassed within the term "antigen-binding fragment" of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')₂ fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) that retains functionality. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv). See, e.g., US patents 5,260,203, 4,946,778, and 4,881,175; Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

Antibody fragments can be obtained using any appropriate technique including conventional techniques known to those with skill in the art.

The term "monospecific antibody molecule" refers to an antibody molecule that displays a single binding specificity and affinity for one particular target, e.g., epitope. This term includes a "monoclonal antibody" or "monoclonal antibody composition," which as used herein refer to a preparation of antibodies or fragments thereof of single molecular composition, irrespective of how the antibody was generated.

The term "monospecific variable region" refers to a variable region that displays a single binding specificity and affinity for one particular target, e.g., epitope.

The term "activin/myo statin-binding antibody molecule" as used herein refers to an antibody molecule that displays binding specificity and affinity for activin and myostatin. As is discussed below, it comprises binding specificity and affinity for two epitopes, e.g., one on activin and one on myostatin.

As used herein, the term "bispecific antibody molecule" refers an antibody molecule that comprises at least two variable regions, or antigen-binding fragments thereof, one of which binds to activin and one of which binds to myostatin. In some embodiments variable each of which differs from the other by at least 1, 2, 3, 4, or 5 amino acid residues within the CDR regions. For example, an activin and myostatin bispecific antibody molecule can comprise at least one variable region, or antigen binding fragment thereof, which binds to activin, and at least one variable region, or antigen binding fragment thereof, which binds to myostatin. In some embodiments, the CDRs of the two variable regions, or antigen binding fragments thereof, differ from one another by at least 1, 2, 3, 4, or 5 amino acid residues within the CDRs. "Specific," as used herein refers to a binding affinity of at least about 10⁵, 10⁶, 10⁷,10⁸, 10⁹, 10¹⁰, or 10¹¹ M^"1.

An "effectively human" immunoglobulin variable region is an immunoglobulin variable region that includes a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. An "effectively human" antibody is an antibody that includes a sufficient number of human amino acid positions such that the antibody does not elicit an immunogenic response in a normal human.

A "humanized" immunoglobulin variable region is an immunoglobulin variable region that is modified to include a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. Descriptions of "humanized" immunoglobulins include, for example, US 6,407,213 and US 5,693,762.

As used herein, "binding affinity" refers to the apparent association constant or K_a. The K_a is the reciprocal of the dissociation constant (Ka). An antibody molecule may, for example, have a binding affinity of at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, or 10¹¹ M^"1 for a particular target molecule, e.g., activin (e.g., activin A and/or activin B) and/or myostatin. Higher affinity binding of an antibody molecule to a first target relative to a second target can be indicated by a higher K_a (or a smaller numerical value Ka) for binding the first target than the K_a (or numerical value ¾) for binding the second target. In such cases, the antibody has specificity for the first target (e.g., a protein in a first conformation or mimic thereof) relative to the second target (e.g., the same protein in a second conformation or mimic thereof; or a second protein). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 5000, 10⁴, or 10⁵ fold.

Binding affinity can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in TRIS-buffer (50 mM TRIS, 150 mM NaCl, 5 mM CaCl₂ at pH 7.5).These techniques can be used to measure the concentration of bound and free antibody molecule as a function of antibody molecule (or target) concentration. The concentration of bound antibody molecule ([Bound]) is related to the concentration of free antibody molecule ([Free]) and the concentration of binding sites for the antibody molecule on the target where (N) is the number of binding sites per target molecule by the following equation:

[Bound] = N · [Free]/((1/Ka) + [Free]).

It is not always necessary to make an exact determination of K_a, though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to K_a, and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay. The inhibition constant (Ki) provides a measure of inhibitor potency; it is the concentration of inhibitor required to reduce enzyme activity by half and is not dependent on enzyme or substrate concentrations. The apparent Ki (Ki,app) is obtained at different substrate concentrations by measuring the inhibitory effect of different concentrations of inhibitor (e.g., inhibitory antibody molecule) on the extent of the reaction (e.g., enzyme activity); fitting the change in pseudo-first order rate constant as a function of inhibitor concentration to the Morrison equation (Equation 1) yields an estimate of the apparent Ki value. The Ki is obtained from the y- intercept extracted from a linear regression analysis of a plot of Ki,app versus substrate concentration.

Equation 1

Where v = measured velocity; vo = velocity in the absence of inhibitor; Κ_ίι£φΡ = apparent inhibition constant; I = total inhibitor concentration; and E = total enzyme concentration.

An "isolated composition" refers to a composition (e.g., protein) that is removed from at least 90% of at least one component of a natural sample from which the isolated composition can be obtained. Compositions produced artificially or naturally can be "compositions of at least" a certain degree of purity if the species or population of species of interests is at least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, 99, or 99.5% pure on a weight-weight basis.

An "epitope" refers to the site on a target compound that is bound by an antibody molecule (e.g., an antibody fragment (e.g., a Fab) or full length antibody). In the case where the target compound is a protein, the site can be entirely composed of amino acid components, entirely composed of chemical modifications of amino acids of the protein (e.g., glycosyl moieties), or composed of combinations thereof. Overlapping epitopes include at least one common amino acid residue, glycosyl group, phosphate group, sulfate group, or other molecular feature.

Calculations of "homology" or "sequence identity" between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.

In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence. For example, the reference sequence may be the length of the immunoglobulin variable domain sequence.

As used herein, the term "substantially identical" (or "substantially homologous") is used herein to refer to a first amino acid or nucleic acid sequence that contains a sufficient number of identical or equivalent (e.g., with a similar side chain, e.g., conserved amino acid substitutions) amino acid residues or nucleotides to a second amino acid or nucleic acid sequence such that the first and second amino acid or nucleic acid sequences have (or encode proteins having) similar activities, e.g., a binding activity, a binding preference, or a biological activity. In the case of antibodies, the second antibody has the same specificity and has at least 50%, at least 25%, or at least 10% of the affinity relative to the same antigen.

Sequences similar or homologous (e.g., at least about 85% sequence identity) to the sequences disclosed herein are also part of this application. In some embodiments, the sequence identity can be about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. In addition, substantial identity exists when the nucleic acid segments hybridize under selective hybridization conditions (e.g., highly stringent hybridization conditions), to the complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.

As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: (1) low stringency hybridization conditions in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0.1% SDS at least at 50°C (the temperature of the washes can be increased to 55°C for low stringency conditions); (2) medium stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; (3) high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C; and (4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified. The disclosure includes nucleic acids that hybridize with low, medium, high, or very high stringency to a nucleic acid described herein or to a complement thereof, e.g., nucleic acids encoding an antibody molecule described herein. The nucleic acids can be the same length or within 30, 20, or 10% of the length of the reference nucleic acid. The nucleic acid can correspond to a region encoding an immunoglobulin variable domain sequence described herein.

An antibody molecule that binds activin and/or myostatin may have mutations (e.g., at least one, two, or four, and/or less than 15, 10, 5, or 3) relative to an antibody molecule described herein (e.g., conservative or non-essential amino acid substitutions), which do not have a substantial effect on protein function. Whether or not a particular substitution will be tolerated, i.e., will not adversely affect biological properties, such as binding activity can be predicted, e.g., by evaluating whether the mutation is conservative or by the method of Bowie, et al. (1990) Science 247: 1306- 1310.

A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). It is possible for many framework and CDR amino acid residues to include one or more conservative substitutions.

A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of the binding agent, e.g., the antibody, without abolishing or more preferably, without substantially altering a biological activity, whereas changing an "essential" amino acid residue results in a substantial loss of activity. Statistical significance can be determined by any art known method. Exemplary statistical tests include: the Students t-test, Mann Whitney U non-parametric test, and Wilcoxon non-parametric statistical test. Some statistically significant relationships have a P value of less than 0.05 or 0.02. Particular antibody molecules may show a difference, e.g., in specificity or binding, that are statistically significant (e.g., P value < 0.05 or 0.02). The terms "induce", "inhibit", "potentiate", "elevate", "increase", "decrease" or the like, e.g., which denote distinguishable qualitative or quantitative differences between two states, and may refer to a difference, e.g., a statistically significant difference, between the two states.

As used herein, the term "subject" or "patient" refers to any organism to which an antibody molecule or a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include humans and animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and domesticated animals). Activin/Myostatin Antibody Molecules

The disclosure provides antibody molecules that bind to activin (e.g., activin A, e.g., human activin A and/or activin B, e.g., human activin B) and/or myostatin (e.g., human myostatin). For example, the antibody molecule that binds activin and/or myostatin includes a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC)

immunoglobulin variable domain sequence. The antibody molecule that binds both activin and myostatin can be, e.g., a bispecific antibody molecule. Exemplary antibody molecules that bind activin and/or myostatin are described herein.

The antibody molecule that binds activin and/or myostatin may be an isolated protein (e.g., at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% free of other proteins).

The antibody molecule that binds activin and/or myostatin may additionally inhibit activin and/or myostatin. The antibody molecule can inhibit an activity of activin and/or myostatin. In one embodiment, the antibody molecule binds an active site of activin, e.g., the antibody molecule contacts residues in or near the active site of activin, and/or the antibody molecule binds an active site of myostatin, e.g., the antibody molecule contacts residues in or near the active site of myostatin. In some embodiments, the antibody molecule does not contact residues in or near the active site of activin but instead binds elsewhere on activin and causes a steric change in activin that affects (e.g., inhibits) its activity. In other embodiments, the antibody molecule does not contact residues in or near the active site of myostatin but instead binds elsewhere on myostatin and causes a steric change in myostatin that affects (e.g., inhibits) its activity. Antibody molecules that bind activin and/or myostatin may be antibodies. Activin- or myostatin-binding antibodies may have their HC and LC variable domain sequences included in a single polypeptide (e.g., scFv), or on different polypeptides (e.g., IgG or Fab). Activin

Activins are dimers composed of two identical or very similar beta subunits linked by one or more disulfide bonds. Activin A is a homodimer of beta-A. Activin B is a homodimer of beta-B. Activin AB is a dimer of beta-A and beta-B. Activin beta-A and beta-B are identical to the two beta subunits of inhibins, which are related proteins that have almost directly opposite biological effects to activins. Activin belongs to the TGF-β protein superfamily.

Activins activate the secretion of follitropin by the pituitary gland. Activins are involved in regulating a number of diverse functions such as hypothalamic and pituitary hormone secretion, gonadal hormone secretion, germ cell development and maturation, erythroid differentiation, insulin secretion, nerve cell survival, embryonic axial development or bone growth, depending on their subunit composition.

An exemplary amino acid sequence of human activin beta-A chain precursor (also referred to herein as "human activin A")is shown below.

GenBank Accession: NP_002183; Version: NP_002183.1 GL4504699

1 mpllwlrgfl lascwiivrs sptpgseghs aapdcpscal aalpkdvpns qpemveavkk 61 hilnmlhlkk rpdvtqpvpk aallnairkl hvgkvgengy veieddigrr aemnelmeqt

121 seiitfaesg tarktlhfei skegsdlsvv eraevwlflk vpkanrtrtk vtirlfqqqk 181 hpqgsldtge eaeevglkge rselllsekv vdarkstwhv fpvsssiqrl ldqgkssldv 241 riaceqcqes gaslvllgkk kkkeeegegk kkgggeggag adeekeqshr pflmlqarqs 301 edhphrrrrr glecdgkvni cckkqffvsf kdigwndwii apsgyhanyc egecpshiag 361 tsgsslsfhs tvinhyrmrg hspfanlksc cvptklrpms mlyyddgqni ikkdiqnmiv

421 eecgcs (SEQ ID NO: 1)

An exemplary amino acid sequence of human activin beta-B chain precursor (also referred to herein as "human activin B") is shown below.

GenBank Accession: NP_002184; Version: NP_002184.2 GI: 154813204

1 mdglpgralg aacllllaag wlgpeawgsp tppptpaapp pppppgspgg sqdtctscgg

61 frrpeelgrv dgdfleavkr hilsrlqmrg rpnithavpk aamvtalrkl hagkvredgr

121 veiphldgha spgadgqerv seiisfaetd glassrvrly ffisnegnqn lfvvqaslwl

181 ylkllpyvle kgsrrkvrvk vyfqeqghgd rwnmvekrvd lkrsgwhtfp lteaiqalfe 241 rgerrlnldv qcdscqelav vpvfvdpgee shrpfvvvqa rlgdsrhrir krglecdgrt

301 nlccrqqffi dfrligwndw iiaptgyygn ycegscpayl agvpgsassf htavvnqyrm

361 rglnpgtvns cciptklstm smlyfddeyn ivkrdvpnmi veecgca (SEQ ID NO: 2)

An exemplary activin protein against which activin antibody molecules may be developed can include the human activin beta-A chain and/or beta-B chain amino acid sequences or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to one of these sequences, or a fragment thereof, e.g., a fragment without the signal sequence or prodomain.

Myostatin

Myostatin (also known as growth differentiation factor 8, abbreviated GDF-8) is a protein that belongs to the bone morphogenetic protein (BMP) family and the TGF-beta superfamily. This group of proteins is characterized by a polybasic proteolytic processing site which is cleaved to produce a mature protein containing seven conserved cysteine residues. The members of this family are regulators of cell growth and differentiation in both embryonic and adult tissues. Myostatin is a secreted growth differentiation factor that inhibits muscle differentiation and growth in the process known as myogenesis. Myostatin is produced primarily in skeletal muscle cells, circulates in the blood and acts on muscle tissue, by binding a cell-bound receptor called the activin type II receptor. Myostatin can bind to activin receptor type lib (actRIIb) and to a lesser extent activin receptor type Ila (actRIIa). Animals lacking myostatin or animals treated with substances that block the activity of myostatin have significantly larger muscles. Mutations in both copies of the human myostatin gene results in individuals that have significantly more muscle mass and hence are considerably stronger than normal.

An exemplary amino acid sequence of human myostatin precursor is shown below.

GenBank Accession: NP_005250; Version: NP_005250.1 GI: 4885259

1 mqklqlcvyi ylfmlivagp vdlnenseqk envekeglcn actwrqntks srieaikiqi

61 lsklrletap niskdvirql lpkapplrel idqydvqrdd ssdgsleddd yhattetiit 121 mptesdflmq vdgkpkccff kfsskiqynk vvkaqlwiyl rpvetpttvf vqilrlikpm 181 kdgtrytgir slkldmnpgt giwqsidvkt vlqnwlkqpe snlgieikal denghdlavt 241 fpgpgedgln pflevkvtdt pkrsrrdfgl dcdehstesr ccrypltvdf eafgwdwiia 301 pkrykanycs gecefvflqk yphthlvhqa nprgsagpcc tptkmspinm lyfngkeqii

361 ygkipamvvd rcgcs

An exemplary myostatin protein against which myostatin antibody molecules may be developed can include the human myostatin amino acid sequences or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to one of these sequences, or a fragment thereof, e.g., a fragment without the signal sequence or prodomain.

Display Libraries

The antibody molecules described herein can be identified, e.g., using a display library, and/or an antibody molecule described herein can be used to identify other antibody molecules with similar biological activity. In one embodiment, using antibodies (e.g., Fabs, scFvs, IgG, or IgM) displayed on genetic packages (such as phage, yeast, E. coli, or ribosomes), antibodies that bind to activin and/or myostatin can be selected. This procedure can have many variations. For example, in one variation, antibodies that bind to activin and/or myostatin can be screened in the presence of one of the antibody molecules described herein and some depleted, and then antibodies that bind to the free activin and/or myostatin depletion can be used only in the second and subsequent rounds, or activin (or myostatin) can be immobilized and soluble myostatin-antibody (or activin- antibody) complexes can be introduced in the solution as decoy for antibodies that bind to sites other than the binding site of the antibody molecules described herein.

In one embodiment, using antibodies that come from mice, the antibody molecules described herein can be used as a screening tool. For example, antibodies that bind activin and/or myostatin in the absence of an antibody molecule described herein but that are blocked by an antibody molecule described herein can then be tested for inhibitory activity.

In one embodiment, the antibody molecule described herein can be used to select or screen antibody molecules that share the multispecific binding, e.g., binding to human activin and human myostatin with high affinity and specificity. In another embodiment, the antibody molecules described herein can be used to select or screen antibody molecules that are specific to human activin, human myostatin, mouse activin, and/or mouse myostatin.

A display library is a collection of entities; each entity includes an accessible antibody molecule component and a recoverable component that encodes or identifies the antibody molecule component. The antibody molecule component is varied so that different amino acid sequences are represented. The antibody molecule component can be of any length, e.g. from three amino acids to over 300 amino acids. A display library entity can include more than one antibody molecule component, for example, the two polypeptide chains of a sFab. In one exemplary implementation, a display library can be used to identify antibody molecules that bind to activin and/or myostatin. For example, the antibody molecule component of each member of the library is probed with activin (e.g., a domain of activin or other fragment) and if the antibody molecule component binds to activin, the display library member is identified, typically by retention on a support. As another example, the antibody molecule component of each member of the library is probed with myostatin (e.g., a domain of myostatin or other fragment) and if the antibody molecule component binds to myostatin, the display library member is identified, typically by retention on a support.

Retained display library members are recovered from the support and analyzed. The analysis can include amplification and a subsequent selection under similar or dissimilar conditions. For example, positive and negative selections can be alternated. The analysis can also include determining the amino acid sequence of the antibody molecule component and purification of the antibody molecule component for detailed characterization.

The retained family members are subjected to a subsequent analysis to recover antibody molecules that also bind to myostatin and/or activin, e.g., using a method described herein.

A variety of formats can be used for display libraries. Examples include the following.

Phage Display: The antibody molecule component is typically covalently linked to a bacteriophage coat protein. The linkage results from translation of a nucleic acid encoding the antibody molecule component fused to the coat protein. The linkage can include a flexible peptide linker, a protease site, or an amino acid incorporated as a result of suppression of a stop codon. Phage display is described, for example, in U.S. 5,223,409; Smith (1985) Science

228: 1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809; de Haard et aZ.(1999) J. Biol. Chem 274: 18218- 30; Hoogenboom et al. (1998) Immunotechnology 4: 1-20; Hoogenboom et a/. (2000) Immunol Today 2:371-8 and Hoet et al. (2005) Nat Biotechnol.23{3)344-&. Bacteriophage displaying the antibody molecule component can be grown and harvested using standard phage preparatory methods, e.g. PEG precipitation from growth media. After selection of individual display phages, the nucleic acid encoding the selected antibody molecule components can be isolated from cells infected with the selected phages or from the phage themselves, after amplification. Individual colonies or plaques can be picked, the nucleic acid isolated and sequenced.

Other Display Formats. Other display formats include cell based display (see, e.g., WO

03/029456), protein-nucleic acid fusions (see, e.g., US 6,207,446), ribosome display (See, e.g., Mattheakis et al. (1994) Proc. Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000) Nat Biotechnol. 18: 1287-92; Hanes et al. (2000) Methods Enzymol. 328:404-30; and Schaffitzel et a/.(1999) J Immunol Methods. 231(1-2): 119-35), and E. coli periplasmic display (Harvey et al. J Immunol Methods. 2006;308(l-2):43-52).

Scaffolds. Scaffolds useful for display include: antibodies {e.g., Fab fragments, single chain Fv molecules (scFV), single domain antibodies, camelid antibodies, and camelized antibodies); T-cell receptors; MHC proteins; extracellular domains {e.g., fibronectin Type III repeats, EGF repeats); protease inhibitors; TPR repeats; trifoil structures; zinc finger domains; DNA-antibody molecules; particularly monomeric DNA antibody molecules; RNA antibody molecules; enzymes, e.g., proteases (particularly inactivated proteases), RNase; chaperones, e.g., thioredoxin and heat shock proteins; intracellular signaling domains (such as SH2 and SH3 domains); linear and constrained peptides; and linear peptide substrates. Display libraries can include synthetic and/or natural diversity. See, e.g., US 2004-0005709. Display technology can also be used to obtain antibody molecules (e.g., antibodies) that bind particular epitopes of a target. This can be done, for example, by using competing non- target molecules that lack the particular epitope or are mutated within the epitope, e.g., with alanine. Such non-target molecules can be used in a negative selection procedure as described below, as competing molecules when binding a display library to the target, or as a pre-elution agent, e.g., to capture in a wash solution dissociating display library members that are not specific to the target.

Iterative Selection. In one preferred embodiment, display library technology is used in an iterative mode. A first display library is used to identify one or more antibody molecules for a target. These identified antibody molecules are then varied using a mutagenesis method to form a second display library. Higher affinity antibody molecules are then selected from the second library, e.g., by using higher stringency or more competitive binding and washing conditions.

In some implementations, the mutagenesis is targeted to regions at the binding interface. If, for example, the identified antibody molecules are antibodies, then mutagenesis can be directed to the CDR regions of the heavy or light chains as described herein. Further, mutagenesis can be directed to framework regions near or adjacent to the CDRs. In the case of antibodies, mutagenesis can also be limited to one or a few of the CDRs, e.g., to make precise step-wise improvements. Exemplary mutagenesis techniques include: error-prone PCR, recombination, DNA shuffling, site-directed mutagenesis and cassette mutagenesis.

In one example of iterative selection, the methods described herein are used to first identify an antibody molecule from a display library that binds activin and/or myostatin with at least a minimal binding specificity for a target or a minimal activity, e.g., an equilibrium dissociation constant for binding of less than 1 nM, 10 nM, or 100 nM. The nucleic acid sequence encoding the initially-identified antibody molecule is used as a template nucleic acid for the introduction of variations, e.g., to identify a second antibody molecule that has enhanced and/or different properties (e.g., binding affinity, kinetics, or stability) relative to the initial antibody molecule. In some embodiments, the initially-identified antibody molecule does not bind to myostatin or activin with at least a minimal binding specificity for a target or a minimal activity, e.g., an equilibrium dissociation constant for binding of less than 1 nM, 10 nM, or 100 nM. In some embodiments, the second antibody molecule binds to activin and myostatin with at least a minimal binding specificity for a target or a minimal activity, e.g., an equilibrium dissociation constant for binding of less than 1 nM, 10 nM, or 100 nM.

Off- Rate Selection. Since a slow dissociation rate can be predictive of high affinity, particularly with respect to interactions between polypeptides and their targets, the methods described herein can be used to isolate antibody molecules with a desired (e.g., reduced) kinetic dissociation rate for a binding interaction to a target.

To select for slow dissociating antibody molecules from a display library, the library is contacted to an immobilized target. The immobilized target is then washed with a first solution that removes non-specifically or weakly bound biomolecules. Then the bound antibody molecules are eluted with a second solution that includes a saturating amount of free target or a target specific high-affinity competing monoclonal antibody, i.e. , replicates of the target that are not attached to the particle. The free target binds to biomolecules that dissociate from the target. Rebinding is effectively prevented by the saturating amount of free target relative to the much lower concentration of immobilized target.

The second solution can have solution conditions that are substantially physiological or that are stringent. Typically, the solution conditions of the second solution are identical to the solution conditions of the first solution. Fractions of the second solution are collected in temporal order to distinguish early from late fractions. Later fractions include biomolecules that dissociate at a slower rate from the target than biomolecules in the early fractions.

Further, it is also possible to recover display library members that remain bound to the target even after extended incubation. These can either be dissociated using chaotropic conditions or can be amplified while attached to the target. For example, phage bound to the target can be contacted to bacterial cells.

Selecting or Screening for Specificity. The display library screening methods described herein can include a selection or screening process that discards display library members that bind to a non-target molecule. Examples of non-target molecules include streptavidin on magnetic beads, blocking agents such as bovine serum albumin, non-fat bovine milk, any capturing or target immobilizing monoclonal antibody, or non-transfected cells which do not express the activin and/or myostatin target.

In one implementation, a so-called "negative selection" step is used to discriminate between the target and a related non-target molecule and a related, but distinct non-target molecule. The display library or a pool thereof is contacted to the non-target molecule.

Members of the sample that do not bind the non-target are collected and used in subsequent selections for binding to the target molecule or even for subsequent negative selections. The negative selection step can be prior to or after selecting library members that bind to the target molecule.

In another implementation, a screening step is used. After display library members are isolated for binding to the target molecule, each isolated library member is tested for its ability to bind to a non-target molecule (e.g., a non-target listed above). For example, a high-throughput ELISA screen can be used to obtain this data. The ELISA screen can also be used to obtain quantitative data for binding of each library member to the target as well as for cross-species reactivity to related targets or subunits of the target (e.g., mouse activin or mouse myostatin) and also under different condition such as pH 6 or pH 7.5. The non-target and target binding data are compared (e.g., using a computer and software) to identify library members that specifically bind to the target.

Other Exemplary Expression Libraries

Other types of collections of proteins (e.g., expression libraries) can be used to identify antibody molecules with a particular property (e.g., ability to bind activin and/or myostatin, and/or ability to modulate activin and/or myostatin), including, e.g., protein arrays of antibodies (see, e.g., De Wildt et al. (2000) Nat. Biotechnol. 18:989-994), lambda gtl 1 libraries, two-hybrid libraries and so forth. Exemplary Libraries

It is possible to immunize a non-human primate and recover primate antibody genes that can be displayed on phage (see below). From such a library, one can select antibodies that bind the antigen used in immunization. See, for example, Vaccine. (2003) 22(2):257-67 or

Immuno genetics. (2005) 57(10):730-738. Thus one could obtain primate antibodies that bind and inhibit activin (or myostatin) by immunizing a chimpanzee or macaque and using a variety of means to select or screen for primate antibodies that bind and inhibit activin (or myostatin). One can also make chimeras of primatized Fabs with human constant regions, see Curr Opin Mol Ther. (2004)6(6):675-83. "PRIMATIZED antibodies, genetically engineered from cynomolgus macaque monkey and human components, are structurally indistinguishable from human antibodies. They may, therefore, be less likely to cause adverse reactions in humans, making them potentially suited for long-term, chronic treatment. See, for example, Curr Opin Investig Drugs. (2001) 2(5):635-8.

One exemplary type of library presents a diverse pool of polypeptides, each of which includes an immunoglobulin domain, e.g., an immunoglobulin variable domain. Of interest are display libraries where the members of the library include primate or "primatized" (e.g., human, non-human primate or "humanized") immunoglobulin domains (e.g., immunoglobulin variable domains) or chimeric primatized Fabs with human constant regions. Human or humanized immunoglobulin domain libraries may be used to identify human or "humanized" antibodies that, for example, recognize human antigens. Because the constant and framework regions of the antibody are human, these antibodies may avoid themselves being recognized and targeted as antigens when administered to humans. The constant regions may also be optimized to recruit effector functions of the human immune system. The in vitro display selection process surmounts the inability of a normal human immune system to generate antibodies against self- antigens.

A typical antibody display library displays a polypeptide that includes a VH domain and a VL domain. An "immunoglobulin domain" refers to a domain from the variable or constant domain of immunoglobulin molecules. Immunoglobulin domains typically contain two β-sheets formed of about seven β-strands, and a conserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay, 1988, Ann. Rev. Immunol. 6:381-405). The display library can display the antibody as a Fab fragment {e.g., using two polypeptide chains) or a single chain Fv {e.g., using a single polypeptide chain). Other formats can also be used.

As in the case of the Fab and other formats, the displayed antibody can include one or more constant regions as part of a light and/or heavy chain. In one embodiment, each chain includes one constant region, e.g., as in the case of a Fab. In other embodiments, additional constant regions are displayed.

Antibody libraries can be constructed by a number of processes (see, e.g., de Haard et al., 1999, J. Biol. Chem. 274: 18218-30; Hoogenboom et al, 1998, Immunotechnology 4: 1-20;

Hoogenboom et al., 2000, Immunol. Today 21:371-378, and Hoet et al. (2005) Nat

Biotechnol.23{3): 344-8. Further, elements of each process can be combined with those of other processes. The processes can be used such that variation is introduced into a single

immunoglobulin domain {e.g., VH or VL) or into multiple immunoglobulin domains {e.g., VH and VL). The variation can be introduced into an immunoglobulin variable domain, e.g., in the region of one or more of CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4, referring to such regions of either and both of heavy and light chain variable domains. The variation(s) may be introduced into all three CDRs of a given variable domain, or into CDR1 and CDR2, e.g., of a heavy chain variable domain. Any combination is feasible. In one process, antibody libraries are constructed by inserting diverse oligonucleotides that encode CDRs into the corresponding regions of the nucleic acid. The oligonucleotides can be synthesized using monomeric nucleotides or trinucleotides. For example, Knappik et al., 2000, J. Mol. Biol. 296:57-86 describe a method for constructing CDR encoding oligonucleotides using trinucleotide synthesis and a template with engineered restriction sites for accepting the oligonucleotides.

In another process, an animal, e.g., a rodent, is immunized with activin or myostatin. The animal is optionally boosted with the antigen to further stimulate the response. Then spleen cells are isolated from the animal, and nucleic acid encoding VH and/or VL domains is amplified and cloned for expression in the display library. In yet another process, antibody libraries are constructed from nucleic acid amplified from naive germline immunoglobulin genes. The amplified nucleic acid includes nucleic acid encoding the VH and/or VL domain. Sources of immunoglobulin-encoding nucleic acids are described below. Amplification can include PCR, e.g., with primers that anneal to the conserved constant region, or another amplification method.

Nucleic acid encoding immunoglobulin domains can be obtained from the immune cells of, e.g., a primate (e.g., a human), mouse, rabbit, camel, or rodent. In one example, the cells are selected for a particular property. B cells at various stages of maturity can be selected. In another example, the B cells are naive.

In one embodiment, fluorescent-activated cell sorting (FACS) is used to sort B cells that express surface-bound IgM, IgD, or IgG molecules. Further, B cells expressing different isotypes of IgG can be isolated. In another preferred embodiment, the B or T cell is cultured in vitro. The cells can be stimulated in vitro, e.g., by culturing with feeder cells or by adding mitogens or other modulatory reagents, such as antibodies to CD40, CD40 ligand or CD20, phorbol myristate acetate, bacterial lipopolysaccharide, concanavalin A, phytohemagglutinin, or pokeweed mitogen.

In another embodiment, the cells are isolated from a transgenic non-human animal that includes a human immunoglobulin locus.

In one preferred embodiment, the cells have activated a program of somatic

hypermutation. Cells can be stimulated to undergo somatic mutagenesis of immunoglobulin genes, for example, by treatment with anti-immunoglobulin, anti-CD40, and anti-CD38 antibodies (see, e.g., Bergthorsdottir et ah, 2001, J. Immunol. 166:2228). In another

embodiment, the cells are naive.

The nucleic acid encoding an immunoglobulin variable domain can be isolated from a natural repertoire by the following exemplary method. First, RNA is isolated from the immune cell. Full length (i.e., capped) mRNAs are separated (e.g. by degrading uncapped RNAs with calf intestinal phosphatase). The cap is then removed with tobacco acid pyrophosphatase and reverse transcription is used to produce the cDNAs.

The reverse transcription of the first (antisense) strand can be done in any manner with any suitable primer. See, e.g., de Haard et ah, 1999, J. Biol. Chem. 274: 18218-30. The primer binding region can be constant among different immunoglobulins, e.g., in order to reverse transcribe different isotypes of immunoglobulin. The primer binding region can also be specific to a particular isotype of immunoglobulin. Typically, the primer is specific for a region that is 3' to a sequence encoding at least one CDR. In another embodiment, poly-dT primers may be used (and may be preferred for the heavy-chain genes). A synthetic sequence can be ligated to the 3' end of the reverse transcribed strand. The synthetic sequence can be used as a primer binding site for binding of the forward primer during PCR amplification after reverse transcription. The use of the synthetic sequence can obviate the need to use a pool of different forward primers to fully capture the available diversity.

The variable domain-encoding gene is then amplified, e.g., using one or more rounds. If multiple rounds are used, nested primers can be used for increased fidelity. The amplified nucleic acid is then cloned into a display library vector.

Secondary Screening Methods

After selecting candidate library members that bind to a target, each candidate library member can be further analyzed, e.g., to further characterize its binding properties for the target, e.g., activin, and/or for binding to another protein, e.g., myostatin. Each candidate library member can be subjected to one or more secondary screening assays. The assay can be for a binding property, a catalytic property, an inhibitory property, a physiological property (e.g., cytotoxicity, renal clearance, immunogenicity), a structural property (e.g., stability,

conformation, oligomerization state) or another functional property. The same assay can be used repeatedly, but with varying conditions, e.g., to determine pH, ionic, or thermal sensitivities.

As appropriate, the assays can use a display library member directly, a recombinant polypeptide produced from the nucleic acid encoding the selected polypeptide, or a synthetic peptide synthesized based on the sequence of the selected polypeptide. In the case of selected Fabs, the Fabs can be evaluated or can be modified and produced as intact IgG proteins.

Exemplary assays for binding properties include the following.

ELISA. Antibody molecules can be evaluated using an ELISA assay. For example, each protein is contacted to a microtitre plate whose bottom surface has been coated with the target, e.g., a limiting amount of the target. The plate is washed with buffer to remove non- specifically bound polypeptides. Then the amount of the antibody molecule bound to the target on the plate is determined by probing the plate with an antibody that can recognize the antibody molecule, e.g., a tag or constant portion of the antibody molecule. The antibody is linked to a detection system (e.g., an enzyme such as alkaline phosphatase or horse radish peroxidase (HRP) which produces a colorimetric product when appropriate substrates are provided).

Homogeneous Binding Assays. The ability of an antibody molecule described herein to bind a target can be analyzed using a homogenous assay, i.e., after all components of the assay are added, additional fluid manipulations are not required. For example, fluorescence resonance energy transfer (FRET) can be used as a homogenous assay (see, for example, Lakowicz et ah, U.S. Patent No. 5,631,169; Stavrianopoulos, et al, U.S. Patent No. 4,868,103). A fluorophore label on the first molecule (e.g., the molecule identified in the fraction) is selected such that its emitted fluorescent energy can be absorbed by a fluorescent label on a second molecule (e.g., the target) if the second molecule is in proximity to the first molecule. The fluorescent label on the second molecule fluoresces when it absorbs to the transferred energy. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay should be maximal. A binding event that is configured for monitoring by FRET can be conveniently measured through standard fluorometric detection means, e.g., using a fluorimeter. By titrating the amount of the first or second binding molecule, a binding curve can be generated to estimate the equilibrium binding constant.

Another example of a homogenous assay is ALPHASCREEN™ (Packard Bioscience, Meriden CT). ALPHASCREEN™ uses two labeled beads. One bead generates singlet oxygen when excited by a laser. The other bead generates a light signal when singlet oxygen diffuses from the first bead and collides with it. The signal is only generated when the two beads are in proximity. One bead can be attached to the display library member, the other to the target.

Signals are measured to determine the extent of binding.

Surface Plasmon Resonance (SPR). The interaction of antibody molecule and a target can be analyzed using SPR. SPR or Biomolecular Interaction Analysis (BIA) detects biospecific interactions in real time, without labeling any of the interactants. Changes in the mass at the binding surface (indicative of a binding event) of the BIA chip result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)). The changes in the refractivity generate a detectable signal, which are measured as an indication of real-time reactions between biological molecules. Methods for using SPR are described, for example, in U.S. Patent No. 5,641,640; Raether, 1988, Surface Plasmons Springer Verlag; Sjolander and Urbaniczky, 1991, Anal. Chem. 63:2338-2345; Szabo et al , 1995, Curr. Opin. Struct. Biol. 5:699-705 and on-line resources provide by BIAcore International AB

(Uppsala, Sweden). BIAcore Flexchip can be used to compare and rank interactions in real time, in terms of kinetics, affinity or specificity without the use of labels.

Information from SPR can be used to provide an accurate and quantitative measure of the equilibrium dissociation constant (Kd), and kinetic parameters, including K_on and K_Qff, for the binding of a antibody molecule to a target. Such data can be used to compare different biomolecules. For example, selected proteins from an expression library can be compared to identify proteins that have high affinity for the target or that have a slow K_Qff. This information can also be used to develop structure- activity relationships (SAR). For example, the kinetic and equilibrium binding parameters of matured versions of a parent protein can be compared to the parameters of the parent protein. Variant amino acids at given positions can be identified that correlate with particular binding parameters, e.g., high affinity and slow K_Qff. This information can be combined with structural modeling (e.g., using homology modeling, energy minimization, or structure determination by x-ray crystallography or NMR). As a result, an understanding of the physical interaction between the protein and its target can be formulated and used to guide other design processes.

Cellular Assays. Antibody molecules can be screened for ability to bind to cells which transiently or stably express and display the target of interest on the cell surface. For example, antibody molecules that bind to activin and/or myostatin can be fluorescently labeled and binding to activin or myostatin in the presence of absence of antagonistic antibody can be detected by a change in fluorescence intensity using flow cytometry, e.g., a FACS machine.

Other Exemplary Methods for Obtaining Antibody Molecules that Bind to Activin and/or Myostatin

In addition to the use of display libraries, other methods can be used to obtain an antibody molecule that bind activin and/or myostatin. For example, activin protein, myostatin protein or a region from either can be used as an antigen in a non-human animal, e.g., a rodent.

In one embodiment, the non-human animal includes at least a part of a human

immunoglobulin gene. For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci. Using the hybridoma

technology, antigen-specific monoclonal antibodies (Mabs) derived from the genes with the desired specificity may be produced and selected. See, e.g., XENOMOUSE™, Green et ah, 1994, Nat. Gen. 7: 13-21; U.S. 2003-0070185, WO 96/34096, published Oct. 31, 1996, and PCT Application No. PCT/US96/05928, filed Apr. 29, 1996. Screening human antibodies from XENOMOUSEs to find a multispecific antibody is possible.

In another embodiment, a monoclonal antibody is obtained from the non-human animal, and then modified, e.g., humanized or deimmunized, and/or used, e.g., as part of a bispecific antibody molecule. Winter describes a CDR-grafting method that may be used to prepare the humanized antibodies (UK Patent Application GB 2188638A, filed on March 26, 1987; US

Patent No. 5,225,539. All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen. Humanized antibodies can be generated by replacing sequences of the Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by

Morrison, S. L., 1985, Science 229: 1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. US Patent Nos. 5,585,089, US 5,693,761 and US 5,693,762. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Numerous sources of such nucleic acid are available. For example, nucleic acids may be obtained from a hybridoma producing an antibody against a predetermined target, as described above. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.

Production of Bispecific Antibody Molecules

Bispecific antibody molecules can be produced by various methods.

General categories of methods which can be used to produce bispecific antibodies include, e.g., (1) coupling the moieties by, e.g., chemical crosslinking; (2) appending heterologous peptides to each of the antigen-binding regions to form fusion or hybrid proteins, and joining the fusion or hybrid proteins via the appended peptides; (3) generating single chain antibodies comprising the two antigenic specificities; or (4) somatic fusion of, e.g., hybridomas. See, e.g., WO 2003/008452.

In the first category of methods to generate bispecific antibodies, a variety of types of moieties can be coupled to form bispecific antibodies. For example, two bivalent antibodies, each specific for a different target, can be separated and the half molecules then rejoined covalently to form a bispecific antibody, using conventional procedures. Such a bispecific antibody comprises a common Fc portion and one Fab portion from each of the parental molecules. Thus, one Fab portion is specific for one target, and the other is specific for a different target. Any of a variety of conventional methods can be used to chemically

couple(cross-link) two polypeptide chains {e.g., antibody moieties). Covalent binding can be achieved either by direct condensation of existing side chains {e.g., the formation of disulfide bonds between cysteine residues) or by the incorporation of external bridging molecules.

In the second category of methods to generate bispecific antibodies, each of two antigen- binding regions, each specific for a different receptor subunit, is appended to another moiety, e.g., any of a variety of heterologous peptides, i.e., peptides which do not occur in immunoglobulins (sometimes designated herein as "peptide linkers" or "fusion domains"), thereby generating hybrid or fusion proteins. The hybrid or fusion proteins are then associated via the appended moieties.

In the third category of methods to generate bispecific antibodies, recombinant techniques are used to generate a single-chain bispecific antibody. Single-chain antibody binding proteins (sFv) are generated for each of two antigen- binding regions of interest by linking the VH and VL chains, or fragments or variants thereof, with a peptide linker; and the two sets of sFv are then joined, also by a peptide linker, to form a bispecific single chain antibody (bsFv).

In the fourth category of methods to generate bispecific antibodies, two different clonal cell lines (e.g., hybridomas or lymphocytes) are fused to form a trioma, quadroma or other polydoma, and the bispecific antibodies which are secreted are isolated. Such bispecific antibodies comprise a common Fc portion and one Fab portion from each of the parental cells (e.g., hybridomas). Conventional methods can be used to fuse such cells are conventional.

Recombinant DNA techniques have also been used to generate "knob into hole" bispecific antibodies. See, e.g., U.S. Patent Application Publication No. 20030078385.

Other methods of generating bispecific antibody molecules are described, e.g., in WO 2013/055958.

In addition to the bispecific antibodies described above, multispecific antibodies can be made by extrapolating any of the above methods, or combinations thereof, to join three or more antibody moieties, in any combination. Some of the possible variations are described as follows. In one embodiment, an Fc region may be modified to include a third antigen-binding region. For example, part or all of an Fc region may be replaced with a third antigen-binding region. Such modifications can be accomplished with conventional genetic engineering techniques. In other embodiments, bivalent mono- or bi-specific antibodies can be cross-linked to one another in a side-by-side, head-to-head or tail -to-tail orientation.

Preferably, a bispecific monoclonal antibody described herein is "isolated," as defined above. Methods to isolate and/or purify a bispecific monoclonal antibody of the invention are conventional and are similar to those described above for the purification of monoclonal antibodies in general. Bispecific monoclonal antibodies prepared by cell fusion can be obtained from either the supernatant of a hybrid hybridoma (or other polydoma) or from the ascites fluid of a mouse injected with the hybrid hybridoma.

If the method of preparation of a bispecific antibody results in the formation of monospecific as well as bispecific antibodies (e.g. , following procedures of chemical coupling), the desired bispecific antibodies can be separated from the monospecific ones by any of a variety of procedures which allow differentiation between the two forms, e.g., passive elution from preparative, non-denaturing acrylamide gels or various conventional chromatographic techniques, e.g., anion- exchange, HPLC, or thiophilic adsorption chromatography. In a most preferred embodiment, each of the antibody moieties is tagged with a different tag, and doubly tagged, bispecific antibodies are separated from singly tagged monospecific antibodies by dual affinity chromatography.

Reducing Immunogenicity of Antibody Molecules that Bind to Activin and/or Myostatin

Antibody molecules that bind activin and/or myostatin (e.g., IgG or Fab antibody molecules) may be modified to reduce immunogenicity. Reduced immunogenicity is desirable in antibody molecules that bind activin and/or myostatin intended for use as therapeutics, as it reduces the chance that the subject will develop an immune response against the therapeutic molecule. Techniques useful for reducing immunogenicity of antibody molecules that bind to activin and/or myostatin include deletion/modification of potential human T cell epitopes and "germlining" of sequences outside of the CDRs (e.g., framework and Fc).

An antibody molecule that binds activin and/or myostatin may be modified by specific deletion of human T cell epitopes or "deimmunization" by the methods disclosed in WO

98/52976 and WO 00/34317. Briefly, the heavy and light chain variable regions of an antibody are analyzed for peptides that bind to MHC Class II; these peptides represent potential T-cell epitopes (as defined in WO 98/52976 and WO 00/34317). For detection of potential T-cell epitopes, a computer modeling approach termed "peptide threading" can be applied, and in addition a database of human MHC class II binding peptides can be searched for motifs present in the VH and VL sequences, as described in WO 98/52976 and WO 00/34317. These motifs bind to any of the 18 major MHC class II DR allotypes, and thus constitute potential T cell epitopes. Potential T-cell epitopes detected can be eliminated by substituting small numbers of amino acid residues in the variable regions, or preferably, by single amino acid substitutions. As far as possible conservative substitutions are made, often but not exclusively, an amino acid common at this position in human germline antibody sequences may be used. Human germline sequences are disclosed in Tomlinson, LA. et al., 1992, J. Mol. Biol. 227:776-798; Cook, G. P. et al, 1995, Immunol. Today Vol. 16 (5): 237-242; Chothia, D. et al, 1992, J. Mol. Bio. 227:799- 817. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, LA. et al. MRC Centre for Protein

Engineering, Cambridge, UK). After the deimmunizing changes are identified, nucleic acids encoding V_R and V_L can be constructed by mutagenesis or other synthetic methods (e.g., de novo synthesis, cassette replacement, and so forth). Mutagenized variable sequence can, optionally, be fused to a human constant region, e.g., human IgGl or κ constant regions. In some cases a potential T cell epitope will include residues which are known or predicted to be important for antibody function. For example, potential T cell epitopes are usually biased towards the CDRs. In addition, potential T cell epitopes can occur in framework residues important for antibody structure and binding. Changes to eliminate these potential epitopes will in some cases require more scrutiny, e.g., by making and testing chains with and without the change. Where possible, potential T cell epitopes that overlap the CDRs are eliminated by substitutions outside the CDRs. In some cases, an alteration within a CDR is the only option, and thus variants with and without this substitution should be tested. In other cases, the substitution required to remove a potential T cell epitope is at a residue position within the framework that might be critical for antibody binding. In these cases, variants with and without this substitution should be tested. Thus, in some cases several variant deimmunized heavy and light chain variable regions are designed and various heavy/light chain combinations tested in order to identify the optimal deimmunized antibody. The choice of the final deimmunized antibody can then be made by considering the binding affinity of the different variants in conjunction with the extent of deimmunization, i.e., the number of potential T cell epitopes remaining in the variable region. Deimmunization can be used to modify any antibody, e.g., an antibody that includes a non-human sequence, e.g., a synthetic antibody, a murine antibody other non-human monoclonal antibody, or an antibody isolated from a display library.

Antibody molecules that bind activin and/or myostatinare "germlined" by reverting one or more non-germline amino acids in framework regions to corresponding germline amino acids of the antibody, so long as binding properties are substantially retained. Similar methods can also be used in the constant region, e.g., in constant immunoglobulin domains.

Antibody molecules that bind to activin and/or myostatin, e.g., an antibody molecule described herein, may be modified in order to make the variable regions of the antibody more similar to one or more germline sequences. For example, an antibody molecule can include one, two, three, or more amino acid substitutions, e.g., in a framework, CDR, or constant region, to make it more similar to a reference germline sequence. One exemplary germlining method can include identifying one or more germline sequences that are similar (e.g., most similar in a particular database) to the sequence of the isolated antibody. Mutations (at the amino acid level) are then made in the isolated antibody, either incrementally or in combination with other mutations. For example, a nucleic acid library that includes sequences encoding some or all possible germline mutations is made. The mutated antibodies are then evaluated, e.g., to identify an antibody that has one or more additional germline residues relative to the isolated antibody and that is still useful (e.g., has a functional activity). In one embodiment, as many germline residues are introduced into an isolated antibody as possible. In one embodiment, mutagenesis is used to substitute or insert one or more germline residues into a framework and/or constant region. For example, a germline framework and/or constant region residue can be from a germline sequence that is similar (e.g., most similar) to the non-variable region being modified. After mutagenesis, activity (e.g., binding or other functional activity) of the antibody molecule can be evaluated to determine if the germline residue or residues are tolerated (i.e. , do not abrogate activity). Similar mutagenesis can be performed in the framework regions.

Selecting a germline sequence can be performed in different ways. For example, a germline sequence can be selected if it meets a predetermined criteria for selectivity or similarity, e.g., at least a certain percentage identity, e.g., at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity. The selection can be performed using at least 2, 3, 5, or 10 germline sequences. In the case of CDR1 and CDR2, identifying a similar germline sequence can include selecting one such sequence. In the case of CDR3, identifying a similar germline sequence can include selecting one such sequence, but may including using two germline sequences that separately contribute to the amino-terminal portion and the carboxy-terminal portion. In other implementations more than one or two germline sequences are used, e.g., to form a consensus sequence.

In one embodiment, with respect to a particular reference variable domain sequence, e.g., a sequence described herein, a related variable domain sequence has at least 30, 40, 50, 60, 70, 80, 90, 95 or 100% of the CDR amino acid positions that are not identical to residues in the reference CDR sequences, residues that are identical to residues at corresponding positions in a human germline sequence (i.e. , an amino acid sequence encoded by a human germline nucleic acid).

In one embodiment, with respect to a particular reference variable domain sequence, e.g., a sequence described herein, a related variable domain sequence has at least 30, 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of the FR regions identical to FR sequence from a human germline sequence, e.g., a germline sequence related to the reference variable domain sequence.

Accordingly, it is possible to isolate an antibody molecule which has similar activity to a given antibody molecule of interest, but is more similar to one or more germline sequences, particularly one or more human germline sequences. For example, an antibody molecule can be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identical to a germline sequence in a region outside the CDRs (e.g., framework regions). Further, an antibody molecule can include at least 1, 2, 3, 4, or 5 germline residues in a CDR region, the germline residue being from a germline sequence of similar (e.g., most similar) to the variable region being modified. Germline sequences of primary interest are human germline sequences. The activity of the antibody molecule (e.g., the binding activity as measured by K_A) can be within a factor or 100, 10, 5, 2, 0.5, 0.1, and 0.001 of the original antibody molecule.

Germline sequences of human immunoglobulin genes have been determined and are available from a number of sources, including the international ImMunoGeneTics information system® (IMGT), available via the world wide web at imgt.cines.fr, and the V BASE directory (compiled by Tomlinson, LA. et al. MRC Centre for Protein Engineering, Cambridge, UK, available via the world wide web at vbase.mrc-cpe.cam.ac.uk).

A germline reference sequence for the HC variable domain can be based on a sequence that has particular canonical structures, e.g., 1-3 structures in the HI and H2 hypervariable loops. The canonical structures of hypervariable loops of an immunoglobulin variable domain can be inferred from its sequence, as described in Chothia et al, 1992, J. Mol. Biol. 227:799-817;

Tomlinson et al, 1992, J. Mol. Biol. 227:776-798); and Tomlinson et al, 1995, EMBO J.

14(18):4628-38.

Protein Production

Standard recombinant nucleic acid methods can be used to express an antibody molecule that binds to activin and/or myostatin. Generally, a nucleic acid sequence encoding the protein is cloned into a nucleic acid expression vector. Of course, if the protein includes multiple polypeptide chains, each chain can be cloned into an expression vector, e.g., the same or different vectors, that are expressed in the same or different cells.

Antibody Production. Some antibodies, e.g., Fabs, can be produced in bacterial cells, e.g., E. coli cells. For example, if the Fab is encoded by sequences in a phage display vector that includes a suppressible stop codon between the display entity and a bacteriophage protein (or fragment thereof), the vector nucleic acid can be transferred into a bacterial cell that cannot suppress a stop codon. In this case, the Fab is not fused to the gene III protein and is secreted into the periplasm and/or media.

Antibodies can also be produced in eukaryotic cells. In one embodiment, the antibodies (e.g., scFv's) are expressed in a yeast cell such as Pichia (see, e.g., Powers et al, 2001, J.

Immunol. Methods. 251: 123-35), Hanseula, or Saccharomyces.

In one preferred embodiment, antibodies are produced in mammalian cells. Preferred mammalian host cells for expressing the clone antibodies or antigen-binding fragments thereof include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, 1982, Mol. Biol. 159:601 621), lymphocytic cell lines, e.g., NSO myeloma cells and SP2 cells, COS cells, HEK293T cells (J. Immunol. Methods (2004) 289(l-2):65-80.), and a cell from a transgenic animal, e.g., a transgenic mammal. For example, the cell is a mammary epithelial cell.

In addition to the nucleic acid sequence encoding the diversified immunoglobulin domain, the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells {e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

In an exemplary system for recombinant expression of an antibody, or antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/ AdMLP promoter regulatory element or an SV40 enhancer/ AdMLP promoter regulatory element) to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity

chromatography with a Protein A or Protein G coupled matrix.

For antibody molecules that include an Fc domain, the antibody production system may produce antibodies in which the Fc region is glycosylated. For example, the Fc domain of IgG molecules is glycosylated at asparagine 297 in the CH2 domain. This asparagine is the site for modification with biantennary-type oligosaccharides. It has been demonstrated that this glycosylation is required for effector functions mediated by Fcg receptors and complement Clq (Burton and Woof, 1992, Adv. Immunol. 51: 1-84; Jefferis et al., 1998, Immunol. Rev. 163:59- 76). In one embodiment, the Fc domain is produced in a mammalian expression system that appropriately glycosylates the residue corresponding to asparagine 297. The Fc domain can also include other eukaryotic post-translational modifications.

Antibody molecules can also be produced by a transgenic animal. For example, U.S. Patent No. 5,849,992 describes a method of expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter and nucleic acids encoding the antibody of interest and a signal sequence for secretion. The milk produced by females of such transgenic mammals includes, secreted-therein, the antibody molecule of interest. The antibody molecules can be purified from the milk, or for some applications, used directly.

Characterization of Antibody Molecules That Bind to Activin and/or Myostatin

Binding of antibody molecules that bind to activin and/or myostatin to cells expressing activin and/or myostatin can be characterized in a number assays known in the art, including FACS (Fluorescence Activated Cell Sorting), immunofluorescence, and immunocytochemistry. Antibody molecule that binds activin and/or myostatin is contacted with cells and/or tissues which express or contain activin and/or myostatin, and binding is detected in accordance with the method being used. For example, a fluorescent detection system (e.g., fluorescent-labeled secondary antibody) employed for FACS and immunofluorescence analysis, or an enzymatic system is used for immunocytochemistry are generally used in these assays can be performed on non-perm. Antibody molecules that bind activin and/or myostatin can be characterized as to cellular binding by FACS (Fluorescence Activated Cell Sorting) using cells expressing activin and/or myostatin. Individual cells held in a thin stream of fluid are passed through one or more laser beams cause light to scatter and fluorescent dyes to emit light at various frequencies.

Photomultiplier tubes (PMT) convert light to electrical signals and cell data is collected. Forward and side scatter are used for preliminary identification of cells. Forward and side scatter are used to exclude debris and dead cells. Fluorescent labeling allows investigation of cell structure and function. Cell autofluorescence is generated by labeling cell structures with fluorescent dyes. FACS collects fluorescence signals in one to several channels corresponding to different laser excitation and fluorescence emission wavelength. Immunofluorescence, the most widely used application, involves the staining of cells with antibodies conjugated to fluorescent dyes such as fluorescein and phycoerythrin (PE). This method can be used to label activin and/or myostatin on the cell surface of cells using biotinylated antibody molecules that bind activin and/or myostatin. Biotin is used in these two-step detection systems in concert with conjugated steptavidin. Biotin is typically conjugated to proteins via primary amines (i.e., lysines). Usually, between 1.5 and 3 biotin molecules are conjugated to each antibody. A second fluorescently conjugated antibody (streptavidin/PE) is added which is specific for biotin.

Antibody molecules that bind activin and/or myostatin can be characterized in cultured cells expressing the activin antigen and/or the myostatin antigen. The method generally used is immunocytochemistry. Immunocytochemistry involves the use of antibodies that recognize parts of the receptor that are exposed to the outside environment when expressed at the cell surface (the "primary antibody"). If the experiment is carried out in intact cells, such an antibody will only bind to surface expressed receptors. Biotinylated or non-biotinylated antibody molecules that bind activin and/or myostatin can be used. The secondary antibody can be either a streptavidin/HRP antibody (for biotinylated antibody molecule) or an anti-human IgG/HRP (for non-biotinylated antibody molecule). The staining can then be detected using an inverted microscope. The assay can be performed in the absence of the antibody molecule that binds activin and/or myostatin or in the presence of lC^g/mL of the antibody molecule that binds activin and/or myostatin.

Antibody molecules that bind activin and/or myostatin can be characterized in assays that measure their modulatory activity toward activin, myostatin, or fragments thereof, in vitro or in vivo. The assay is performed in the absence of the antibody molecule that binds activin and/or myostatin, and in the presence of increasing concentrations of the antibody molecule that binds activin and/or myostatin. The concentration of antibody molecule at which 50% of the activin activity (or myostatin activity) (e.g., binding activity) is inhibited is the IC₅₀ value (Inhibitory Concentration 50%) or EC₅₀ (Effective Concentration 50%) value for that antibody molecule. Within a series or group of antibody molecules, those having lower IC₅₀ or EC ₅₀ values are considered more potent inhibitors of activin (or myostatin) than those antibody molecules having higher IC₅₀ or EC₅₀ values. Exemplary antibody molecules have an IC₅₀ value of less than 800 nM, 400 nM, 100 nM, 25 nM, 5 nM, or 1 nM, e.g., as measured in an in vitro assay for inhibition of activin activity (or myostatin activity) when the activin (or myostatin) is at 2 pM.

The antibody molecules can also be evaluated for selectivity toward activin or myostatin. For example, an antibody molecule can be assayed for its potency toward activin, myostatin and a panel of other proteins, e.g., human and/or mouse proteins, e.g., GDF-5, GDF-6, GDF-7, BMP- 2, BMP-4, and BMP-7, and an IC₅₀ value or EC₅₀ value can be determined for each of these proteins. In one embodiment, a compound that demonstrates a low IC₅₀ value or EC₅₀ value for the activin and/or myostatin, and a higher IC₅₀ value or EC₅₀ value, e.g., at least 2-, 5-, or 10- fold higher, for another protein within the test panel (e.g., GDF-5, GDF-6, GDF-7, BMP-2, BMP-4, and BMP-7) is considered to be selective toward activin and/or myostatin. Antibody molecules that bind to activin and/or myostatin can be evaluated for their ability to inhibit activin (or myostatin) in a cell based assay.

A pharmacokinetics study in rat, mice, or monkey can be performed with antibody molecules that bind activin and/or myostatin for determining activin and/or myostatin half-life in the serum. Likewise, the effect of the antibody molecule can be assessed in vivo, e.g., in an animal model for a disease or condition, for use as a therapeutic, for example, to treat a disease or condition described herein.

Pharmaceutical Compositions

In another aspect, the disclosure provides compositions, e.g., pharmaceutically acceptable compositions or pharmaceutical compositions, which include an antibody molecule that binds activin and/or myostatin, e.g., an antibody molecule, other polypeptide or peptide identified as binding to activin and/or myostatin described herein. The antibody molecule that binds activin and/or myostatin can be formulated together with a pharmaceutically acceptable carrier.

Pharmaceutical compositions include therapeutic compositions and diagnostic compositions, e.g., compositions that include labeled antibody molecules that bind activin and/or myostatin for in vivo imaging.

A pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for parenteral administration, e.g., subcutaneous, intramuscular, or intravenous administration (e.g., by injection or infusion), although carriers suitable for spinal, epidermal, inhalation, and intranasal administration are also contemplated. Depending on the route of administration, the antibody molecule that binds activin and/or myostatin may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

A pharmaceutically acceptable salt is a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, e.g., Berge, S.M., et ah, 1977, J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, as well as from nontoxic organic amines, such as Ν,Ν'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

The compositions may be in a variety of forms. These include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The form can depend on the intended mode of administration and therapeutic application. Many compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for administration of humans with antibodies. An exemplary mode of administration is parenteral (e.g., subcutaneous, intramuscular, intravenous, intraperitoneal). In one embodiment, the antibody molecule that binds activin and/or myostatin is administered by subcutaneous injection. In one embodiment, the antibody molecule that binds activin and/or myostatin is administered by intramuscular injection. In one embodiment, the antibody molecule that binds activin and/or myostatin is administered by intravenous infusion or injection.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the antibody molecule in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

An antibody molecule that binds activin and/or myostatin can be administered by a variety of methods, although for many applications, typical route/mode of administration are subcutaneous or intramuscular injection or intravenous injection or infusion. For example, for therapeutic applications, the antibody molecule that that binds activin and/or myostatin can be administered by subcutaneous or intramuscular injection at a dose of about 10 to 1000 mg (e.g., about 10 to 500, 10 to 250, 10 to 100, 100 to 1000, 250 to 1000, 500 to 1000, 50 to 500, or 100 to 250 mg) per injection, e.g., at a concentration of about 1 to 500 mg/mL (e.g., about 5 to 250, 10 to 100, or 20 to 80 mg/mL). As another example, for therapeutic applications, the antibody molecule that binds activin and/or myostatin can be administered by intravenous infusion at a rate of less than 30, 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 100 mg/m or 7 to 25 mg/m . The antibody molecule that that binds activin and/or myostatin can be administered twice weekly, weekly, bi-weekly, or monthly. The route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate,

polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are available. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., 1978, Marcel Dekker, Inc., New York.

Pharmaceutical compositions can be administered with medical devices. For example, in one embodiment, a pharmaceutical composition disclosed herein can be administered with a device, e.g., a needleless hypodermic injection device, a pump, or implant. In some

embodiments, medical devices suitable for subcutaneous, intramuscular, or intravenous administration can be used.

In certain embodiments, an antibody molecule that binds activin and/or myostatin can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds disclosed herein cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patent Nos. 4,522,811 ; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties that are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade, 1989, J. Clin. Pharmacol. 29:685).

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody disclosed herein is 0.1-20 mg/kg, more preferably 1-10 mg/kg. An antibody molecule that binds activin and/or myostatin can be administered, e.g., by subcutaneous or intramuscular injection, e.g., at a dose of about 10 to 1000 mg (e.g., about 10 to 500, 10 to 250, 10 to 100, 100 to 1000, 250 to 1000, 500 to 1000, 50 to 500, or 100 to 250 mg) per injection, e.g., at a concentration of about 1 to 500 mg/mL (e.g., about 5 to 250, 10 to 100, or 20 to 80 mg/mL). An antibody molecule that binds activin and/or myostatin can also be

administered, e.g., by intravenous infusion, e.g., at a rate of less than 30, 20, 10, 5, or 1 mg/min

2 2

to reach a dose of about 1 to 100 mg/m or about 5 to 30 mg/m . The antibody molecule that that binds activin and/or myostatin can be administered twice a day, daily, every other day, weekly, bi-weekly, or monthly. For antibody molecules smaller in molecular weight than an antibody, appropriate amounts can be proportionally less. Dosage values may vary with the type and severity of the condition to be alleviated. For a particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

The pharmaceutical compositions disclosed herein may include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody molecule that binds activin and/or myostatin disclosed herein. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result (e.g., at least a 20% reduction in at least one symptom of the disease or condition being treated). A therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the protein to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effect of the composition is outweighed by the therapeutically beneficial effects.

A "therapeutically effective dosage" preferably modulates a measurable parameter by a statistically significant degree or at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of an antibody molecule described herein to modulate a measurable parameter, e.g., a disease-associated parameter, can be evaluated in an animal model system predictive of efficacy in human disorders and conditions, e.g., a disorder or condition described herein. Alternatively, this property of a composition can be evaluated by examining the ability of the antibody molecule described herein to modulate a parameter in vitro.

A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, because a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

Stabilization and Retention

In one embodiment, an antibody molecule that binds activin and/or myostatin is physically associated with a moiety that improves its stabilization and/or retention in circulation, e.g., in blood, serum, lymph, or other tissues, e.g., by at least 1.5, 2, 5, 10, or 50 fold. For example, an antibody molecule that binds activin and/or myostatin can be associated with a polymer, e.g., a substantially non-antigenic polymer, such as polyalkylene oxides or

polyethylene oxides. Suitable polymers will vary substantially by weight. Polymers having molecular number average weights ranging from about 200 to about 35,000 (or about 1,000 to about 15,000, and 2,000 to about 12,500) can be used. For example, an antibody molecule that binds activin and/or myostatin can be conjugated to a water soluble polymer, e.g., hydrophilic polyvinyl polymers, e.g., polyvinylalcohol and polyvinylpyrrolidone. A non-limiting list of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained.

An antibody molecule that binds activin and/or myostatin can also be associated with a carrier protein, e.g., a serum albumin, such as a human serum albumin. For example, a translational fusion can be used to associate the carrier protein with the antibody molecule that binds activin and/or myostatin. Kits

An antibody molecule that binds activin and/or myostatin described herein can be provided in a kit, e.g., as a component of a kit. For example, the kit includes (a) an antibody molecule that binds activin and an antibody molecule that binds myostatin, or an antibody molecule that binds both activin and myostatin, e.g., a composition that includes an antibody molecule that binds activin and an antibody molecule that binds myostatin, or an antibody molecule that binds activin and myostatin, and, optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of an antibody molecule that binds activin and/or myostatin for the methods described herein.

The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to using the antibody molecule to treat, prevent, or diagnosis of disorders and conditions, e.g., a disorder or condition described herein.

In one embodiment, the informational material can include instructions to administer an antibody molecule that binds activin and/or myostatin in a suitable manner to perform the methods described herein, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). In another embodiment, the informational material can include instructions to administer an antibody molecule that binds activin and/or myostatin to a suitable subject, e.g., a human, e.g., a human having, or at risk for, a disorder or condition described herein. For example, the material can include instructions to administer an antibody molecule that binds activin and/or myostatin to a patient with a disorder or condition described herein. The informational material of the kits is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in print but may also be in other formats, such as computer readable material.

An antibody molecule that binds activin and/or myostatin can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that an antibody molecule that binds activin and/or myostatin be substantially pure and/or sterile. When an antibody molecule that binds activin and/or myostatin is provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred. When an antibody molecule that binds activin and/or myostatin is provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit.

The kit can include one or more containers for the composition containing an antibody molecule that binds activin and/or myostatin. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained association with the container. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of an antibody molecule that binds activin and/or myostatin. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of an antibody molecule that binds activin and/or myostatin. The containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.

The kit optionally includes a device suitable for administration of the composition, e.g., a syringe, inhalant, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device. In one embodiment, the device is for subcutaneous, intramuscular, or intravenous administration. In one embodiment, the device is an implantable device that dispenses metered doses of the antibody molecule. The disclosure also features a method of providing a kit, e.g., by combining components described herein.

Treatments

Antibody molecules that bind to activin and/or myostatin and identified by the method described herein and/or detailed herein have therapeutic and prophylactic utilities, particularly in human subjects. These antibody molecules are administered to a subject to treat, prevent, and/or diagnose a variety of disorders or conditions, including, e.g., a disorder or condition described herein, or even to cells in culture, e.g. in vitro or ex vivo. Treating includes administering an amount effective to alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder or condition, the symptoms of the disorder or condition, or the predisposition toward the disorder or condition. The treatment may also delay onset, e.g., prevent onset, or prevent deterioration of a disease or condition. As described herein, the methods can include administering a bispecific antibody molecule which targets both activin and myostatin. In other aspects, the methods can include administering at least two different antibody molecules, e.g., one antibody molecule that binds activin and one antibody molecule that binds myostatin. Thus, in some embodiments, an antibody molecule that binds activin can be administered in combination with an antibody molecule that binds myostatin.

Administered "in combination," as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder or diagnosed as being at risk for the disorder and before the disorder has been cured or eliminated, or before the symptom or symptoms associated with risk for the disorder have been alleviated or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as

"simultaneous" or "concurrent delivery". In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

As used herein, an amount of an target-binding agent effective to prevent a disorder, or a prophylactically effective amount of the binding agent refers to an amount of a target binding agent, e.g., an antibody molecule that binds activin and/or myostatin, e.g., an antibody molecule described herein, which is effective, upon single- or multiple-dose administration to the subject, for preventing or delaying the occurrence of the onset or recurrence of a disorder or condition, e.g., a disorder or condition described herein.

The agent (e.g., an antibody molecule that binds activin and/or myostatin, e.g., an antibody molecule described herein) may be administered multiple times (e.g., at least two, three, five, or ten times) before a therapeutically effective amount is attained.

Methods of administering antibody molecules that bind activin and/or myostatin and other agents are also described in "Pharmaceutical Compositions." Suitable dosages of the molecules used can depend on the age and weight of the subject and the particular drug used. The antibody molecules can be used as competitive agents to inhibit, reduce an undesirable interaction, e.g., between a natural or pathological agent and activin and/or myostatin. The dose of the antibody molecule that binds activin and/or myostatin can be the amount sufficient to block at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.9% of the activity of activin and/or myostatin in the patient, especially at the site of disease. Depending on the disease, this may require 0.1, 1.0, 3.0, 6.0, or 10.0 mg/Kg. For an IgG having a molecular mass of 150,000 g/mole (two binding sites), these doses correspond to approximately 18 nM, 180 nM, 540 nM, 1.08 μΜ, and 1.8 μΜ of binding sites for a 5 L blood volume. In one embodiment, the antibody molecules that bind activin and/or myostatin are used to inhibit an activity of a cell, e.g., in vivo. The method can include: administering the antibody molecule to a subject requiring such treatment.

Because antibody molecules that bind activin and/or myostatin recognize activin- expressing cells and myostatin expressing cells and can bind to cells that are associated with (e.g., in proximity of or intermingled with) diseased cells, e.g., diseased cells described herein, antibody molecules that bind activin and/or myostatin can be used to inhibit (e.g., inhibit at least one activity of) any such cells and inhibit the disease progression. Reducing activin activity and/or myostatin activity near a diseased tissue can indirectly inhibit (e.g., inhibit at least one activity of) the diseased cells which may be dependent on the activin activity and/or myostatin activity, or up- or down-regulation of other genes related to the disease, and so forth.

Alternatively, the antibody molecules bind to cells in the vicinity of the diseased cells, but are sufficiently close to the diseased cells to directly or indirectly inhibit (e.g., inhibit at least one activity of) the diseased cells. Thus, antibody molecules that bind activin and/or myostatin (e.g., modified with a toxin, e.g., a cytotoxin) can be used to selectively inhibit cells in diseased tissue (including the diseased cells themselves and cells associated with the disease).

Methods of administering antibody molecules that bind activin and/or myostatin are described in "Pharmaceutical Compositions." Suitable dosages of the molecules used will depend on the age and weight of the subject and the particular drug used. The antibody molecules can be used as competitive agents to inhibit or reduce an undesirable interaction, e.g., between a natural or pathological agent and activin or myostatin.

Exemplary Diseases and Conditions

The antibody molecules described herein are useful to treat diseases or conditions in which activin and/or myostatin are implicated, e.g., a disease or condition described herein, or to treat one or more symptoms associated therewith. In some embodiments, the antibody molecule that binds activin and/or myostatin (e.g., activin- and/or myostatin-binding IgG or Fab) inhibits activin activity, e.g., an activity described herein, and/or myostatin activity, e.g., an activity described herein.

Metabolic Disorders

Exemplary disorders or conditions that can be treated or prevented by the compounds and methods described herein include, e.g., metabolic disorders, e.g., obesity or diabetes, a comorbidity of obesity or diabetes, an obesity or diabetes related disorder, non-alcoholic fatty liver disease (NAFLD) (e.g., non-alcoholic steatohepatitis (NASH)), or a disorder in which one or more symptoms can be alleviated by exercise or diet. The subject to whom the compound is administered may be overweight, for example, obese. For example, the subject can have a body mass index (BMI) at least about 25, e.g., from about 25 to about 30, from about 30 to about 35, from about 35 to 40, or greater than about 40. Alternatively, or in addition, the subject may be diabetic, for example having insulin resistance or glucose intolerance, or both. The subject may have diabetes mellitus, for example, the subject may have Type II diabetes. The subject may be overweight, for example, obese and have diabetes mellitus, for example, Type II diabetes.

In addition, or alternatively, the subject may have, or may be at risk of having, a disorder in which obesity or being overweight is a risk factor. Such disorders include, but are not limited to, cardiovascular disease, for example hypertension, atherosclerosis, congestive heart failure, and dyslipidemia; stroke; gallbladder disease; osteoarthritis; sleep apnea; reproductive disorders for example, polycystic ovarian syndrome; cancers, for example breast, prostate, colon, endometrial, kidney, and esophagus cancer; varicose veins; acanthosis nigricans; eczema;

exercise intolerance; insulin resistance; hypertension; hypercholesterolemia; cholithiasis;

osteoarthritis; orthopedic injury; insulin resistance, for example, type 2 diabetes and syndrome X; metabolic syndrome; and thromboembolic disease (see Kopelman (2000), Nature 404:635-43; Rissanen et al., British Med. J. 301, 835, 1990).

Other disorders associated with obesity include depression, anxiety, panic attacks, migraine headaches, PMS, chronic pain states, fibromyalgia, insomnia, impulsivity, obsessive - compulsive disorder, irritable bowel syndrome (IBS), and myoclonus. Furthermore, obesity is a recognized risk factor for increased incidence of complications of general anesthesia. (See e.g., Kopelman, Nature 404:635-43, 2000). In general, obesity reduces life span and carries a serious risk of co-morbidities such as those listed above.

Other diseases or disorders associated with obesity are birth defects, maternal obesity being associated with increased incidence of neural tube defects, carpal tunnel syndrome (CTS); chronic venous insufficiency (CVI); daytime sleepiness; deep vein thrombosis (DVT); end stage renal disease (ESRD); gout; heat disorders; impaired immune response; impaired respiratory function; infertility; liver disease; lower back pain; obstetric and gynecologic complications; pancreatititis; as well as abdominal hernias; acanthosis nigricans; endocrine abnormalities;

chronic hypoxia and hypercapnia; dermatological effects; elephantitis; gastroesophageal reflux; heel spurs; lower extremity edema; mammegaly which causes considerable problems such as bra strap pain, skin damage, cervical pain, chronic odors and infections in the skin folds under the breasts, etc.; large anterior abdominal wall masses, for example abdominal panniculitis with frequent panniculitis, impeding walking, causing frequent infections, odors, clothing difficulties, lower back pain; musculoskeletal disease; pseudo tumor cerebri (or benign intracranial hypertension), and sliding hiatil hernia.

Conditions or disorders associated with increased caloric intake include, but are not limited to, insulin resistance, glucose intolerance, obesity, diabetes, including type 2 diabetes, eating disorders, insulin-resistance syndromes, metabolic syndrome X, and Alzheimer' s disease.

Disorders in which one or more symptoms can be alleviated by exercise include, but not limited to, cardiovascular disease, neurodegenerative diseases, e.g., multiple sclerosis,

Parkinson's disease and Alzheimer's disease; certain cancers, e.g., prostate, breast, colon; certain intestinal disorders, e.g., ulcers, irritable bowel syndrome, indigestion, diverticulosis, gastrointestinal bleeding; certain emotional disorders, e.g., depression, menopause related emotional symptoms, e.g., anxiety, stress, depression.

The disclosure also features compounds, e.g., antibody molecules that bind activin and/or myostatin, for use as a medicament for the prevention or treatment of diseases and disorders described herein, e.g., a metabolic disorder, e.g., obesity, or obesity related disorders, or disorders in which exercise can alleviate one or more symptoms, or age-related disorders.

The antibody molecules described herein can also be administered in combination with another agent, for example, a treatment for lipolysis, an antihypertension treatment, a treatment for dyslipidemia, and/or a treatment for type 2 diabetes.

Exemplary treatment for lipolysis includes, but not limited to, a beta 3 agonist (e.g., Amibegron (SR-58611A), Solabegron (GW-427,353), Nebivolol, L-796,568, CL-316,243, LY- 368,842, Ro40-2148 and Octopamine).

Exemplary anti-hypertension treatment include, but not limited to, diuretic, e.g., hydrochlorothiazide, Acetazolamide, Chlorthalidone, Hydrochlorothiazide, Indapamide, Metolazone, Amiloride hydrochloride, Bumetanide, Ethacrynic acid Furosemide,

Spironolactone, Torsemide, Triamterene; beta-blockere.g., Acebutolol, Atenolol, Betaxolol, Bisoprolol, Carteolol, Carvedilol, Labetalol, Metoprolol Nadolol, Penbutolol, Propranolol, Timolol; angiotensin II receptor blockers, e.g., Candesartan, Irbesartan, Losartan, Telmisartan, Valsartan; angiotensin-converting enzyme inhibitors e.g., perindropril, Benazepril, Captopril, Enalapril, Fosinopril, Lisinopril, Moexipril, Quinapril, Ramipril, Trandolapril; alpha blockers, centrally acting drugs, vasodialators, rennin inhibitors, calcium channel blockers, e.g.,

Amlodipine, Diltiazem, Felodipine, Isradipine, Nicardipine, Nifedipine, Nisoldipine, Verapamil hydrochloride; combination therapies, e.g., Amiloride and hydrochlorothiazide, Amlodipine and benazepril, Atenolol and chlorthalidone, Benazepril and hydrochlorothiazide, Bisoprolol and hydrochlorothiazide, Captopril and hydrochlorothiazide, Enalapril and hydrochlorothiazide, Felodipine and enalapril, Hydralazine and hydrochlorothiazide, Lisinopril and hydrochlorothiazide, Losartan and hydrochlorothiazide, Methyldopa and hydrochlorothiazide, Metoprolol and hydrochlorothiazide, Nadolol and bendrofhimethiazide, Propranolol and hydrochlorothiazide, Spironolactone and hydrochlorothiazide, Triamterene and

hydrochlorothiazide, Verapamil and trandolapril.

Exemplary treatment for dyslipidemia includes, but not limited to, statins, e.g.,

Atorvastatin (Lipitor®), Fluvastatin (Lescol®), Lovastatin (Mevacor®), Pravastatin

(Pravachol®), Simvastatin (Zocor®), Rosuvastatin (Crestor®); Bile acid sequestrants, e.g., Questran® and Questran Light®, Colestid®, WelChol®; Niacin, e.g., Nicolar®, Niaspan®; Fibrates, e.g., Atromid®, Tricor®, Lopid®, Lofibra® (fenofibrate); Ezetimibe; Omega-3 fatty acids, e.g., Lovaza®; Combination statin and niacin,e.g., Advicor® (niacin-lovastatin);

Combination cholesterol absorption inhibitor and statin,e.g., Vytorin® (ezetimibe-simvastatin).

Exemplary treatment for type 2 diabetes include, but not limited to, Meglitinides, e.g., Repaglinide (Prandin®), Nateglinide (Starlix®); Sulfonylureas, e.g., Glipizide (Glucotrol®), Glimepiride (Amaryl®), Glyburide (DiaBeta®, Glynase®); Dipeptidy peptidase-4 (DPP-4) inhibitors, e.g., Saxagliptin (Onglyza®), Sitagliptin (Januvia®), Linagliptin (Tradjenta®);

Biguanides, e.g., Metformin (Fortamet®, Glucophage®, etc); Thiazolidinediones, e.g.,

Rosiglitazone (Avandia®), Pioglitazone (Actos®); Alpha-glucosidase inhibitors, e.g., Acarbose (Precose®), Miglitol (Glyset®); Amylin mimetics, e.g., Pramlintide (Symlin®); Incretin mimetics, e.g., Exenatide (Byetta®), Liraglutide (Victoza®).

Non-alcoholic fatty liver disease (NAFLD) is one cause of a fatty liver, occurring when fat is deposited (steatosis) in the liver not due to excessive alcohol use. It is related to insulin resistance and the metabolic syndrome (obesity, combined hyperlipidemia, diabetes mellitus (type II) and high blood pressure). NAFLD can also be caused by some medications, e.g., amiodarone, antiviral drugs (nucleoside analogues), aspirin rarely as part of Reye's syndrome in children, corticosteroids, methotrexate, tamoxifen, or tetracycline. Non-alcoholic steatohepatitis (NASH) is the most extreme form of NAFLD, and is regarded as a major cause of cirrhosis of the liver of unknown cause.

Most patients with NAFLD have few or no symptoms. Patients may complain of fatigue, malaise, and dull right-upper-quadrant abdominal discomfort. Mild jaundice may be noticed although this is rare. More commonly NAFLD is diagnosed following abnormal liver function tests during routine blood tests.

NAFLD can be treated, e.g., by nutritional counseling, weight loss, insulin sensitizers (e.g., metformin and thiazolidinediones), ursodeoxycholic acid and lipid-lowering drugs, Vitamin E, statin, and modest wine drinking. NAFLD may also respond to respond to treatments originally developed for other insulin-resistant states (e.g., diabetes mellitus type 2), e.g., a treatment described herein.

In certain embodiments, the antibody molecule that binds activin and/or myostatin is administered as a single agent treatment. In other embodiments, the antibody molecule that binds activin and/or myostatin is administered in combination with an additional therapy described above.

Also provided are methods of preventing or reducing risk of developing a disorder described above, by administering an effective amount of an antibody molecule that binds activin and/or myostatin to a subject at risk of developing such disorder, thereby reducing the subject's risk of developing said disorder.

Guidance for determination of a therapeutically effective amount of an antibody molecule that binds activin and/or myostatin may be obtained from an animal model of metabolic disorders. For example, animal models for metabolic disorders are described, e.g., in Rees et al. Diabet Med. 2005; 22(4):359-370, Vickers et al. Br J Pharmacol. 2011; 164(4): 1248-1262, and Anstee et al. Int J Exp Pathol. 2006;87(1): 1-16.

Other disorders or conditions that can be treated or prevented by the compounds or methods described herein include, but not limited to, disorders or conditions characterized by a need for increased fat browning, increased brown adipose tissue (BAT), or increased

thermogenesis; disorders or conditions characterized by a need for increased muscle mass; frailty, e.g., frailty associated with or arising from decreased muscle mass or strength; disorders or conditions characterized by loss of muscle mass or function, e.g., arising from insufficient use of muscle, e.g., associated with or arising from bed rest or other inactivity arising from or associated with age, disability, or medical condition, or a medical or surgical procedure; acquired or inherited disorders or conditions of the muscles, e.g., a muscular dystrophy, e.g., Duchenne muscular dystrophy, or spinal muscular atrophy, or amyotrophic lateral sclerosis (ALS).

Muscular Dystrophy

The antibody molecules that bind activin and/or myostatin can be used to treat muscular dystrophy (MD). MD is a group of muscle diseases that weaken the musculoskeletal system and hamper locomotion. Muscular dystrophies are characterized, e.g., by progressive skeletal muscle weakness, defects in muscle proteins, and the death of muscle cells and tissue.

Signs and symptoms of MD include, but not limited to, progressive muscular wasting, poor balance, drooping eyelids, atrophy, scoliosis (curvature of the spine and the back), inability to walk, frequent falls, waddling gait, calf deformation, limited range of movement, respiratory difficulty, joint contractures, cardiomyopathy, arrhythmias, and muscle spasms. The most common and severe form of the disease is Duchenne muscular dystrophy (DMD). Other major forms include, e.g., Becker, limb-girdle, congenital, facioscapulohumeral, myotonic, oculopharyngeal, distal, and Emery-Dreifuss muscular dystrophy. Most types of MD are multi-system disorders with manifestations in body systems including the heart,

gastrointestinal system, nervous system, endocrine glands, eyes and brain. In addition, several MD-like conditions have also been identified.

These conditions are generally inherited, and the different muscular dystrophies follow various inheritance patterns. Mutations of the dystrophin gene and nutritional defects (with no genetics history) at the prenatal stage are also possible in about 33% of people affected by DMD. The main cause of the Duchenne and Becker types of muscular dystrophy is the muscle tissue's cytoskeletal impairment to properly create the functional protein dystrophin and dystrophin- associated protein complex. Dystrophin protein is found in muscle fibre membrane. Its helical nature allows it to act like a spring or shock absorber. Dystrophin links actin (cytoskeleton) and dystroglycans of the muscle cell plasma membrane, known as the sarcolemma (extracellular). In addition to mechanical stabilization, dystrophin also regulates calcium levels.

Duchenne muscular dystrophy (DMD). DMD is the most common childhood form of muscular dystrophy. The DMD gene encoding dystrophin is involved in this type of MD. The amount of dystrophin correlates with the severity of the disease (i.e. the less dystrophin present, the more severe the phenotype). Dystrophin is part of a complex structure involving several other protein components. The "dystrophin- glycoprotein complex" helps anchor the structural skeleton (cytoskeleton) within the muscle cells, through the outer membrane (sarcolemma) of each cell, to the tissue framework (extracellular matrix) that surrounds each cell. Due to defects in this assembly, contraction of the muscle leads to disruption of the outer membrane of the muscle cells and eventual weakening and wasting of the muscle.

Becker muscular dystrophy (BMD). BMD is a less severe variant of DMD and is caused by the production of a truncated, but partially functional form of dystrophin. The DMD gene encoding dystrophin is also involved in this type of MD.

Congenital muscular dystrophy. Congenital muscular dystrophy includes several disorders with a range of symptoms. Symptoms include, e.g., general muscle weakness and possible joint deformities. Muscle degeneration may be mild or severe. Problems may be restricted to skeletal muscle, or muscle degeneration may be paired with effects on the brain and other organ systems. A number of the forms of the congenital muscular dystrophies are caused by defects in proteins that are thought to have some relationship to the dystrophin-glycoprotein complex and to the connections between muscle cells and their surrounding cellular structure. Some forms of congenital muscular dystrophy show severe brain malformations, such as lissencephaly and hydrocephalus.

Distal muscular dystrophy. Symptoms include, e.g., weakness and wasting of muscles of the hands, forearms, and lower legs. Miyoshi myopathy, one of the distal muscular dystrophies, causes initial weakness in the calf muscles, and is caused by defects in the same gene responsible for one form of LGMD (Limb Girdle Muscular Dystrophy). The DYSF gene encoding dysferlin (also known as dystrophy-associated fer-l-like protein) is involved in this type of MD.

Emery-Dreifuss Muscular Dystrophy. Clinical signs include, e.g., muscle weakness and wasting, starting in the distal limb muscles and progressing to involve the limb-girdle muscles. Most patients also suffer from cardiac conduction defects and arrhythmias which, if left untreated, increase the risk of stroke and sudden death. There are three subtypes of Emery- Dreifuss Muscular Dystrophy, distinguishable by their pattern of inheritance: X-Linked, autosomal dominant and autosomal recessive. The disease is caused by mutations in the LMNA gene, or more commonly, the EMD gene. Both genes encode for protein components of the nuclear envelope.

Facioscapulohumeral muscular dystrophy (FSHD). FSHD initially affects, e.g., the muscles of the face, shoulders, and upper arms with progressive weakness. The pattern of inheritance is autosomal dominant, but there are a significant number of spontaneous mutations. It has been shown that two defects are needed for FSHD. The first is the deletion of D4Z4 repeats and the second is a "toxic gain of function" of the DUX4 gene.

Limb-girdle muscular dystrophy (LGMD). Symptoms include, e.g., muscle weakness, affecting both upper arms and legs. Many forms have been identified, showing different patterns of inheritance (autosomal recessive vs. autosomal dominant). Some of the recessive forms have been associated with defects in proteins that make up the dystrophin-glycoprotein complex.

Myotonic muscular dystrophy. It is an autosomal dominant condition that presents with myotonia (delayed relaxation of muscles) as well as muscle wasting and weakness. Myotonic dystrophy varies in severity and manifestations and affects many body systems in addition to skeletal muscles, including, e.g., the heart, endocrine organs, eyes, and gastrointestinal tract. Myotonic muscular dystrophy type 1 (DM1), also known as Steinert disease, is the most common adult form of muscular dystrophy. It results from the expansion of a short (CTG) repeat in the DNA sequence of the DMPK (myotonic dystrophy protein kinase) gene. Myotonic muscular dystrophy type 2 (DM2) is much rarer and is a result of the expansion of the CCTG repeat in the ZNF9 (zinc finger protein 9) gene. These molecular changes may interfere with the production of important muscle proteins. Oculopharyngeal muscular dystrophy. Symptoms affect, e.g., muscles of eyelids, face, and throat followed by pelvic and shoulder muscle weakness. It has been attributed to a short repeat expansion in the genome which regulates the translation of some genes into functional proteins.

MD can be diagnosed based on the results of muscle biopsy, increased creatine phosphokinase (CpK3), electromyography, electrocardiography, and DNA analysis. A physical examination and the patient's medical history will help the doctor determine the type of muscular dystrophy. Specific muscle groups are affected by different types of MD. Pseudohypertrophy may be observed when loss of muscle mass (wasting) has caused a buildup of fat and connective tissue that makes the muscle appear larger.

MD can be treated, e.g., by physical therapy, aerobic exercise, occupational therapy, orthotic intervention {e.g., ankle-foot orthosis), corrective orthopedic surgery, medications {e.g., quinine, phenytoin, or mexiletine), low intensity anabolic steroids, prednisone supplements, speech therapy, and orthopedic instruments {e.g., wheelchairs and standing frames), and antisense oligonucleotides.

In certain embodiments, the antibody molecule that binds activin and/or myostatin is administered as a single agent treatment. In other embodiments, the antibody molecule that binds activin and/or myostatin is administered in combination with an additional MD therapy.

Also provided are methods of preventing or reducing risk of developing MD, by administering an effective amount of an antibody molecule that binds activin and/or myostatin to a subject at risk of developing MD, thereby reducing the subject's risk of developing MD.

Guidance for determination of a therapeutically effective amount of an antibody molecule that binds activin and/or myostatin may be obtained from an animal model of MD. For example, animal models for MD are described, e.g., in Ng et al. Prog Mol Biol Transl Sci. 2012; 105: 83- 111.

Spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by a genetic defect in the SMN1 gene that codes SMN, a protein widely expressed in all eukaryotic cells. SMN1 is apparently selectively necessary for survival of motor neurons, as diminished abundance of the protein results in death of neuronal cells in the anterior horn of the spinal cord and subsequent system-wide muscle wasting (atrophy).

Spinal muscular atrophy manifests in various degrees of severity which all have in common general muscle wasting and mobility impairment. Other body systems may be affected as well, particularly in early-onset forms. The term spinal muscular atrophy is used as both a specific term for the genetic disorder caused by deficient SMN, and a general label for a larger number of rare disorders having in common a genetic cause and slow progression of weakness without sensory impairment caused by disease of motor neurons in the spinal cord and brainstem.

SMA manifests over a wide range of severity affecting infants through adults. The disease spectrum is variously divided into 3-5 types, in accordance either with the age of onset of symptoms or with the highest attained milestone of motor development.

The symptoms vary greatly depending on the SMA type involved, the stage of the disease and individual factors and commonly include, e.g., areflexia (particularly in extremities), overall muscle weakness, poor muscle tone, limpness or a tendency to flop (the "floppy baby"

syndrome), difficulty achieving developmental milestones, difficulty sitting/standing/walking, in infants adopting of a frog-leg position when sitting (hips abducted and knees flexed), loss of strength of the pulmonary muscles (e.g., weak cough, weak cry (infants), accumulation of secretions in the lungs or throat, respiratory distress), bell-shaped torso (caused by using only abdominal muscles for respiration), clenched fists with sweaty hands, head often tilted to one side even when lying down, fasciculations (twitching) of the tongue, difficulty sucking or swallowing, poor feeding, arthrogryposis (multiple congenital contractures), and weight lower than normal.

SMA can be treated, e.g., bypalliative care, gene therapy, stem cell therapy, medications that cause SMN2 activation (e.g., growth hormone, histone deacetylase inhibitors,

hydroxycarbamide (hydroxyurea), natural polyphenol compounds, prolactin, salbutamol

(albuterol)), SMN stabilization (e.g., aminoglycosides), or neuroprotection (e.g., p-lactam antibiotics (e.g. , ceftriaxone) ,follistatin, olesoxime, Riluzole).

In certain embodiments, the antibody molecule that binds activin and/or myostatin is administered as a single agent treatment. In other embodiments, the antibody molecule that binds activin and/or myostatin is administered in combination with an additional SMA therapy.

Also provided are methods of preventing or reducing risk of developing SMA, by administering an effective amount of an antibody molecule that binds activin and/or myostatin to a subject at risk of developing SMA, thereby reducing the subject's risk of developing SMA.

Guidance for determination of a therapeutically effective amount of an antibody molecule that binds activin and/or myostatin may be obtained from an animal model of SMA. For example, animal models for SMA are described, e.g., in Monani et al. Hum Mol Genet.

2000;9(16):2451-2457, and Zanoteli et al. Funct Neurol. 2010;25(2):73-79. Amyotrophic Lateral Sclerosis

The antibody molecules that bind activin and/or myo statin can be used to treat amyotrophic lateral sclerosis (ALS). ALS, also referred to as Lou Gehrig's disease, is a debilitating disease with varied etiology characterized, e.g., by rapidly progressive weakness, muscle atrophy and fasciculations, muscle spasticity, difficulty speaking (dysarthria), difficulty swallowing (dysphagia), and difficulty breathing (dyspnea).

Signs and symptoms include, e.g., muscle weakness and atrophy throughout the body caused by the degeneration of the upper and lower motor neurons. Unable to function, the muscles weaken and atrophy. Individuals affected by the disorder may ultimately lose the ability to initiate and control all voluntary movement, although bladder and bowel sphincters and the muscles responsible for eye movement are usually, but not always, spared until the terminal stages of the disease. The defining feature of ALS is the death of both upper and lower motor neurons in the motor cortex of the brain, the brain stem, and the spinal cord. Prior to their destruction, motor neurons develop proteinaceous inclusions in their cell bodies and axons. This may be partly due to defects in protein degradation.

There is a known hereditary factor in familial ALS (FALS). A defect on chromosome 21, which codes for superoxide dismutase, is associated with approximately 20% of familial cases of ALS, or about 2% of ALS cases overall. To date, a number of genetic mutations have been associated with various types of ALS (e.g., ALS 1 - ALS 18). The currently known genes associated with ALS include, e.g., SOD1, ALS2, SETX, FUS, VAPB, ANG, TARDBP, FIG4, OPTN, ATXN2, VCP, UBQLN2, SIGMAR1, CHMP2B, and PFN1.

ALS can be diagnosed, e.g., primarily based on the symptoms and signs the physician observes in the patient and a series of tests to rule out other diseases. The presence of upper and lower motor neuron signs in a single limb is strongly suggestive. Other diagnostic approaches include, e.g., neurologic examinations at regular intervals to assess whether symptoms such as muscle weakness, atrophy of muscles, hyperreflexia, and spasticity are getting progressively worse.

ALS can be treated, e.g., by medications (e.g., Riluzole (RILUTEK®), or medications to help, e.g., reduce fatigue, ease muscle cramps, control spasticity, and reduce excess saliva and phlegm), physical therapy, occupational therapy, speech therapy, and breathing support.

In certain embodiments, the antibody molecule that binds activin and/or myostatin is administered as a single agent treatment. In other embodiments, the antibody molecule that binds activin and/or myostatin is administered in combination with an additional ALS therapy. Also provided are methods of preventing or reducing risk of developing ALS, by administering an effective amount of an antibody molecule that binds activin and/or myostatin to a subject at risk of developing ALS, thereby reducing the subject's risk of developing ALS.

Guidance for determination of a therapeutically effective amount of an antibody molecule that binds activin and/or myostatin may be obtained from an animal model of ALS. For example, animal models for ALS are described, e.g., in Pioro et al. Clin Neurosci. 1995-1996;3(6):375- 385, and Moser et al. Mol Genet Genomics. 2013;288(5-6):207-229.

Frailty

The antibody molecules that bind activin and/or myostatin can be used to treat frailty.

Frailty is a common geriatric syndrome that embodies an elevated risk of catastrophic declines in health and function among older adults.

The risk factors for frailty include, e.g., chronic diseases {e.g., cardiovascular disease, diabetes, chronic kidney disease, depression, and cognitive impairment), physiologic

impairments {e.g., activation of inflammation and coagulation systems, anemia, atherosclerosis, autonomic dysfunction, hormonal abnormalities, obesity, and hypovitaminosis D), and

environment-related factors.

The syndrome of geriatric frailty can be assessed, e.g., by Fried / Johns Hopkins Frailty Criteria (Friedei al. J Gerontol A Biol Sci Med Sci. 2001; 56 (3): M146-56), and Rockwood Frailty Index (Rockwoodei al. J Gerontol A Biol Sci Med Sci. 2007; 62 (7): 722-727).

The components of frailty include, e.g., sarcopenia (loss of muscle mass), osteoporosis, muscle weakness, and post-surgical complications.

Sarcopenia is characterized first by a decrease in muscle mass, which causes weakness and frailty. During sarcopenia, there is a replacement of muscle fibres with fat and an increase in fibrosis. Muscle weakness, also known as muscle fatigue, refers to the inability to exert force with one's skeletal muscles. Weakness often follows muscle atrophy and a decrease in activity, such as after a long bout of bed rest as a result of an illness. There is also a gradual onset of muscle weakness as a result of sarcopenia, which is the age-related loss of skeletal muscle. A test of strength can be used during a diagnosis of a muscular disorder before the etiology can be identified. Such etiology depends on the type of muscle weakness, which can be true or perceived as well as variable topically. True weakness is substantial, while perceived rather is a sensation of having to put more effort to do the same task. On the other hand, various topic locations for muscle weakness are central, neural and peripheral. Central muscle weakness is an overall exhaustion of the whole body, while peripheral weakness is an exhaustion of individual muscles. Neural weakness is somewhere between. Methods for treating or managing frailty are described, e.g., in Fairhall et al. BMC Med. 2011;9:83.

In certain embodiments, the antibody molecule that binds activin and/or myostatin is administered as a single agent treatment. In other embodiments, the antibody molecule that binds activin and/or myostatin is administered in combination with an additional frailty therapy or management.

Also provided are methods of preventing or reducing risk of developing frailty, by administering an effective amount of an antibody molecule that binds activin and/or myostatin to a subject at risk of developing frailty, thereby reducing the subject's risk of developing frailty.

Guidance for determination of a therapeutically effective amount of an antibody molecule that binds activin and/or myostatin may be obtained from an animal model of frailty. For example, animal models for frailty are described, e.g., in Walstonei al.J Gerontol A Biol Sci Med Sci. 2008;63(4):391-398.

A therapeutically effective amount of an antibody molecule that binds activin and/or myostatinis administered to a subject having or suspected of having a disease or condition in which activin and/or myostatin are implicated, thereby treating {e.g., ameliorating or improving a symptom or feature of a disease or condition, slowing, stabilizing or halting disease progression) the disease or condition.

The antibody molecule that binds activin and/or myostatin is administered in a therapeutically effective amount. A therapeutically effective amount of an antibody molecule that binds activin and/or myostatin is the amount which is effective, upon single or multiple dose administration to a subject, in treating a subject, e.g., curing, alleviating, relieving or improving at least one symptom of a disease or condition in a subject to a degree beyond that expected in the absence of such treatment. A therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effect of the composition is outweighed by the therapeutically beneficial effects.

A therapeutically effective amount can be administered, typically an amount of the compound which is effective, upon single or multiple dose administration to a subject, in treating a subject, e.g., curing, alleviating, relieving or improving at least one symptom of a disorder or condition in a subject to a degree beyond that expected in the absence of such treatment. A therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effect of the composition is outweighed by the therapeutically beneficial effects. A therapeutically effective dosage preferably modulates a measurable parameter, favorably, relative to untreated subjects. The ability of a compound to inhibit a measurable parameter can be evaluated in an animal model system predictive of efficacy in a human disorder or condition.

Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Combination Therapies

The antibody molecules described herein, e.g., activin- or myo statin-binding Fabs or IgGs, can be administered in combination with one or more of the other therapies for treating a disease or condition associated with activin activity and/or myostatin activity, e.g., a disease or condition described herein. For example, an antibody molecule that binds activin and/or myostatin can be used therapeutically or prophylactically with surgery, another activin and/or myostatin inhibitor (e.g., a small molecule inhibitor, another antibody molecule that binds activin and/or myostatin (e.g., another Fab or IgG described herein), peptide inhibitor, or small molecule inhibitor).

One or more small-molecule activin or myostatin inhibitors can be used in combination with one or more antibody molecules described herein. For example, the combination can result in a lower dose of the small-molecule inhibitor being needed, such that side effects are reduced.

The antibody molecules that bind activin and/or myostatin described herein can be administered in combination with one or more of the other therapies for treating a disorder or condition described herein. For example, proteins that inhibit activin, myostatin or that inhibit a downstream event of activin activity or myostatin activity can also be used in combination with other therapies for a disorder or condition described herein, or administration of a second agent. For example, the second agent can be an agent described herein. As another example, the second agent can be an additional anti-activin antibody molecule (e.g., IgG or Fab) or an anti-myostatin antibody molecule (e.g., IgG or Fab). Two or more agents or therapies can be used in combination to treat the same patient. For example, the use or actions of the agents or therapies overlap in time. The agents or therapies can be administered at the same time (e.g., as a single formulation that is administered to a patient or as two separate formulations administered concurrently) or sequentially in any order. Sequential administrations are administrations that are given at different times. The time between administration of the one agent and another agent can be minutes, hours, days, or weeks. The use of an antibody molecule that binds activin and/or myostatin described herein can also be used to reduce the dosage of another therapy, e.g., to reduce the side-effects associated with another agent that is being administered. Accordingly, a combination can include administering a second agent at a dosage at least 10, 20, 30, or 50% lower than would be used in the absence of the antibody molecule that binds activin and/or myostatin.

In addition, a subject can be treated for a disease or condition described herein, by administering to the subject a first and second agent. For example, the first agent modulates early stage of the disease or condition, and the second agent modulates a subsequent stage of the disease or condition, or also modulates early stage of the disease or condition. The first and second agents can be administered using a single pharmaceutical composition or can be administered separately. For example, the first or second agent can be an antibody molecule that binds activin and/or myostatin described herein.

When administered in combination, two or more agents can be administered to a subject at the same time or within an interval such that there is overlap of an effect of each agent on the patient. In some embodiments, they are administered within about 480, 360, 240, 120, 60, 30, 15, 10, 5, or 1 minute of one another. In some embodiments, the administrations of the agents are spaced sufficiently close together such that a combinatorial (e.g., a synergistic) effect is achieved.

The agents can be administered simultaneously, for example in a combined unit dose

(e.g., providing simultaneous delivery of both agents). Alternatively, the agents can be administered at a specified time interval, for example, an interval of minutes, hours, days or weeks. Generally, the agents are concurrently bioavailable, e.g., detectable, in the subject. In some embodiments, the agents are administered essentially simultaneously, for example two unit dosages administered at the same time, or a combined unit dosage of the two agents. In other embodiments, the agents are delivered in separate unit dosages. The agents can be administered in any order, or as one or more preparations that includes two or more agents. In a preferred embodiment, at least one administration of one of the agents, e.g., the first agent, is made within minutes, one, two, three, or four hours, or even within one or two days of the other agent, e.g., the second agent. In some embodiments, combinations can achieve synergistic results, e.g., greater than additive results, e.g., at least 25, 50, 75, 100, 200, 300, 400, or 500% greater than additive results.

The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. The following examples provide further illustrate and are not limiting.

EXAMPLES

The following examples provide further illustration and are not limiting.

Example 1: Combination Therapy Using Anti-Activin A and Anti-Myostatin Antibodies

Methods: Forty 8-week old, male C57BL/6 mice were randomized by body weight into treatment groups. Each group was dosed intraperitoneally with vehicle, 3mg/kg anti-activin A antibody, 3mg/kg anti-myostatin antibody or 3mg/kg of both anti-activin A and anti-myostatin (N=10/group). Mice were dosed twice weekly for 2 weeks for a total of 4 doses. 14 days after the first dose, body composition (lean and fat mass) was determined using whole body NMR (Bruker Instruments). Additionally, muscle and fat tissues were collected and weighed. Fat tissue was processed for RNA and analyzed for gene expression of thermogenesis and adipogenesis.

Results:

Body weight changes: Treatment with neutralizing antibody to activin A (anti-activin A) failed to show any changes in body weight, while neutralizing antibody myostatin (anti- myostatin) increased body weight (FIGs. 1 and 2). Surprisingly, treatment with a combination of the anti-activin A and anti-myostatin antibodies showed larger increase in body weight (FIGs. 1 and 2).

Lean mass and skeletal muscle: The changes in body weight correlated with changes in lean mass determined by whole body NMR. As shown in FIG. 3, anti-activin failed show any increase in lean mass as reflective of the body weight. Anti-myostatin treatment increased lean mass and but the largest increases were observed with combination treatment using anti-activin A and anti-myostatin antibodies (FIG. 3). The increase in body weight and lean mass resulted from an increase in skeletal muscle. The changes in lean mass correlated well with the weights of individual muscle groups isolated at the end of the treatment failed (FIG. 4). Once again, anti- activin A did not increase the weight of the isolated muscles whereas anti-myostatin treatment increased the size of the isolated muscle. The combination treatment with anti-activin A and anti- myostatin antibodies together demonstrated the largest increase in muscle weight. Fat mass: The effects of the three treatments resulted in alteration in the adipose tissue of mice. Anti-activin A antibody treatment did not alter the fat mass of lean mice (FIG. 5). The anti-myostatin and combination treatment showed a reduction in overall fat mass. Anti-activin A treatment showed a slight increase in the mass of the inguinal fat pad (reflective of subcutaneous fat in humans) and a reduction in the mass of the epididymal fat pad (FIGs. 6 and 7). Anti-myostatin or the combination treatment showed no effect on the inguinal fat but significantly reduced the weight of the epididymal (visceral) fat pad (FIGs. 6 and 7).

Thermo genesis: Expression profiling of the adipose tissue from mice treated mice showed changes in the brown fat program indicative of altered energy expenditure. Treatment with the anti-activin A antibody showed small increase in the expression of UCP1 and cidea in the inguinal, epididymal and BAT tissue. Anti-myostatin treatment also demonstrated increase in UCP1 and cidea in all three tissues examined. Combination treatment also showed increase in UCP-1, cidea and FGF21. Surprisingly the effect of combination treatment resulted in synergistic increase in UCP-1, cidea and FGF21. The results are shown in FIGs. 8-10.

REFERENCES

The contents of all cited references including literature references, issued patents, published or non-published patent applications cited throughout this application are hereby expressly incorporated by reference in their entireties. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

WHAT IS CLAIMED IS:

Claims

1. A method of treating a subject having a disorder or condition comprising administering to said subject, an effective amount of a bispecific antibody molecule that binds both activin and myostatin, or a combination of an anti-activin antibody molecule and an anti- myostatin antibody molecule; wherein the first variable region (e.g., the first antigen-binding site) binds activin specifically, e.g., does not bind or substantially bind myostatin and wherein the second variable region (e.g., the second antigen-binding site) binds myostatin specifically, e.g., does not bind or substantially bind activin.

2. The method of claim 1, wherein said antibody molecule binds one or more (e.g., 1, 2, 3, 4, 5, or all) of growth differentiation factor (GDF)-5, GDF-6, GDF-7, bone morphogenetic factor (BMP)-2, BMP-4, and BMP-7.

3. The method of claim 2, wherein said antibody molecule binds one, two, or all of GDF- 5, GDF-6, GDF-7.

4. The method of claim 2, wherein said antibody molecule binds one, two, or all of BMP-2, BMP-4, and BMP-7.

5. The method of any one of claims 1-4, wherein said antibody molecule binds to activin and/or myostatin with a binding affinity of at least about 10⁵, 10⁶, 10⁷,10⁸, 10⁹, 10¹⁰, or 10¹¹ M ¹.

6. The method of any one of claims 1-5, wherein said antibody molecule binds to activin and/or myostatin with a K_Qff slower than about 1 x 10^~3, 5 x 10^~4, or 1 x 10^~4 s^"1.

7. The method of any one of claims 1-6, wherein said antibody molecule binds to activin and/or myostatin with a K_on faster than about 1 x 10², 1 x 10³, or 5 x 10³ M^'V¹.

8. The method of any one of claims 1-7, wherein said antibody molecule inhibits activin activity and/or myostatin activity, e.g., with a Ki of less than about 10^~5, 10^~6, 10^~7, 10^~8, 5xl0^~9, 10^"9, 5xl0^"10 and 10^"10 M.

9. The method of any one of claims 1-8, wherein said antibody molecule has an IC50 for activin and/or myostatin of less than about 100, 10, or 1 nM.

10. The method of any one of claims 1-9, wherein said antibody molecule has an affinity for activin and/or myostatin characterized by a KD of less than about 100 or 10 nM, or about 3, 5, 7, 9, or 10 nM.

11. The method of any one of claims 1- 10, wherein said antibody molecule can be, e.g., an IgGl, IgG2, IgG3, IgG4, or Fab2'.

12. The method of any one of claims 1- 11, wherein said antibody molecule comprises a human CDR or human framework region.

13. The method of any one of claims 1- 12, wherein said antibody molecule binds an epitope bound by an antibody molecule described herein, or an epitope that overlaps with such epitope.

14. The method of any one of claims 1- 13, wherein said antibody molecule is

administered subcutaneously, intramuscularly, or intravenously, e.g., twice weekly, weekly, biweekly, or monthly.

15. The method of claim 1, comprising administering a combination of an anti-activin antibody molecule and an anti-myostatin antibody molecule.

16. The method of claim 15, wherein said anti-activin antibody molecule and an anti- myostatin antibody molecule are administered in the same dosage form.

17. The method of claim 15, wherein said anti-activin antibody molecule and an anti- myostatin antibody molecule are administered as separate dosage forms.

18. The method of claim 15, wherein said anti-activin antibody molecule and said anti- myostatin antibody molecule are administered to said subject at the same time or within about 240, 180, 120, 90, 60, 30, 15, 10, 5, or 1 minute of one another, e.g., such that there is overlap of an effect of each antibody molecule on said subject.

19. The method of claim 15, wherein said anti-activin antibody molecule and said anti- myostatin antibody molecule are administered sufficiently close together such that a combinatorial (e.g., synergistic) effect is achieved, e.g., greater than an additive effect, e.g., at least 25, 50, 75, 100, 200, 300, 400, or 500% greater than an additive effect.

20. The method of any one of claims 15- 19, wherein said anti-activin antibody molecule and/or anti-myostatin antibody molecule binds one or more (e.g., 1, 2, 3, 4, 5, or all) of GDF-5, GDF-6, GDF-7, BMP-2, BMP-4, and BMP-7.

21. The method of any one of claims 15-20, wherein said anti-activin antibody molecule binds to activin with a binding affinity of at least about 10⁵, 10⁶, 10⁷,10⁸, 10⁹, 10¹⁰, or 10¹¹ M ¹.

22. The method of any one of claims 15-21, wherein said anti-myostatin antibody molecule binds to myostatin with a binding affinity of at least about 10⁵, 10⁶, 10⁷,10⁸, 10⁹, 10¹⁰, or lO¹^^"1.

23. The method of any one of claims 15-22, wherein said anti-activin antibody molecule binds to activin with a K_off slower than about 1 x 10^"3, 5 x 10^"4, or 1 x 10^"4 s ¹.

24. The method of any one of claims 15-23, wherein said anti-myostatin antibody molecule binds to myostatin with a K_Qff slower than about 1 x 10^"3, 5 x 10^"4, or 1 x 10^"4 s^"1.

25. The method of any one of claims 15-24, wherein said anti-activin antibody molecule binds to activin with a K_on faster than about 1 x 10², 1 x 10³, or 5 x 10³ M^'V¹.

26. The method of any one of claims 15-25, wherein said anti-myostatin antibody molecule binds to myostatin with a K_on faster than about 1 x 10², 1 x 10³, or 5 x 10³ M^'V¹.

27. The method of any one of claims 15-26, wherein said anti-activin antibody molecule inhibits activin activity, e.g., with a Ki of less than about 10^"5, 10^"6, 10^"7, 10^~8, 5xl0^"9, 10^"9, 5x10^" ¹⁰, or l0-¹⁰ M.

28. The method of any one of claims 15-27, wherein said anti-myostatin antibody molecule inhibits myostatin activity, e.g., with a Ki of less than about 10^"5, 10^"6, 10^"7, 10^"8, 5x10^" ⁹, 10-⁹, 5xl0-¹⁰, or l0-¹⁰ M.

29. The method of any one of claims 15-28, wherein said anti-activin antibody molecule has an IC50 of less than about 100, 10, or 1 nM.

30. The method of any one of claims 15-29, wherein said anti-myostatin antibody molecule has an IC50 of less than about 100, 10, or 1 nM.

31. The method of any one of claims 15-30, wherein said anti-activin antibody molecule has an affinity characterized by a K_D of less than about 100 or 10 nM, or about 3, 5, 7, 9, or 10 nM.

32. The method of any one of claims 15-31, wherein said anti-myostatin antibody molecule has an affinity characterized by a K_D of less than about 100 or 10 nM, or about 3, 5, 7, 9, or 10 nM.

33. The method of any one of claims 15-32, wherein said anti-activin antibody molecule has a ti/2 of at least about 10, 20, 30, 40, 50, 60, 120, 240, or 360 minutes.

34. The method of any one of claims 15-33, wherein said anti-myostatin antibody molecule has a t_m of at least about 10, 20, 30, 40, 50, 60, 120, 240, or 360 minutes.

35. The method of any one of claims 15-34, wherein said anti-activin antibody molecule and/or anti-myostatin antibody molecule can be, e.g., an IgGl, IgG2, IgG3, IgG4, Fab, Fab2', scFv, minibody, or scFv-Fc fusion.

36. The method of any one of claims 15-35, wherein said anti-activin antibody molecule and/or anti-myostatin antibody molecule comprises a human CDR or human framework region.

37. The method of claims 15-36, wherein said anti-activin antibody molecule and/or anti-myostatin antibody molecule binds an epitope bound by an antibody molecule described herein, or an epitope that overlaps with such epitope.

38. The method of any one of claims 15-37, wherein said anti-activin antibody molecule and/or anti-myostatin antibody molecule is administered subcutaneously, intramuscularly, or intravenously, e.g., twice weekly, weekly, bi-weekly, or monthly.

39. The method of any one of claims 1-38, wherein said disorder or condition is a metabolic disorder or a symptom thereof.

40. The method of claim 39, wherein said metabolic disorder is diabetes, e.g., Type II diabetes.

41. The method of claim 39, wherein said metabolic disorder is obesity.

42. The method of claim 39, wherein the subject has one or more symptoms of: high blood sugar, insulin resistance, glucose intolerance, abnormal lipid levels (e.g., decreased high- density lipoprotein (HDL) level, increased levels of triglycerides and low-density lipoprotein (LDL)), and high blood pressure.

43. The method of claim 39, wherein said metabolic disorder is non-alcoholic fatty liver disease (NAFLD), e.g., non-alcoholic steatohepatitis (NASH).

44. The method of any one of claims 1-38, wherein said disorder or condition is characterized by a need for increased fat browning, increased brown adipose tissue (BAT), or increased thermogenesis.

45. The method of any one of claims 1-38, wherein said disorder or condition is characterized by a need for increased muscle mass.

46. The method of any of one of claims 1-38, wherein said disorder or condition comprises frailty, e.g., frailty associated with or arising from decreased muscle mass or strength.

47. The method of any one of claims 1-38, wherein said disorder or condition is characterized by loss of muscle mass or function, e.g., arising from insufficient use of muscle, e.g., associated with or arising from bed rest or other inactivity arising from or associated with age, disability, or medical condition, or a medical or surgical procedure.

48. The method of any one of claims 1-38, wherein said disorder or condition comprises an acquired or inherited disorder or condition of the muscles, e.g., a muscular dystrophy, e.g., Duchenne muscular dystrophy, spinal muscular atrophy, or amyotrophic lateral sclerosis (ALS).

49. The method of any one of claims 1-48, wherein the subject is a human.

50. The method of any one of claims 1-49, wherein the activin is activin A.

51. The method of any one of claims 1-49, wherein the activin is activin B.

52. A bispecific antibody molecule that binds activin and myostatin, e.g., an antibody molecule described herein.

53. An isolated preparation comprising an anti-activin antibody molecule and an anti- myostatin antibody molecule, e.g., an anti-activin antibody molecule described herein and an anti-myostatin antibody molecule described herein.

54. A composition, e.g., a pharmaceutical composition, comprising a bispecific antibody molecule that binds activin and myostatin, e.g., a bispecific antibody molecule that binds activin and myostatin described herein, e.g., for treating a disorder or condition described herein.

55. A composition, e.g., a pharmaceutical composition, comprising an anti-activin antibody molecule and an anti-myostatin antibody molecule, e.g., a combination of an anti- activin antibody molecule and an anti-myostatin antibody molecule described herein, e.g., for treating a disorder or condition described herein.

56. Use of a bispecific antibody molecule that binds activin and myostatin, e.g., a bispecific antibody molecule that binds activin and myostatin described herein, for the preparation of a medicament for the treatment of a disorder or condition described herein.

57. Use of an anti-activin antibody molecule and an anti-myostatin antibody molecule, e.g., a combination of an anti-activin antibody molecule and an anti-myostatin antibody molecule described herein, for the preparation of a medicament for the treatment of a disorder or condition described herein.

58. A kit comprising a bispecific antibody molecule that binds activin and myostatin.

59. A kit comprising an anti-activin antibody molecule and an anti-myostatin antibody molecule, e.g., a combination of an anti-activin antibody molecule and an anti-myostatin antibody molecule described herein.

60. An article of manufacture comprising a bispecific antibody molecule that binds activin and myostatin.

61. An article of manufacture comprising an anti-activin antibody molecule and an anti- myostatin antibody molecule, e.g., a combination of an anti-activin antibody molecule and an anti-myostatin antibody molecule described herein.