Anti-promyostatin / latent myostatin antibodies and uses thereof

Abstract

Aspects of the present disclosure relate to antibodies that specifically bind promyostatin and / or latent myostatin and uses thereof.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201680065184.7, filed on September 15, 2016, and entitled “Anti-promyostatin / latent myostatin antibodies and their uses”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 219,094, filed on September 15, 2015, and entitled “Anti-promyostatin / latent myostatin antibodies and uses thereof,” the contents of which are incorporated herein for all purposes. Technical Field

[0004] Embodiments of the present disclosure may include modulators of growth factor activity. In some embodiments, such modulators may include antibodies and may modulate the activity and / or biology of a TGF-β family member. Background Art

[0005] Myostatin is a secreted growth factor that negatively regulates muscle mass. Disabling mutations in the myostatin gene (resulting in a hypermuscular phenotype) have been described in cattle, sheep, fish, dogs, and humans. Myostatin expression is generally restricted to skeletal muscle, with low expression levels reported in adipose and cardiac tissues. Inhibition of myostatin signaling leads to an increase in muscle size. Summary of the Invention

[0006] In some embodiments, aspects of the present disclosure relate to antibodies that specifically bind to forms of myostatin (e.g., promyostatin and / or latent myostatin). For example, the antibodies provided herein specifically bind to one or more of the pro-form and / or latent-form of myostatin, such as promyostatin and / or latent myostatin. In certain aspects, the present disclosure is based on the surprising discovery that the antibodies provided herein specifically bind to pure or substantially pure proGDF8 (also known as promyostatin). In some embodiments, the antibodies provided herein inhibit myostatin signaling. In some embodiments, inhibition of myostatin signaling can be used to increase muscle mass or prevent muscle atrophy. In some embodiments, the antibodies provided herein bind to myostatin and prevent myostatin from being cleaved by proprotein convertase and / or tolloid proteases. In some embodiments, preventing cleavage of promyostatin or latent myostatin prevents myostatin activation. Further aspects of the present disclosure relate to antibodies having an affinity for an antigen that is sensitive to pH. In some embodiments, such pH-sensitive antibodies are effective in clearing the antigen from serum. Furthermore, in some embodiments, the antibodies provided herein are sweeping antibodies that can effectively clear an antigen (e.g., promyostatin and / or latent myostatin) from serum.

[0007] Aspects of the present disclosure include antibodies comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises a complementarity determining region 3 (CDRH3) comprising the sequence set forth in any one of SEQ ID NOs: 10-11. In some embodiments, the antibody specifically binds promyostatin / latent myostatin. In some embodiments, the light chain variable domain comprises a complementarity determining region 3 (CDRL3) comprising the sequence set forth in any one of SEQ ID NOs: 22-23. In another embodiment, the antibody comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, wherein CDRH1 comprises the sequence as shown in any one of SEQ ID NOs: 1-3, CDRH2 comprises the sequence as shown in any one of SEQ ID NOs: 4-9, CDRH3 comprises the sequence as shown in any one of SEQ ID NOs: 10-11, CDRL1 comprises the sequence as shown in any one of SEQ ID NOs: 12-17, CDRL2 comprises the sequence as shown in any one of SEQ ID NOs: 18-21, and CDRL3 comprises the sequence as shown in any one of SEQ ID NOs: 22-23.

[0008] In some embodiments, the CDRH1 comprises the sequence shown in SEQ ID NO: 1 or 2, CDRH2 comprises the sequence shown in SEQ ID NO: 4 or 5, CDRH3 comprises the sequence shown in SEQ ID NO: 10, CDRL1 comprises the sequence shown in SEQ ID NO: 12 or 13, CDRL2 comprises the sequence shown in SEQ ID NO: 18 or 19, and CDRL3 comprises the sequence shown in SEQ ID NO: 22.

[0009] In another embodiment, the CDRH1 comprises the sequence shown in SEQ ID NO: 1 or 3, CDRH2 comprises the sequence shown in SEQ ID NO: 6 or 7, CDRH3 comprises the sequence shown in SEQ ID NO: 11, CDRL1 comprises the sequence shown in SEQ ID NO: 14 or 15, CDRL2 comprises the sequence shown in SEQ ID NO: 20 or 21, and CDRL3 comprises the sequence shown in SEQ ID NO: 23.

[0010] In other embodiments, CDRH1 comprises the sequence set forth in SEQ ID NO:1 or 3, CDRH2 comprises the sequence set forth in SEQ ID NO:8 or 9, CDRH3 comprises the sequence set forth in SEQ ID NO:11, CDRL1 comprises the sequence set forth in SEQ ID NO:16 or 17, CDRL2 comprises the sequence set forth in SEQ ID NO:20 or 21, and CDRL3 comprises the sequence set forth in SEQ ID NO:23.

[0011] In another embodiment, the antibody comprises a heavy chain variable domain sequence as shown in any one of SEQ ID NOs: 25 to 29. In some embodiments, the antibody comprises a light chain variable domain sequence as shown in any one of SEQ ID NOs: 30 to 35.

[0012] Other aspects of the present disclosure include antibodies that specifically bind to promyostatin / latent myostatin and comprise a heavy chain variable domain and a light chain variable domain, wherein the light chain variable domain comprises a complementarity determining region 3 (CDRL3) comprising the sequence set forth in any one of SEQ ID NOs: 22-23. In some embodiments, the antibody comprises the light chain variable domain sequence of SEQ ID NO: 30.

[0013] Some aspects of the present disclosure relate to polypeptides having a sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO 29. In some embodiments, the polypeptide is a heavy chain variable domain. In some embodiments, the polypeptide is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO 29.

[0014] Some aspects of the present disclosure relate to polypeptides having a sequence selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO 35. In some embodiments, the polypeptide is a light chain variable domain. In some embodiments, the polypeptide is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of the amino acid sequences set forth in SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO 35.

[0015] Another aspect of the present disclosure includes antibodies that compete with the above antibodies for binding to promyostatin / latent myostatin. In some embodiments, the antibodies bind to promyostatin / latent myostatin at the same epitope as the above antibodies. In another embodiment, the antibodies bind to promyostatin / latent myostatin at a distance of less than 10 μg / cm2 from the antibody to promyostatin / latent myostatin. -6 In other embodiments, the antibody has an equilibrium dissociation constant Kd of 10 -11 M to 10 -6 Within the range of M.

[0016] In some embodiments, the antibody is a humanized antibody, a diabody, a chimeric antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. In another embodiment, the antibody is a humanized antibody. In another embodiment, the antibody is a human antibody. In some embodiments, the antibody comprises a framework with a human germline sequence. In another embodiment, the antibody comprises a heavy chain constant domain selected from IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE constant domains. In some embodiments, the antibody comprises an IgG4 constant domain. In other embodiments, the antibody comprises an IgG4 constant domain with a Ser to Pro backbone substitution, which produces an IgG1-like hinge and allows the formation of interchain disulfide bonds. In another embodiment, the antibody is coupled to a reagent selected from a fluorescent agent, a luminescent agent, an enzyme reagent, and a radioactive agent.

[0017] In another embodiment, the antibody specifically binds promyostatin / latent myostatin compared to mature myostatin. In some embodiments, the antibody specifically binds promyostatin / latent myostatin compared to another member of the transforming growth factor beta family. In another embodiment, the member is GDF11 or activin.

[0018] Further aspects of the present disclosure include antibodies that specifically bind to promyostatin / latent myostatin and inhibit the formation of mature myostatin by tolloid protease proteolysis. In some embodiments, the antibodies inhibit the formation of mature myostatin by tolloid protease proteolysis with an IC50 of less than 1 μM. In some embodiments, the antibodies are cross-reactive with human and murine promyostatin / latent myostatin. In other embodiments, the antibodies specifically bind to promyostatin / latent myostatin compared to GDF11 or activin. In another embodiment, the antibodies specifically bind to promyostatin / latent myostatin compared to mature myostatin.

[0019] Another aspect of the present disclosure includes a method for reducing myostatin receptor activation in cells present in a culture medium containing promyostatin / latent myostatin, the method comprising delivering to the culture medium an amount effective to inhibit proteolytic activation of promyostatin / latent myostatin. In some embodiments, the culture medium further comprises a proprotein convertase. In other embodiments, the culture medium further comprises a tolloid protease. In another embodiment, the antibody is delivered to the culture medium in an amount effective to inhibit proteolytic activation of promyostatin / latent myostatin by the tolloid protease. In some embodiments, the cells are in vitro. In other embodiments, the cells are in vivo.

[0020] Another aspect of the present disclosure includes a method for treating a subject suffering from a myopathy, the method comprising administering to the subject an effective amount of the above-mentioned antibody. In some embodiments, the myopathy is a primary myopathy. In another embodiment, the primary myopathy comprises disuse atrophy. In other embodiments, disuse atrophy is associated with hip fracture, selective joint replacement, critical illness myopathy, spinal cord injury or stroke. In some embodiments, the myopathy is a secondary myopathy, wherein muscle loss is secondary to disease pathology. In other embodiments, the secondary myopathy comprises denervation, hereditary muscle weakness or cachexia. In another embodiment, the secondary myopathy is denervation associated with amyotrophic lateral sclerosis or spinal muscular atrophy. In some embodiments, the secondary myopathy is hereditary muscle weakness associated with muscular dystrophy. In other embodiments, the secondary myopathy is cachexia associated with renal failure, AIDS, heart disease, cancer or aging.

[0021] Another aspect of the present disclosure includes methods of treating a subject having a disease or condition associated with aging. Exemplary diseases or conditions associated with aging include, but are not limited to, sarcopenia (age-related muscle loss), frailty, and androgen deficiency.

[0022] Another aspect of the present disclosure includes a method of treating a subject having a disease or condition associated with disuse atrophy / trauma. Exemplary diseases or conditions associated with disuse atrophy / trauma include, but are not limited to, muscle weakness associated with time spent in an intensive care unit (ICU), hip / joint replacement, hip fracture, stroke, bed rest, SCI, rotator cuff injury, knee replacement, bone fracture, and burns.

[0023] Another aspect of the present disclosure includes methods of treating a subject having a neurodegenerative disease or condition.Exemplary neurodegenerative diseases or conditions include, but are not limited to, spinal muscular atrophy and amyotrophic lateral sclerosis (ALS).

[0024] Another aspect of the present disclosure includes methods of treating a subject having a disease or condition associated with cachexia. Exemplary diseases or conditions associated with cachexia include, but are not limited to, cancer, chronic heart failure, acquired immunodeficiency syndrome (AIDS), chronic obstructive pulmonary disease (COPD), and chronic kidney disease (CKD).

[0025] Another aspect of the present disclosure includes methods of treating a subject suffering from a disease or condition associated with a rare disease. Exemplary rare diseases and conditions include, but are not limited to, osteogenesis imperfecta, sporadic inclusion body myositis, and acute lymphoblastic leukemia.

[0026] Another aspect of the present disclosure includes treating a subject suffering from a disease or condition related to dysmetabolic and / or body composition. In some embodiments, the disease or condition is obesity (e.g., severe obesity), Prader-Willi, type II diabetes, or anorexia. However, other diseases or conditions related to dysmetabolic and / or body composition are within the scope of the present disclosure.

[0027] Another aspect of the present disclosure includes methods of treating a subject having a disease or condition associated with a congenital myopathy. Exemplary congenital myopathies include, but are not limited to, X-linked myotubular myopathy, autosomal dominant centronuclear myopathy, autosomal recessive centronuclear myopathy, nematode myopathy, and congenital fiber-type disproportionate myopathy.

[0028] Another aspect of the present disclosure includes methods of treating a subject having a disease or condition associated with a muscular dystrophy. Exemplary muscular dystrophy include, but are not limited to, Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy (FSH), and limb-girdle muscular dystrophy.

[0029] Another aspect of the present disclosure includes methods of treating a subject having a urogynecological-related disease or disorder, speech disorder (stenosis), extraocular myopathy, carpal tunnel syndrome, Guillain-Barré syndrome, or osteosarcoma.

[0030] In some embodiments, treatment results in improved muscle strength in the subject. In other embodiments, treatment results in improved metabolic state in the subject.

[0031] In some embodiments, the antibody is administered at a dose in the range of 0.1 mg / kg-100 mg / kg. In another embodiment, the antibody is administered at a dose in the range of 0.3 mg / kg-30 mg / kg.

[0032] In some embodiments, the antibody is administered intravenously to the subject. In other embodiments, the antibody is administered subcutaneously to the subject. In another embodiment, the antibody is administered to the subject on multiple occasions. In some embodiments, the multiple administrations are performed at least monthly. In another embodiment, the multiple administrations are performed at least weekly.

[0033] Further aspects of the present disclosure include compositions comprising any of the above antibodies and a carrier. In some embodiments, the carrier is a pharmaceutically acceptable carrier. In other embodiments, the antibody and carrier are in lyophilized form. In another embodiment, the antibody and carrier are in solution. In some embodiments, the antibody and carrier are frozen. In other embodiments, the antibody and carrier are frozen at a temperature of less than or equal to -65°C.

[0034] Other aspects of the present disclosure include isolated nucleic acids encoding a protein comprising three complementarity determining regions (CDRs): CDRH1, CDRH2, and CDRH3, wherein CDRH3 comprises the sequence set forth in SEQ ID NO: 10 or 11. In some embodiments, the CDRH1 comprises the sequence set forth in SEQ ID NO: 1, 2, or 3. In other embodiments, CDRH2 comprises the sequence set forth in any one of SEQ ID NOs: 4-9.

[0035] Another aspect of the present disclosure includes isolated nucleic acids encoding a protein comprising three complementarity determining regions (CDRs): CDRL1, CDRL2, and CDRL3, wherein CDRL3 comprises the sequence set forth in SEQ ID NO: 22. In some embodiments, the CDRL1 comprises the sequence set forth in any one of SEQ ID NOs: 12-17. In other embodiments, CDRL2 comprises the sequence set forth in any one of SEQ ID NOs: 18-21.

[0036] Further aspects of the present disclosure include an isolated nucleic acid comprising a sequence as set forth in any one of SEQ ID NOs: 38-49.

[0037] Another aspect of the present disclosure includes isolated cells comprising the isolated nucleic acid described above.

[0038] In some aspects, the present disclosure includes methods for evaluating a biological sample obtained from a subject suffering from a myopathy. In some embodiments, the method comprises: preparing an immunoreaction mixture comprising a protein from a biological sample obtained from a subject and an antibody that specifically binds to promyostatin / latent myostatin; maintaining the immunoreaction mixture under conditions that allow formation of a binding complex between the antibody and promyostatin / latent myostatin; and determining the level of binding complex formation. In some embodiments, the method comprises: preparing an immunoreaction mixture comprising a protein from a biological sample obtained from a subject and an antibody that specifically binds to promyostatin; maintaining the immunoreaction mixture under conditions that allow formation of a binding complex between the antibody and promyostatin; and determining the level of binding complex formation. In some embodiments, the method comprises: preparing an immunoreaction mixture comprising a protein from a biological sample obtained from a subject and an antibody that specifically binds to latent myostatin; maintaining the immunoreaction mixture under conditions that allow formation of a binding complex between the antibody and latent myostatin; and determining the level of binding complex formation. In some embodiments, the method comprises: preparing an immunoreaction mixture comprising a protein from a biological sample obtained from a subject and an antibody that specifically binds to mature myostatin; maintaining the immunoreaction mixture under conditions that permit formation of a binding complex between the antibody and mature myostatin; and determining the level of binding complex formation.

[0039] In one aspect, disclosed herein is an isolated antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 31. In one embodiment, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 50. In another embodiment, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 51.

[0040] In another aspect, disclosed herein is an isolated antibody comprising a heavy chain variable region comprising a CDRH1 sequence comprising SEQ ID NO: 1, a CDRH2 sequence comprising SEQ ID NO: 6, and a CDRH3 sequence comprising SEQ ID NO: 11; and a light chain variable region comprising a CDRL1 sequence comprising SEQ ID NO: 14, a CDRL2 sequence comprising SEQ ID NO: 20, and a CDRL3 sequence comprising SEQ ID NO: 23.

[0041] In one embodiment, the heavy chain variable region comprises the sequence of SEQ ID NO: 26. In another embodiment, the light chain variable region comprises the sequence of SEQ ID NO: 32.

[0042] In one embodiment, the heavy chain variable region comprises the sequence of SEQ ID NO: 27. In another embodiment, the light chain variable region comprises the sequence of SEQ ID NO: 33.

[0043] In another aspect, disclosed herein is an isolated antibody comprising a heavy chain variable region comprising a CDRH1 sequence comprising SEQ ID NO: 1, a CDRH2 sequence comprising SEQ ID NO: 8, and a CDRH3 sequence comprising SEQ ID NO: 11; and a light chain variable region comprising a CDRL1 sequence comprising SEQ ID NO: 16, a CDRL2 sequence comprising SEQ ID NO: 20, and a CDRL3 sequence comprising SEQ ID NO: 23.

[0044] In one embodiment, the heavy chain variable region comprises the sequence of SEQ ID NO: 28. In another embodiment, the light chain variable region comprises the sequence of SEQ ID NO: 34.

[0045] In one embodiment, the heavy chain variable region comprises the sequence of SEQ ID NO: 29. In one embodiment, the light chain variable region comprises the sequence of SEQ ID NO: 35.

[0046] In one embodiment, the antibody is a human antibody. In one embodiment, the antibody comprises an IgG4 constant domain. In one embodiment, the antibody comprises an IgG4 constant domain with a Ser to Pro backbone substitution, which creates an IgG1-like hinge and allows for the formation of interchain disulfide bonds.

[0047] In one embodiment, the antibody specifically binds promyostatin / latent myostatin. In one embodiment, the antibody specifically binds promyostatin. In another embodiment, the antibody specifically binds latent myostatin. In one embodiment, the antibody does not bind mature myostatin.

[0048] In one embodiment, the antibody inhibits the proteolytic formation of mature myostatin by tolloid proteases. In one embodiment, the antibody inhibits the proteolytic formation of mature myostatin by tolloid proteases with an IC50 of less than 1 μM.

[0049] In one embodiment, the antibody is cross-reactive with human and murine promyostatin / latent myostatin. In another embodiment, the antibody binds promyostatin / latent myostatin but does not bind GDF11 or activin.

[0050] In one aspect, disclosed herein is a method for reducing myostatin receptor activation in cells present in a culture medium containing promyostatin / latent myostatin, the method comprising delivering to the culture medium an amount effective to inhibit proteolytic activation of promyostatin / latent myostatin. In one embodiment, the culture medium comprises a proprotein convertase. In another embodiment, the culture medium comprises a tolloid protease. In one embodiment, the cells are in vitro. In another embodiment, the cells are in vivo.

[0051] In another aspect, disclosed herein are methods of treating a subject suffering from a myopathy, the method comprising administering to the subject an effective amount of an antibody disclosed herein.

[0052] In one embodiment, the myopathy is a primary myopathy. In another embodiment, the primary myopathy is disuse atrophy. In one embodiment, the disuse atrophy is associated with hip fracture, selective joint replacement, critical illness myopathy, spinal cord injury, and / or stroke.

[0053] In another embodiment, the myopathy is a secondary myopathy, wherein muscle loss is secondary to a disease pathology. In one embodiment, the secondary myopathy comprises denervation, hereditary myasthenia, or cachexia. In another embodiment, the secondary myopathy is denervation associated with amyotrophic lateral sclerosis or spinal muscular atrophy. In yet another embodiment, the secondary myopathy is hereditary myasthenia associated with muscular dystrophy. In one embodiment, the secondary myopathy is cachexia associated with renal failure, AIDS, heart disease, cancer, or aging.

[0054] In one embodiment, administration results in improved muscle strength in the subject. In one embodiment, administration results in improved metabolic state in the subject.

[0055] In one embodiment, the antibody is administered at a dose in the range of 0.1 mg / kg-100 mg / kg. In another embodiment, the antibody is administered at a dose in the range of 0.3 mg / kg-30 mg / kg.

[0056] In one embodiment, the antibody is administered to the subject intravenously. In another embodiment, the antibody is administered to the subject subcutaneously.

[0057] In one embodiment, the antibody is administered to the subject on multiple occasions. In one embodiment, the multiple administrations are performed at least monthly. In another embodiment, the multiple administrations are performed at least weekly.

[0058] In another aspect, disclosed herein are pharmaceutical compositions comprising an antibody disclosed herein and a pharmaceutically acceptable carrier. In one embodiment, the composition is a lyophilized composition. In another embodiment, the composition is a liquid composition. In one embodiment, the composition is frozen. In one embodiment, the composition is frozen at a temperature of less than or equal to -65°C.

[0059] In another aspect, disclosed herein is a syringe comprising a pharmaceutical composition described herein.

[0060] In another aspect, disclosed herein is an isolated nucleic acid encoding an antibody comprising a heavy chain variable region comprising the nucleic acid sequence of SEQ ID NO: 39 and a light chain variable region comprising the nucleic acid sequence of SEQ ID NO: 45.

[0061] In another aspect, disclosed herein is an isolated nucleic acid encoding an antibody comprising a heavy chain variable region comprising a CDRH1 sequence comprising SEQ ID NO: 1, a CDRH2 sequence comprising SEQ ID NO: 6, and a CDRH3 sequence comprising SEQ ID NO: 11; and a light chain variable region comprising a CDRL1 sequence comprising SEQ ID NO: 14, a CDRL2 sequence comprising SEQ ID NO: 20, and a CDRL3 sequence comprising SEQ ID NO: 23.

[0062] In one embodiment, the heavy chain variable region comprises the sequence of SEQ ID NO: 40. In one embodiment, the light chain variable region comprises the sequence of SEQ ID NO: 46.

[0063] In one embodiment, the heavy chain variable region comprises the sequence of SEQ ID NO: 41. In another embodiment, the light chain variable region comprises the sequence of SEQ ID NO: 47.

[0064] In another embodiment, disclosed herein is an isolated nucleic acid encoding an antibody comprising a heavy chain variable region comprising a CDRH1 sequence comprising SEQ ID NO: 1, a CDRH2 sequence comprising SEQ ID NO: 8, and a CDRH3 sequence comprising SEQ ID NO: 11; and a light chain variable region comprising a CDRL1 sequence comprising SEQ ID NO: 16, a CDRL2 sequence comprising SEQ ID NO: 20, and a CDRL3 sequence comprising SEQ ID NO: 23.

[0065] In one embodiment, the heavy chain variable region comprises the sequence of SEQ ID NO: 42. In another embodiment, the light chain variable region comprises the sequence of SEQ ID NO: 48.

[0066] In one embodiment, the heavy chain variable region comprises the sequence of SEQ ID NO: 43. In another embodiment, the light chain variable region comprises the sequence of SEQ ID NO: 49.

[0067] In another aspect, disclosed herein is an isolated cell comprising the isolated nucleic acid described herein.

[0068] In particular, the present invention relates to the following:

[0069] 1. An isolated antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 31.

[0070] 2. The isolated antibody of item 1, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 50.

[0071] 3. The isolated antibody of item 1 or 2, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 51.

[0072] 4. An isolated antibody comprising:

[0073] a heavy chain variable region comprising a CDRH1 sequence comprising SEQ ID NO: 1, a CDRH2 sequence comprising SEQ ID NO: 6, and a CDRH3 sequence comprising SEQ ID NO: 11; and

[0074] The light chain variable region comprises a CDRL1 sequence comprising SEQ ID NO: 14, a CDRL2 sequence comprising SEQ ID NO: 20, and a CDRL3 sequence comprising SEQ ID NO: 23.

[0075] 5. The isolated antibody of item 4, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 26.

[0076] 6. The isolated antibody of item 4 or 5, wherein the light chain variable region comprises the sequence of SEQ ID NO: 32.

[0077] 7. The isolated antibody of item 4, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 27.

[0078] 8. The isolated antibody of item 4 or item 7, wherein the light chain variable region comprises the sequence of SEQ ID NO: 33.

[0079] 9. An isolated antibody comprising

[0080] a heavy chain variable region comprising a CDRH1 sequence comprising SEQ ID NO: 1, a CDRH2 sequence comprising SEQ ID NO: 8, and a CDRH3 sequence comprising SEQ ID NO: 11; and

[0081] The light chain variable region comprises a CDRL1 sequence comprising SEQ ID NO: 16, a CDRL2 sequence comprising SEQ ID NO: 20, and a CDRL3 sequence comprising SEQ ID NO: 23.

[0082] 10. The isolated antibody of item 9, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 28.

[0083] 11. The isolated antibody of item 9 or item 10, wherein the light chain variable region comprises the sequence of SEQ ID NO: 34.

[0084] 12. The isolated antibody of item 9, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 29.

[0085] 13. The isolated antibody of item 9 or item 12, wherein the light chain variable region comprises the sequence of SEQ ID NO: 35.

[0086] 14. The isolated antibody of any one of items 1 to 13, wherein the antibody is a human antibody.

[0087] 15. The isolated antibody of any one of items 1-14, wherein the antibody comprises an IgG4 constant domain.

[0088] 16. The isolated antibody of any one of items 1-14, wherein the antibody comprises an IgG4 constant domain with a Ser to Pro backbone substitution that creates an IgG1-like hinge and allows for interchain disulfide bond formation.

[0089] 17. The isolated antibody of any one of items 1 to 16, wherein the antibody specifically binds promyostatin / latent myostatin.

[0090] 18. The isolated antibody of any one of items 1-17, wherein the antibody does not bind to mature myostatin.

[0091] 19. The isolated antibody of any one of items 1 to 18, wherein the antibody inhibits proteolytic formation of mature myostatin by tolloid protease.

[0092] 20. The isolated antibody of item 19, wherein the antibody inhibits proteolytic cleavage of tolloid protease to form mature myostatin with an IC50 of less than 1 μM.

[0093] 21. The isolated antibody of item 19 or 20, wherein the antibody is cross-reactive with human and murine promyostatin / latent myostatin.

[0094] 22. The isolated antibody of any one of items 19-21, wherein the antibody binds to promyostatin / latent myostatin but does not bind to GDF11 or activin.

[0095] 23. A method of reducing myostatin receptor activation in cells present in a culture medium comprising promyostatin / latent myostatin, the method comprising delivering to the culture medium the antibody of any one of items 1 to 22 in an amount effective to inhibit proteolytic activation of the promyostatin / latent myostatin.

[0096] 24. A method of treating a subject suffering from a myopathy, the method comprising administering to the subject an effective amount of the antibody of any one of items 1-22.

[0097] 25. A pharmaceutical composition comprising the antibody according to any one of items 1 to 22 and a pharmaceutically acceptable carrier.

[0098] 26. The pharmaceutical composition according to item 25, wherein the composition is a lyophilized composition.

[0099] 27. The pharmaceutical composition according to item 25, wherein the composition is a liquid composition.

[0100] 28. The pharmaceutical composition according to item 25, wherein the composition is frozen.

[0101] 29. The pharmaceutical composition according to item 28, wherein the composition is frozen at a temperature lower than or equal to -65°C.

[0102] 30. A syringe comprising the pharmaceutical composition according to any one of items 25 to 29.

[0103] 31. An isolated nucleic acid encoding an antibody comprising a heavy chain variable region comprising the nucleic acid sequence of SEQ ID NO: 39 and a light chain variable region comprising the nucleic acid sequence of SEQ ID NO: 45.

[0104] 32. An isolated nucleic acid encoding an antibody comprising:

[0105] a heavy chain variable region comprising a CDRH1 sequence comprising SEQ ID NO: 1, a CDRH2 sequence comprising SEQ ID NO: 6, and a CDRH3 sequence comprising SEQ ID NO: 11; and

[0106] The light chain variable region comprises a CDRL1 sequence comprising SEQ ID NO: 14, a CDRL2 sequence comprising SEQ ID NO: 20, and a CDRL3 sequence comprising SEQ ID NO: 23.

[0107] 33. The isolated nucleic acid of item 32, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 40.

[0108] 34. The isolated nucleic acid of item 32 or item 33, wherein the light chain variable region comprises the sequence of SEQ ID NO: 46.

[0109] 35. The isolated nucleic acid of item 32, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 41.

[0110] 36. The isolated nucleic acid of item 32 or item 35, wherein the light chain variable region comprises the sequence of SEQ ID NO: 47.

[0111] 37. An isolated nucleic acid encoding an antibody comprising:

[0112] a heavy chain variable region comprising a CDRH1 sequence comprising SEQ ID NO: 1, a CDRH2 sequence comprising SEQ ID NO: 8, and a CDRH3 sequence comprising SEQ ID NO: 11; and

[0113] The light chain variable region comprises a CDRL1 sequence comprising SEQ ID NO: 16, a CDRL2 sequence comprising SEQ ID NO: 20, and a CDRL3 sequence comprising SEQ ID NO: 23.

[0114] 38. The isolated nucleic acid of item 37, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 42.

[0115] 39. The isolated nucleic acid of item 37 or item 38, wherein the light chain variable region comprises the sequence of SEQ ID NO: 48.

[0116] 40. The isolated nucleic acid of item 37, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 43.

[0117] 41. The isolated nucleic acid of item 37 or item 40, wherein the light chain variable region comprises the sequence of SEQ ID NO: 49.

[0118] 42. An isolated cell comprising the isolated nucleic acid of any one of items 31-41. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figures 1A-1B The myostatin domain structure and promyostatin assembly are shown. Figure 1A Myostatin was shown to be secreted as a proprotein with an inhibitory prodomain followed by a C-terminal growth factor domain, the proprotein existing as a disulfide-linked dimer. Figure 1B The precursor protein is shown assembled in an inactive conformation, with the prodomain (dark grey) surrounding the growth factor (light grey) in a "straightjacket" assembly. This figure is adapted from the structure of latent TGFβ1 (Shi et al. Nature 2011).

[0120] Figure 2 The activation of myostatin has been shown to involve two distinct protease events, resulting in the production of three major myostatin species. The biosynthetic precursor protein, promyostatin, is processed by two separate proteases. Cleavage of promyostatin (and proGDF11) is carried out by proprotein convertases such as Furin / PACE3 (Paired Basic Amino acid Cleaving Enzyme 3) or PCSK5 (Proprotein Convertase Subtilisin / Kexin Type 5), which cleave the conserved RXXR site between the prodomain and the mature growth factor. This cleavage generates a latent complex in which the mature growth factor is shielded from binding to its receptor by the prodomain. Activation and release of the active growth factor are accomplished following cleavage by additional proteases from the BMP / tolloid family, such as TLL-2 (Tolloid-like protein 2) or BMP1 (bone morphogenetic protein 1). These cleavage events produce the mature form of myostatin, which can be referred to as active myostatin or mature myostatin.

[0121] Figures 3A-3C Ab1 was shown to block the cleavage of promyostatin by members of the tolloid family of proteases. Latent myostatin samples pre-incubated with increasing amounts of Ab1 were analyzed in a myostatin activation assay. Figure 3A ) After analysis of myostatin release, samples were then run under reducing conditions and probed by Western blotting using an antibody raised against the prodomain of myostatin ( Figure 3B). The ~18 kDa band (box), corresponding to the ARM portion of the prodomain generated after tolloid cleavage, decreases proportionally with increasing Abl dose. Latent and promyostatin standards (45 ng loaded) show migration of promyostatin at ~50 kDa and the prodomain at ~37 kDa. Figure 3C The activation of myostatin has been shown to involve two distinct protease events, resulting in the production of three major myostatin species. The biosynthetic precursor protein, promyostatin, is processed by two separate proteases. Cleavage of promyostatin (and proGDF11) is carried out by proprotein convertases such as Furin / PACE3 (coupled essential amino acid cleavage enzyme 3) or PCSK5 (proprotein convertase subtilisin / Kexin type 5), which cleave the conserved RXXR site between the prodomain and the mature growth factor. This cleavage generates a latent complex in which the mature growth factor is shielded from binding to its receptor by the prodomain. See Figure 3B , which shows potential inhibition of proteases, thereby blocking further cleavage of promyostatin. Activation and release of the active growth factor is accomplished after cleavage by additional proteases from the BMP / tolloid family, such as TLL-2 (Tolloid-like protein 2) or BMP1 (bone morphogenetic protein 1).

[0122] Figure 4 The performance of the parental Ab1 antibody and other candidates in a cell-based reporter assay is shown. Following an overnight proteolysis reaction with enzymes from both the proprotein convertase and tolloid protease families, the release of mature growth factor was measured in 293T cells using a CAGA-based reporter assay. Results were compared to control reactions to calculate the fraction of promyostatin or proGDF11 released in the assay. The standard deviation of the mean of three replicates is shown, but for most data points, they are not visible on the graph due to their low amplitude.

[0123] Figure 5 The graphic shows that Ab1, Ab2, Ab4 and Ab6 antibodies did not inhibit proGDF11 activation.

[0124] Figure 6 Results of experiments evaluating mean percent body weight change are shown. Animals were weighed daily, and the percent weight change from day 0 was calculated. Data represent group mean ± SEM. Mean percent change data for each group on study day 42 were analyzed relative to the PBS control group using one-way ANOVA followed by a Holm-Sidak's post-hoc test. **p < 0.01.

[0125] Figures 7A-7D Results of experiments evaluating tissue weight are shown. Figure 7AMean gastrocnemius muscle weights are shown. Figure 7B Displays average pectoral muscle weight. Figure 7C Average soleus muscle weights are shown. Figure 7D Mean triceps weights are shown. Statistical evaluation was performed using a one-way ANOVA followed by a Holm-Sidak's post hoc test relative to the vehicle control group (Group 1). Data represent group means ± SEM. **p < 0.01. Bars indicate Groups 1-5 from left to right.

[0126] Figures 8A-8C Results of experiments evaluating tissue weight are shown. Figure 8A Mean tibialis anterior muscle weights are shown. Figure 8B Displays the average diaphragm weight. Figure 8C Mean quadriceps muscle weights are shown. Statistical evaluation was performed using a one-way ANOVA followed by a Holm-Sidak's post hoc test relative to the vehicle control group (Group 1). Data represent group means ± SEM. *p < 0.05. Bars indicate Groups 1-5 from left to right.

[0127] Figures 9A-9B Results of trials evaluating mean percent changes in body weight and lean body mass are shown. Figure 9A is a graph showing the percent weight change from Day 0 calculated for animals weighed twice weekly throughout the study. Figure 9B Animals underwent echoMRI (QNMR) to measure body composition on days -4, 7, 14, 21, and 28, and the percent lean body mass change from day 0 was calculated. Data represent group means ± SEM. For both body weight and lean body mass, the mean percent change data for each group on day 28 of the study were analyzed relative to the IgG control group (Group 2) using a one-way ANOVA followed by a Holm-Sidak's post-hoc test. ***p < 0.0005, **p < 0.005, *p < 0.05, ns (not significant).

[0128] Figures 10A-10D is a graph showing the results of a test to evaluate muscle mass. Figure 10A Displays average quadriceps weight, Figure 10B Displays the average gastrocnemius muscle weight, Figure 10C Shows the average tibialis anterior muscle weight, and Figure 10D Mean diaphragm muscle weights are shown. The percentage difference in mean muscle weight between the Ab1-treated group and the IgG control group is indicated above each bar. Statistical evaluation was performed using a one-way ANOVA followed by a Holm-Sidak's post-hoc test relative to the IgG control group (Group 2). Data represent group means ± SEM. ****p < 0.0001, ***p < 0.0005, **p < 0.005, *p < 0.05, ns (not significant).

[0129] Figures 11A-11B Results of trials evaluating mean percent changes in body weight and lean body mass are shown. Figure 11A Shown are percent weight changes from day 0 calculated from animals weighed twice weekly throughout the study. Figure 11B Animals underwent echoMRI (QNMR) to measure body composition on days -1, 6, and 13, and to calculate percent lean body mass change from day -1. PBS = phosphate-buffered saline, Dex = dexamethasone, IgG(20) = IgG control antibody administered at 20 mg / kg / wk, Abl(20) = Abl antibody administered at 20 mg / kg / wk, and Abl(2) = Abl antibody administered at 2 mg / kg / wk. Data represent group means ± SEM. Data for mean percentage change for each group on day 14 (for body weight) and day 13 (for lean body mass) were analyzed using one-way ANOVA followed by Dunnett's multiple comparison test relative to Group 1 (****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05) and relative to Group 5 (++++p<0.0001, +++p<0.0005, ++p<0.005, +p<0.05). ns (not significant).

[0130] Figures 12A-12D is a graph showing the results of a test evaluating the weight of different muscles. Figure 12A Displays the average gastrocnemius muscle weight (g), Figure 12B Displays average quadriceps weight (g), Figure 12C Shown are the mean percent gastrocnemius muscle weight changes relative to control animals treated with PBS (IP) and normal drinking water (Group 1), and Figure 12D Shown are the mean percent quadriceps weight changes relative to control animals treated with PBS (IP) and normal drinking water (Group 1). PBS = phosphate buffered saline, Dex = dexamethasone, IgG(20) = IgG control antibody dosed at 20 mg / kg / wk, Abl(20) = Abl antibody dosed at 20 mg / kg / wk, and Abl(2) = Abl antibody dosed at 2 mg / kg / wk. Figures 12A-12B , error bars represent standard deviation (SD). Figures 12C-12D, error bars represent standard error of the mean (SEM). Statistical evaluation was performed using a one-way ANOVA followed by Dunnett's multiple comparison test relative to group 1 (****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05) and relative to group 5 (++++p<0.0001, +++p<0.0005, ++p<0.005, +p<0.05). ns (not significant). Bars indicate from left to right PBS, water; PBS, dex; IgG control; Ab1(20); and Ab1(2).

[0131] Figures 13A-13B Results of trials evaluating mean percent changes in body weight and lean body mass are shown. Figure 13A Shown are percent weight changes from day 0 calculated for animals weighed twice weekly throughout the study. Figure 13B Shown are percent lean body mass changes from day -1 calculated from animals that underwent EchoMRI (QNMR) to measure body composition on days -1, 7, and 14. PBS = phosphate-buffered saline, IgG(20) = IgG control antibody dosed at 20 mg / kg / wk, Abl(20) = Abl antibody dosed at 20 mg / kg / wk, and Abl(2) = Abl antibody dosed at 2 mg / kg / wk. Data represent group means ± SEM.

[0132] Figures 14A-14D Showing the results of a test assessing muscle mass. Figure 14A Shown are the average gastrocnemius muscle weights (g) for the casted leg. Figure 14B Shows the average quadriceps weight (g) of the casted leg, Figure 14C Shown are the mean percent gastrocnemius muscle weight changes relative to control animals treated with PBS (IP) and not casted (Group 1), and Figure 14D Shown are the mean percent quadriceps weight changes relative to control animals treated with PBS (IP) and not casted (Group 1). PBS = phosphate buffered saline, IgG(20) = IgG control antibody dosed at 20 mg / kg / wk, Abl(20) = Abl antibody dosed at 20 mg / kg / wk, and Abl(2) = Abl antibody dosed at 2 mg / kg / wk. Figures 14A-14B , error bars represent standard deviation (SD). Figures 14C-14D, error bars represent standard error of the mean (SEM). Statistical evaluation was performed using one-way ANOVA followed by Dunnett's multiple comparison test relative to group 1 (****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05) and relative to group 5 (++++p<0.0001, +++p<0.0005, ++p<0.005, +p<0.05). ns (not significant). Bars indicate from left to right PBS, no plaster; PBS, plaster; IgG control (2), plaster; Abl (20), plaster; and Abl (2), plaster.

[0133] Figure 15 Results of an experiment evaluating changes in lean body mass on day 21 (upper right) and day 28 (upper left) are shown. Also depicted are the percent changes in lean body mass at three different doses of the test antibody (20 mg / kg / wk (lower left), 2 mg / kg / wk (lower center), and 0.5 mg / kg / wk (lower right)), a PBS control, and an IgG control. Statistical evaluation was performed using a one-way ANOVA followed by Dunnett's multiple comparison test relative to Group 1 (****p<0.0001, ***p<0.005, **p<0.01, *p<0.05) and relative to the IgG control. For the two top figures, bars from left to right are: PBS; IgG Ctrl 20 mg / kg / wk; Abl 20 mg / kg / wk; Abl 2 mg / kg / wk; Abl 0.5 mg / kg / wk; Ab2 20 mg / kg / wk; Ab2 2 mg / kg / wk; Ab2 0.5 mg / kg / wk; Ab4 20 mg / kg / wk; Ab4 2 mg / kg / wk; Ab4 0.5 mg / kg / wk; Ab6 20 mg / kg / wk; Ab6 2 mg / kg / wk; and Ab6 0.5 mg / kg / wk. For the lower left figure (20 mg / kg / wk), the data points corresponding to day 28 post-dose correspond from top to bottom to Abl, Ab4, Ab2, Ab6, IgG control, and PBS. For the lower middle graph (2 mg / kg / wk), the data points corresponding to day 28 after administration correspond to Ab2, Ab1, Ab6, Ab4, IgG control, and PBS from top to bottom. For the lower right graph (0.5 mg / kg / wk), the data points corresponding to day 28 after administration correspond to IgG control Ab1, Ab2, PBS, Ab4, and Ab6 from top to bottom.

[0134] Figures 16A-16B The domain structure and evaluation of the proform of myostatin are shown. Figure 16A The domain structures of promyostatin and latent myostatin are shown, and the protease cleavage sites are indicated. Figure 16B A portion of proprotein convertase-cleaved promyostatin run on an SDS PAGE gel is shown. Under reducing conditions, the protein bands consist of the promyostatin monomer (~50 kD), the prodomain (~37 kD), and the growth factor (12.5 kD).

[0135] Figures 17A-17B Abl was shown to be specific for myostatin. Figure 17A Ab1 was shown to specifically bind to promyostatin and latent myostatin, with no observed binding to other members of the TGFB superfamily, most notably the corresponding form of GDF11. Ab1 was applied at high concentration (50 ug / mL) to Forte-Bio BLI tips coated with the indicated antigens and the association and dissociation rates were measured to obtain approximate Kd values. The magnitude of the biosensor response (indicating binding events) is graphically represented by black bars, and the calculated Kd is indicated in orange. Figure 17B Ab1 was shown to block the activation of promyostatin, but not proGDF11. Following an overnight proteolysis reaction with enzymes from both the proprotein convertase and tolloid protease families, the release of mature growth factors was measured in 293T cells using a CAGA-based reporter assay. Results were compared to control reactions to calculate the fraction of promyostatin or proGDF11 released in the assay.

[0136] Figures 18A-18C SCID dose responses of candidate antibodies are shown. Figure 18A Shows the gastrocnemius muscle weight and Figure 18B Shows the muscle mass of the quadriceps. Figure 18C Shown are the percent changes in mean muscle weight compared to PBS controls. Figures 18A-18B The bars in the figure are from left to right: PBS; IgG Ctrl 20 mg / kg / wk; Abl 20 mg / kg / wk; Abl 2 mg / kg / wk; Abl 0.5 mg / kg / wk; Ab2 20 mg / kg / wk; Ab2 2 mg / kg / wk; Ab2 0.5 mg / kg / wk; Ab4 20 mg / kg / wk; Ab4 2 mg / kg / wk; Ab4 0.5 mg / kg / wk; Ab6 20 mg / kg / wk; Ab6 2 mg / kg / wk; and Ab6 0.5 mg / kg / wk.

[0137] Figure 19 Results of a duration of action study comparing Ab1 with an existing myostatin antibody (AbMyo) are shown. PBS was used as a negative control; IgG was used as a positive control. Changes in lean body mass were examined after 21 days at different dosing regimens.

[0138] Figure 20 is a schematic diagram illustrating an assay for reconstitution of myostatin activation in vitro.

[0139] Figures 21A-21B The heavy chain of a humanized monoclonal antibody (Ab2) of the IgG4 subtype having a proline substituted for a serine is shown ( Figure 21A ; SEQ ID NO: 50) and light chain ( Figure 21B ; SEQ ID NO: 51). This creates an IgG1-like hinge sequence and minimizes incomplete formation of interchain disulfide bridges, a characteristic of IgG4. The complementarity determining regions (CDRs) are underlined. The NST sequence in bold is a consensus site for N-linked glycosylation; the DP sequence in bold is a potential cleavage site; the NX sequence in bold, where X can be S, T, or G, is a potential deamidation site; the DX sequence in bold, where X can be G, S, T, or SDG, is a potential isomerization site; the methionine in bold is a potential methionine oxidation site; and the Q in bold is the expected N-terminal pyroglutamate.

[0140] Figure 22 is a schematic diagram showing the reduction of immunogenicity risk by germlining. 24H4 (WT) contains 5 non-germline amino acids within the framework region, as indicated in the schematic diagram.

[0141] Figures 23A-23C Optimization of Ab1 was shown. Optimization candidates that specifically bind to promyostatin were selected, resulting in dozens of clones with improved affinity. FACS was performed to show affinity with Ab1 ( Figure 23A ) compared to yeast clones ( Figure 23B ) of the increased combination. Figure 23C Variants showing affinity maturation also had slower off-rates through the octet.

[0142] Figures 24A-24B The heavy chain variable regions of parent Abl and affinity-optimized variants Ab3 and Ab5 are shown ( Figure 24A ) and light chain variable region ( Figure 24B ) sequence alignment. The sequence identifiers correspond to SEQ ID NOs: 24, 26, 28 ( Figure 24A ). The sequence identifiers correspond to SEQ ID NOs: 30, 32, 34 ( Figure 24B ). Complementarity determining regions (CDRs) are defined using Kabat (underlined) and IMGT nomenclature (bold). Substitutions relative to parental Abl are shown in light grey.

[0143] Figure 25Shown is the expression of pro-myostatin and latent-myostatin in muscle and plasma from normal and muscle atrophic mice.

[0144] Figure 26 Quantification of changes in promyostatin and latent myostatin in muscle and plasma is shown. Bars show, from left to right, promyostatin, latent myostatin, promyostatin, latent myostatin, and latent myostatin.

[0145] Figure 27 Ab2 is shown to uniquely recognize both promyostatin and latent myostatin, binding to the major form of myostatin in both serum and muscle. Non-reduced Western blot of the prodomain (dark grey) and mature growth factor (light grey). Recombinant promyostatin (rProMyostatin) shows the migration of promyostatin and the myostatin prodomain (latent myostatin) on the gel, which are highlighted by arrows. In serum, both Ab2 and AbMyo bind to latent myostatin (prodomain band) and various partially processed precursors, but only Ab2 recognizes promyostatin (upper band). In muscle, Ab2 precipitates promyostatin, while there is no interaction of AbMyo with promyostatin in muscle tissue.

[0146] Figures 28A-28B Provides a model of myostatin flux in normal and atrophic muscle. In normal muscle ( Figure 28A ), promyostatin is produced in muscle and converted to latent myostatin by cleavage by the furin protease (which can be present intracellularly or extracellularly). Some fraction of latent myostatin in muscle is then released into the circulation, forming a circulating pool of latent myostatin. In muscle atrophy ( Figure 28B ), an increase in active myostatin growth factor is caused by upregulation of promyostatin levels in muscle and increased conversion of latent myostatin to active growth factor. As a result, circulating latent myostatin decreases as the muscle pool of latent myostatin is redirected to form mature myostatin through mTLL2 cleavage.

[0147] Figure 29 Detection of Ab2 (upper line) and IgG control (lower line) antibodies in serum from dosed rats is shown. Ab2 showed elevated levels in the circulation compared to the IgG control, with an average of 17.1 μg / ml of Ab2 in serum at the end of the study. Ab2 levels were determined by human IgG-specific ELISA using known amounts of each antibody used as a reference standard.

[0148] Figures 30A-30BThe pharmacodynamic effects of Ab2 in treated rats are shown. Figure 30A Rats treated with Ab2 showed increased lean body mass compared to PBS- or IgG control-treated animals. Ab2 and IgG were administered intravenously at a dose of 10 mg / kg on day 0. Lean body mass was measured by qNMR 7, 14, 21, and 28 days after dosing (N=8 / group). Figure 30B Rectus femoris and tibilais anterior muscles were collected from all groups at the end of the study (N=8 / group) and weighed to determine muscle mass. Rats treated with Ab2 showed a 14% and 11% increase in rectus femoris and tibilais anterior muscle mass, respectively.

[0149] Figures 31A-31B Promyostatin / latentmyostatin levels in Ab2-treated rats are shown. Figure 31A Treatment with Ab2 (upper line) is shown to increase latent myostatin levels in rat serum by -20-fold. Figure 31B Ab2 treatment results in a 1.9x increase in the latent form of myostatin in rat muscle (rectus femoris). Bars correspond from left to right to promyostatin, latent myostatin, promyostatin, and latent myostatin. No statistically significant changes in promyostatin were observed in rat muscle. These data are from quantitative Western blot analysis of n=3 samples / group.

[0150] Figure 32 Treatment with Ab2 (Ab2) or a comparative antibody (AbMyo) is shown to result in increased lean body mass as early as 7 days after antibody administration. The increase in lean body mass was equivalent for Ab2 and AbMyo until 21 days after administration. However, by 28 days after administration, the increase in lean body mass was lost in the AbMyo-treated group, while the increase in the Ab2-treated group was maintained throughout the study. The upper line corresponds to Ab2, the middle line corresponds to AbMyo, and the lower line corresponds to the IgG control (5 mg / kg).

[0151] Figure 33 Shown are serum levels of drug measured using an anti-human IgG ELISA following a single 5 mg / kg dose of Ab2 (upper line) or a comparator antibody (AbMyo; lower line). Drug was detected in serum as early as 1 hour after dosing, and levels of >1 μg / ml for both antibodies were detectable throughout the study. However, Ab2 exhibited a significantly longer half-life and extrapolated area under the curve (AUCINF) than AbMyo, indicating that at similar doses, Ab2 exhibited significantly higher exposure than AbMyo.

[0152] Figure 34Serum myostatin was measured using fluorescent Western blot in drug-treated mice and controls. Despite increased serum exposure to Ab2, serum latent myostatin levels were similar in both Ab2- and AbMyo-treated mice. These data suggest that circulating levels of free drug are in sufficient excess relative to target levels such that increased serum exposure to Ab2 does not result in a greater increase in circulating latent myostatin than observed in the AbMyo group. Data sets from left to right correspond to IgG, Ab2, AbMyo, IgG, Ab2, and AbMyo.

[0153] Figures 35A-35B Shown are the relative levels of latent and promyostatin measured in mouse muscle lysates by fluorescent Western blotting. Figure 35A Latent myostatin was shown to be elevated in both Ab2- and AbMyo-treated muscles. However, the elevations in latent myostatin in AbMyo-treated muscles returned to baseline by day 28, whereas those in Ab2-treated muscles remained elevated until at least this time (P < 0.003, relative to AbMyo treatment). Figure 35B A similar trend was observed for promyostatin, although the difference between the Ab2 and AbMyo treated groups was not statistically significant at day 28 (P = 0.068).

[0154] Figure 36A The effects of Ab2 treatment on muscle mass and function in mice are shown.

[0155] Figure 36B The effect of Ab2 treatment on maximal force generation in mice is shown. DETAILED DESCRIPTION

[0156] Myostatin is a member of the TGFβ superfamily and belongs to a subfamily that includes two members: myostatin (also known as GDF8) and GDF11. Like other members of the TGFβ superfamily, myostatin and GDF11 are initially expressed as inactive precursor polypeptides (respectively called promyostatin and proGDF11). The domain structure and nomenclature are shown in Figure 1A middle. Figure 1B A cartoon model showing the overall structure of promyostatin, in which the mature growth factor remains locked in a cage consisting of two alpha helices connected by a loop called the "latency lasso."

[0157] Activation and release of mature growth factors occur through several discrete protease cleavage events, as outlined in Figure 2The first cleavage step of promyostatin and proGDF11 is performed by proprotein convertase, which cleaves at the conserved RXXR site between the prodomain and the mature growth factor. This cleavage generates a latent complex in which the mature growth factor is shielded from binding to its receptor by the prodomain. Activation and release of the mature, active myostatin growth factor is completed after cleavage by additional proteases from the BMP / tolloid family, such as mTLL-2 ( Figure 2 ).

[0158] Exemplary proGDF8 sequences in humans, rats, mice, and cynomolgus monkeys are provided below. In these proGDF8 sequences, the proprotein convertase cleavage site is indicated in bold and the tolloid protease site is indicated by underlining. In some embodiments, the proprotein convertase cleavage site comprises amino acid residues 240-243 of SEQ ID NOs: 52-55. In some embodiments, the tolloid protease site comprises amino acid residues 74-75 of SEQ ID NOs: 52-55. It should be understood that the exemplary proGDF8 sequences provided herein are not intended to be limiting, and additional proGDF8 sequences from other species (including any isoforms thereof) are within the scope of this disclosure.

[0159] proGDF8 (human):

[0160]

[0161] proGDF8 (rat):

[0162]

[0163] proGDF8 (mouse):

[0164]

[0165] proGDF8 (cynomolgus monkey):

[0166]

[0167] Myostatin and GDF11 share a relatively high level of conservation between their mature growth factor domains, with 90% identity, but much less conservation in their prodomain regions, with less than 50% amino acid identity between the two. Myostatin and GDF11 bind to and signal through the same receptor, consisting of type I receptors (ALK4 / 5) bound to type II receptors (ACTRIIA / B). Engagement of myostatin with both type I and type II receptors initiates a signaling cascade that leads to SMAD phosphorylation and transcriptional activation of muscle atrophy genes. The relatively high level of conservation in the mature growth factors makes it challenging to identify reagents, such as monoclonal antibodies, that can distinguish between mature myostatin and GDF11.

[0168] In some embodiments, provided herein are promyostatin / latent myostatin antibodies that specifically bind to a chimeric construct comprising a growth factor domain and the N-terminal propeptide portion of GDF11 and the C-terminal portion of the propeptide of GDF8. This chimeric construct (as shown below) is referred to as GDF11Arm8.

[0169] >GDF11Arm8 (SEQ ID NO:65)

[0170] MDMRVPAQLLGLLLLWFSGVLGDYKDDDDKHHHHHHLEVLFQGPAEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLRELIDQ YDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKA

[0171] LDENGHDLAVTPFPGPGEDGLNPFLEVKVTDTPKRSRRNLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS

[0172] The role of myostatin in myopathies

[0173] Skeletal muscle accounts for approximately 40% of body weight and is a dynamic organ, updated at a rate of 1-2% per day. Muscle atrophy is a highly regulated catabolic process that occurs during periods of disuse (e.g., disuse atrophy) and / or in response to elevated systemic inflammation (cachexia). In disuse atrophy (which can occur during extended periods of immobilization, such as during bed rest), muscle loss occurs rapidly. For example, during a one-week hospital stay, patients lose an average of ~1.3 kg of muscle mass.

[0174] Muscle atrophy causes significant morbidity in a wide range of clinical conditions. In denervation diseases such as amyotrophic lateral sclerosis (ALS) or spinal muscular atrophy (SMA) and hereditary diseases including muscular dystrophy, the loss of muscle strength and function is a highly disabling clinical manifestation for which there is no adequate treatment. In cachexia syndromes due to renal failure, AIDS, heart disease or cancer, muscle atrophy usually impairs the successful treatment of the primary condition. Muscle loss is also caused by the natural aging process, and in its most severe form, is classified as sarcopenia, a common condition in the elderly, which is increasingly recognized as a pathology requiring intervention. Finally, the main driving factor for muscle atrophy is disuse. Immobilization causes rapid and significant muscle loss in a large class of conditions such as hip fracture, selective joint replacement, spinal cord injury, critical illness myopathy and stroke. Although its cause of disease is variable, these indications have the characteristic of muscle weakness, which causes severe disability, long physical rehabilitation and recovery time and impaired quality of life.

[0175] There is an unmet medical need in muscle wasting conditions. Therefore, in some embodiments, provided herein is a method for treating muscle wasting. In some embodiments, provided herein is a method for treating primary myopathy. In some embodiments, provided herein is a method for treating a primary myopathy (wherein muscle loss is secondary to the condition of disease pathology), such as, for example, denervation disease, hereditary myasthenia and cachexia. In some embodiments, provided herein is a method for treating a primary myopathy such as disuse atrophy (for example, relevant to hip fracture or spinal cord injury (SCI)) resulting in a raising of muscle mass, strength and function in a subject.

[0176] Myostatin pathway inhibition

[0177] There are several myostatin pathway antagonists in various stages of clinical development for the treatment of muscle-related disorders. Such pathway antagonists target the mature growth factor or its type II receptor, and most antagonize the signaling of multiple TGFβ family members. For example, many current clinical candidates block additional growth factors such as activin A, GDF11, and BMP9 and 10, which are regulators of reproductive biology, wound healing, erythropoiesis, and angiogenesis, respectively. Aspects of the present disclosure relate to the recognition that blocking these factors in addition to myostatin would potentially limit the patient population that can be safely treated due to unacceptable side effects.

[0178] Thus, provided herein are antibodies capable of binding to promyostatin and / or latent myostatin (thereby inhibiting myostatin activity), and their use for treating diseases and disorders associated with myopathies. In some embodiments, given the ubiquity of latent complexes in circulation, provided herein are treatments that specifically target the more abundant and longer-lived myostatin precursors (e.g., promyostatin and latent myostatin) rather than the mature growth factor. Without wishing to be bound by any particular theory, the antibodies provided herein can prevent the proteolytic activation of promyostatin and / or latent myostatin into mature myostatin (which is considered the "active" form of myostatin) that is capable of activating the myostatin pathway (e.g., by binding to type I (ALK4 / 5) and type II (ACTRIIA / B) receptors).

[0179] As used herein, the term "promyostatin / latent myostatin" refers to promyostatin, latent myostatin, or both. In some embodiments, an anti-promyostatin / latent myostatin antibody specifically binds to promyostatin. In some embodiments, an anti-promyostatin / latent myostatin antibody specifically binds to latent myostatin. In some embodiments, an anti-promyostatin / latent myostatin antibody specifically binds to both latent myostatin and promyostatin. It should be understood that "latent myostatin" and "promyostatin" may also be referred to herein as "latent GDF8" and "proGDF8," respectively.

[0180] As used herein, the term "mature myostatin" refers to the mature, biologically active form of myostatin. In some embodiments, mature myostatin is capable of myostatin receptor binding and / or activation. Activation and release of mature myostatin from its pro-myostatin form in vivo is accomplished by several separate protease cleavage events. First, "pro-myostatin" is cleaved by a proprotein convertase, generating "latent-myostatin," in which mature myostatin is shielded from binding to its receptor by a portion of the prodomain. Activation and release of mature myostatin is accomplished following cleavage of the latent myostatin by additional proteases from the BMP / tolloid family, such as mTLL-2. See, e.g., Figure 1A 、 1B and 2. As used herein, the term "mature myostatin" can refer to both full-length mature myostatin and fragments of full-length mature myostatin that retain biological activity. Exemplary mature myostatin sequences, variants thereof, and methods of producing mature myostatin are well known in the art and described in more detail herein.

[0181] The term "promyostatin," also referred to as "proGDF8," refers to an inactive form of mature myostatin comprising a disulfide-linked homodimer, each molecule of which comprises an amino-terminal prodomain covalently bound to a carboxy-terminal mature myostatin domain. In one embodiment, "promyostatin" is not cleaved by proprotein convertases or proteases from the BMP / tolloid family. Exemplary promyostatin sequences, variants thereof, and methods of producing promyostatin are known in the art and described in more detail herein.

[0182] As used herein, the term "latent myostatin" refers to an inactive precursor of mature myostatin comprising a disulfide-linked homodimer, each molecule of which comprises an amino-terminal prodomain non-covalently bound to a carboxyl-terminal mature myostatin domain. In one embodiment, "latent myostatin" is produced from promyostatin that has been cleaved by a proprotein convertase but not by a protease from the BMP / tolloid family. In another embodiment, "latent myostatin" can be produced by combining the prodomain and the carboxyl-terminal mature myostatin domain in vitro and allowing them to fold correctly. See, for example, Sengle et al., J. Biol. Chem., 286(7):5087-5099, 2011. Exemplary latent myostatin sequences, variants thereof, and methods of generating latent myostatin are well known in the art and are described in more detail herein.

[0183] As used herein, the term "promyostatin / latent myostatin" refers to promyostatin, latent myostatin, or both promyostatin and latent myostatin. In one embodiment, the antibodies disclosed herein bind to promyostatin. In another embodiment, the antibodies disclosed herein bind to latent myostatin. In another embodiment, the antibodies disclosed herein bind to both promyostatin and latent myostatin.

[0184] As used herein, the term "purified promyostatin" or "purified pro-GDF8" refers to a composition comprising promyostatin that is free or substantially free of other forms of myostatin, such as latent myostatin and mature myostatin. In one embodiment, the antibodies disclosed herein specifically bind to purified promyostatin. In other words, such antibodies bind to promyostatin in a composition that lacks other forms of myostatin, latent myostatin, and mature myostatin.

[0185] As used herein, the term "proprotein convertase cleavage site" refers to the site where promyostatin is cleaved by a proprotein convertase. In one embodiment, the proprotein convertase cleavage site is a conserved RXXR site between the prodomain and the biologically active domain or mature myostatin. See, for example, Figure 1A 、 1B and 2.

[0186] As used herein, the term "BMP / tolloid protease family cleavage site" refers to a site where latent myostatin is cleaved by a member of the BMP / tolloid protease family. In one embodiment, the BMP / tolloid protease family member is mTLL-2. See, e.g., Figure 1A 、 1B and 2.

[0187] Antibodies that bind promyostatin / latent myostatin

[0188] The present disclosure is based, at least in part, on the unexpected discovery that certain promyostatin / latent myostatin-specific antibodies (e.g., antibodies referred to herein as Abl) prevent the proteolytic activation of promyostatin / latent myostatin to mature myostatin. Furthermore, inhibition of myostatin activation using such antibodies effectively increases muscle mass in a dexamethasone and cast-induced muscle atrophy mouse model. Aspects of the present disclosure provide antibodies (e.g., antibodies and antigen-binding fragments) that bind to promyostatin / latent myostatin and inhibit the proteolytic activation of promyostatin / latent myostatin to mature myostatin.

[0189] Antibodies (used interchangeably in the plural) are immunoglobulin molecules that can specifically bind to targets (such as carbohydrates, polynucleotides, lipids, polypeptides, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses not only complete (e.g., full-length) polyclonal or monoclonal antibodies, but also includes antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chains (scFv), mutants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, double antibodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and immunoglobulin molecules comprising any other modified configurations of antigen recognition sites with desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies include antibodies of any kind, such as IgD, IgE, IgG, IgA, or IgM (or its subclass), and antibodies do not need to be of any particular kind. Depending on the amino acid sequence of the constant domain of the antibody heavy chain, immunoglobulins can be assigned to different kinds. There are five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0190] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds promyostatin / latent myostatin is substantially free of antibodies that specifically bind antigens other than promyostatin / latent myostatin). However, an isolated antibody that specifically binds promyostatin / latent myostatin may have cross-reactivity with other antigens, such as promyostatin / latent myostatin molecules from other species. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0191] The term "human antibody" as used herein is intended to include antibodies and fragments thereof having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo), such as in CDRs and particularly CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[0192] The term "epitope" includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0193] The antibodies described herein can bind to promyostatin / latent myostatin, thereby inhibiting the proteolytic activation of promyostatin / latent myostatin to mature myostatin. In some cases, the antibodies described herein can inhibit the proteolytic activation of promyostatin / latent myostatin by at least 20%, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some cases, the antibodies described herein can inhibit the proteolytic cleavage of promyostatin by a proprotein convertase (e.g., furin) by at least 20%, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some cases, the antibodies described herein can inhibit the proteolytic cleavage of promyostatin or latent myostatin by a tolloid protease (e.g., mTLL2) by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. The inhibitory activity of anti-promyostatin / latent myostatin antibodies can be measured by conventional methods, e.g., by Western blot analysis as described in Example 1 and Figure 3. However, it will be appreciated that additional methods can be used to measure the inhibitory activity of anti-promyostatin / latent myostatin antibodies against the proteolytic cleavage of promyostatin / latent myostatin. In some embodiments, inhibition of promyostatin / latent myostatin cleavage (e.g., by proprotein convertase and / or tolloid protease) can be reflected as an inhibition constant (Ki), which provides a measure of antagonist potency and is the concentration of antagonist (e.g., anti-promyostatin / latent myostatin antibody) required to reduce protease activity (e.g., proprotein convertase or tolloid protease activity) by half and is independent of enzyme or substrate concentration.

[0194] In some embodiments, the proprotein convertase comprises (i) a catalytic domain that hydrolyzes peptide bonds of proteins containing a proprotein convertase cleavage site, and (ii) a binding pocket that binds to rTGF having a proprotein convertase cleavage site. Examples of proprotein convertases used in accordance with the present disclosure include, but are not limited to, PCSK5 / 6, PACE4, PACE7, and PACE3 (e.g., furin). In some embodiments, the proprotein convertase is obtained from any mammal, including but not limited to humans, monkeys, or rodents (e.g., mice, rats, hamsters).

[0195] In some embodiments, the proprotein convertase is homologous to a proprotein convertase selected from PCSK5 / 6, PACE4, PACE7, and PACE3 (e.g., furin). For example, the proprotein convertase can be at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least about 99.9% identical to PCSK5 / 6, PACE4, PACE7, or PACE3 (e.g., furin).

[0196] In some embodiments, the proprotein convertase cleavage site is an amino acid sequence that can be cleaved by a proprotein convertase (e.g., PCSK5 / 6, PACE4, PACE7, and PACE3). In some embodiments, the proprotein convertase cleavage site comprises the amino acid sequence RXXR, wherein R is arginine and X is any amino acid. In some embodiments, the proprotein convertase cleavage site comprises the amino acid sequence RX-(K / R)-R, wherein R is arginine, K is lysine, and X is any amino acid. In some embodiments, the proprotein convertase cleavage site comprises the amino acid sequence RVRR (SEQ ID NO: 57), wherein R is arginine and V is valine. Exemplary proprotein convertase cleavage sites for human, rat, mouse, and cynomolgus monkey myostatin are shown in bold in SEQ ID NOs: 52-55. In some embodiments, the proprotein convertase cleavage site comprises the amino acid sequence RSRR (SEQ ID NO: 56).

[0197] In some embodiments, the tolloid protease used according to the present disclosure includes, but is not limited to BMP-1, mTLL-1 and mTLL-2. The tolloid protease can be obtained from any mammal, including but not limited to humans, monkeys or rodents (e.g., mice, rats, hamsters). In some embodiments, the tolloid protease is homologous to a tolloid protease selected from: BMP-1, mTLL-1 and mTLL-2. For example, the tolloid protease can be at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical or at least about 99.9% identical to BMP-1, mTLL-1 and mTLL-2.

[0198] In some embodiments, the tolloid protease cleavage site is an amino acid sequence that can be cleaved by a tolloid (e.g., BMP-1, mTLL-1, and mTLL-2). Exemplary tolloid protease cleavage sites for human, rat, mouse, and cynomolgus monkey myostatin are underlined in SEQ ID NOs: 52-55. In some embodiments, the tolloid cleavage site comprises the amino acid sequence QR, wherein Q is glutamine and R is arginine.

[0199] In some embodiments, the antibodies described herein are capable of binding to promyostatin / latent myostatin, thereby inhibiting myostatin activity. In some cases, the antibodies described herein can inhibit myostatin signaling by at least 20%, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, inhibition of myostatin signaling can be measured by conventional methods, for example, using a myostatin activation assay as described in Example 1. However, it will be appreciated that additional methods can be used to measure myostatin signaling activity.

[0200] It will be appreciated that the extent of proteolytic cleavage of myostatin by, for example, proprotein convertases and / or tolloid proteases can be measured and / or quantified using any suitable method. In some embodiments, the extent of proteolytic cleavage of myostatin is measured and / or quantified using an enzyme-linked immunosorbent assay (ELISA). For example, an ELISA can be used to measure the level of released growth factor (e.g., mature myostatin). As another example, antibodies that specifically bind to promyostatin, latent myostatin, and / or mature myostatin can be used in an ELISA to measure the level of a particular form of myostatin (e.g., pro / latent / mature-myostatin) to quantify the extent of proteolytic cleavage of myostatin. In some embodiments, the extent of proteolytic cleavage of myostatin is measured and / or quantified using immunoprecipitation followed by SDS-PAGE or mass spectrometry of tryptic peptides, anisotropic fluorescence-based techniques, FRET analysis, hydrogen-deuterium exchange mass spectrometry, and / or NMR spectroscopy.

[0201] In some embodiments, antibodies, also known as immunoglobulins, are tetrameric glycosylated proteins composed of two light chains (L) of approximately 25 kDa each and two heavy chains (H) of approximately 50 kDa each. Two types of light chains can be found in antibodies, called λ and κ. Depending on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, and several of these can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Each light chain typically includes an N-terminal variable (V) domain (V L ) and constant (C) domains (C L Each heavy chain typically includes an N-terminal V domain (V H ), three or four C domains (C H 1-3) and hinge region. Closest to V H C H The domain is named C H 1. V H and V LThe domain is composed of four regions of relatively conserved sequences called framework regions (FR1, FR2, FR3 and FR4), which form a backbone for three regions of hypervariable sequences (complementarity determining regions, CDRs). The CDRs contain most of the residues responsible for the specific interaction of the antibody with the antigen. The CDRs are referred to as CDR1, CDR2 and CDR3. Thus, the CDR components on the heavy chain are referred to as CDRH1, CDRH2 and CDRH3, while the CDR components on the light chain are referred to as CDRL1, CDRL2 and CDRL3. CDRs are generally referred to as Kabat CDRs, as described in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (1991), eds. Kabat et al. Another standard for characterizing antigen binding sites refers to the hypervariable loops described by Chothia. See, e.g., Chothia, D. et al. (1992) J. Mol. Biol. 227: 799-817; and Tomlinson et al. (1995) EMBO J. 14: 4628-4638. Yet another standard is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S and Kontermann, R., Springer-Verlag, Heidelberg). The embodiments described for Kabat CDRs may alternatively be implemented using the similar relationships described with respect to Chothia hypervariable loops or AbM-defined loops, or any combination of these methods.

[0202] In some embodiments, the anti-promyostatin / latentmyostatin antibodies of the disclosure and nucleic acid molecules of the disclosure encoding the antibodies comprise the CDR amino acid sequences shown in Table 1.

[0203] Table 1.

[0204]

[0205] In Table 1, the unique sequences of CDRH3 and CDRL3 reflect Kabat and IMGT.

[0206] In some embodiments, the anti-promyostatin / latent myostatin binding agents (e.g., antibodies) of the present disclosure include any antibody (including antigen-binding fragment) comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or a combination thereof, as provided for any of the antibodies shown in Table 1. In some embodiments, the anti-promyostatin / latent myostatin binding agents comprise the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of any of the antibodies shown in Table 1. The present disclosure also includes any nucleic acid sequence encoding a molecule comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3 as provided for any of the antibodies shown in Table 1. The antibody heavy and light chain CDR3 domains can play a particularly important role in the binding specificity / affinity of an antibody for an antigen. Thus, the anti-promyostatin / latentmyostatin binding agents or nucleic acid molecules thereof of the present disclosure may include at least the heavy chain and / or light chain CDR3 of an antibody as shown in Table 1.

[0207] Aspects of the present disclosure relate to monoclonal antibodies or antigen-binding fragments that bind to a promyostatin / latentmyostatin protein and comprise six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3.

[0208] In some embodiments, CDRH1 comprises the sequence set forth in any one of SEQ ID NOs: 1-3. In some embodiments, CDRH2 comprises the sequence set forth in any one of SEQ ID NOs: 4-9. In some embodiments, CDRH3 comprises the sequence set forth in any one of SEQ ID NOs: 10-11. CDRL1 comprises the sequence set forth in any one of SEQ ID NOs: 12-17. In some embodiments, CDRL2 comprises the sequence set forth in any one of SEQ ID NOs: 18-21. In some embodiments, CDRL3 comprises the sequence set forth in any one of SEQ ID NOs: 22-23.

[0209] In some embodiments (e.g., for the anti-promyostatin / latentmyostatin antibody Ab1 shown in Table 1), CDRH1 comprises the sequence as shown in SEQ ID NO: 1 or 2, CDRH2 comprises the sequence as shown in SEQ ID NO: 4 or 5, CDRH3 comprises the sequence as shown in SEQ ID NO: 10, CDRL1 comprises the sequence as shown in SEQ ID NO: 12 or 13, CDRL2 comprises the sequence as shown in SEQ ID NO: 18 or 19, and CDRL3 comprises the sequence as shown in SEQ ID NO: 22, and the antibody binds promyostatin / latentmyostatin.

[0210] In some embodiments (e.g., for anti-promyostatin / latentmyostatin antibody Ab3 shown in Table 1), CDRH1 comprises the sequence as shown in SEQ ID NO: 1 or 3, CDRH2 comprises the sequence as shown in SEQ ID NO: 6 or 7, CDRH3 comprises the sequence as shown in SEQ ID NO: 11, CDRL1 comprises the sequence as shown in SEQ ID NO: 14 or 15, CDRL2 comprises the sequence as shown in SEQ ID NO: 20 or 21, and CDRL3 comprises the sequence as shown in SEQ ID NO: 23, and the antibody binds promyostatin / latentmyostatin.

[0211] In some embodiments (e.g., for anti-myostatin / latent myostatin antibody Ab5 as shown in Table 1), CDRH1 comprises the sequence shown in SEQ ID NO: 1 or 3, CDRH2 comprises the sequence shown in SEQ ID NO: 8 or 9, CDRH3 comprises the sequence shown in SEQ ID NO: 11, CDRL1 comprises the sequence shown in SEQ ID NO: 16 or 17, CDRL2 comprises the sequence shown in SEQ ID NO: 20 or 21, and CDRL3 comprises the sequence shown in SEQ ID NO: 23, and the antibody binds to promyostatin / latent myostatin. In some examples, any of the anti-myostatin / latent myostatin binding agents (e.g., antibodies) of the present disclosure include any antibodies (including antigen-binding fragments) having one or more CDR (e.g., CDRH or CDRL) sequences that are substantially similar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3. For example, the antibody can include one or more CDR sequences as shown in Table 1 (SEQ ID NOs: 1-23) comprising up to 5, 4, 3, 2, or 1 amino acid residue variation compared to the corresponding CDR region in any one of SEQ ID NOs: 1-23. The complete amino acid and nucleic acid sequences of the heavy and light chain variable regions of the antibodies listed in Table 1 are provided below.

[0212] Heavy chain variable region-Ab1 parent

[0213] QIQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHYYGMDVWGQGTTVTVSS(SEQ ID NO:24)CAGATCCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGATTTTTGGAGTGGTCGCACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ IDNO:38)

[0214] Heavy chain variable region - Ab germline

[0215] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHYYGMDVWGQGTTVTVSS(SEQ ID NO:25)

[0216] CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGATTTTTGGAGTGGTCGCACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO:39)

[0217] Heavy chain variable region - Ab3 parent

[0218] QIQLVQSGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVISYDGSIKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHKYGMDVWGQGTTVTVSS(SEQ ID NO:26)

[0219] CAGATCCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCGCCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTATCAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGATTTTTGGAGTGGTCGCACAAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO:40)

[0220] Heavy chain variable region - Ab4 germline

[0221] QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVISYDGSIKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHKYGMDVWGQGTTVTVSS(SEQ ID NO:27)

[0222] CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCGCCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTATCAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGATTTTTGGAGTGGTCGCACAAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO:41)

[0223] Heavy chain variable region - Ab5 parent

[0224] QIQLVQSGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHKYGMDVWGQGTTVTVSS(SEQ ID NO:28)

[0225] CAGATCCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCGCCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAATAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGATTTTTGGAGTGGTCGCACAAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO:42)

[0226] Heavy chain variable region - Ab6 germline

[0227] QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHKYGMDVWGQGTTVTVSS(SEQ ID NO:29)

[0228] CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCGCCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAATAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGATTTTTGGAGTGGTCGCACAAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO:43)

[0229] Light chain variable region - Ab1 parent

[0230] QPVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVHWYQQLPGTAPKLLIYSDNQRPSGVPDRFSGSKSGTSASLVISGLQSDDEADYYCAAWDDSLNGVFGGGTKLTVL(SEQ ID NO:30)

[0231] CAGCCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATACTGTCCACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATAATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGTCATCAGTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA(SEQ ID NO:44)

[0232] Light chain variable region - Ab2 germline

[0233] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVHWYQQLPGTAPKLLIYSDNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGVFGGGTKLTVL(SEQ ID NO:31)

[0234] CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATACTGTCCACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATAATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA(SEQ ID NO:45)

[0235] Light chain variable region - Ab3 parent

[0236] QPVLTQPPSASGTPGQRVTISCSGSTSNIGSNTVHWYQQLPGTAPKLLIYSDDQRPSGVPDRFSGSKSGTSASLVISGLQSDDEADYYCAAWDESLNGVFGGGTKLTVL(SEQ ID NO:32)

[0237] CAGCCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTAATACTGTCCACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATGATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGTCATCAGTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGATGAGAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA(SEQ ID NO:46)

[0238] Light chain variable region - Ab4 germline

[0239] QSVLTQPPSASGTPGQRVTISCSGSTSNIGSNTVHWYQQLPGTAPKLLIYSDDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDESLNGVFGGGTKLTVL(SEQ ID NO:33)

[0240] CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTAATACTGTCCACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATGATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGAGAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA(SEQ ID NO:47)

[0241] Light chain variable region - Ab5 parental

[0242] QPVLTQPPSASGTPGQRVTISCSGSSSNIGGNTVHWYQQLPGTAPKLLIYSDDQRPSGVPDRFSGSKSGTSASLVISGLQSDDEADYYCAAWDESLNGVFGGGTKLTVL(SEQ ID NO:34)

[0243] CAGCCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAGGAAATACTGTCCACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATGATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGTCATCAGTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGATGAGAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA(SEQ ID NO:48)

[0244] Light chain variable region - Ab6 germline

[0245] QSVLTQPPSASGTPGQRVTISCSGSSSNIGGNTVHWYQQLPGTAPKLLIYSDDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDESLNGVFGGGTKLTVL(SEQ ID NO:35)

[0246] CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAGGAAATACTGTCCACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATGATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGAGAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA(SEQ ID NO:49)

[0247] Ab2 - heavy chain

[0248] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:50)

[0249] Ab2 - light chain

[0250] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGSNTVHWYQQLPGTAPKLLIYSDNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGVFGGGTKLTVLG QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQID NO:51)

[0251] In some embodiments, the anti-promyostatin / latent myostatin antibodies of the present disclosure include any antibody comprising a heavy chain variable domain of any one of SEQ ID NOs: 24 to 29 or a light chain variable domain of any one of SEQ ID NOs: 30 to 35. In some embodiments, the anti-promyostatin / latent myostatin antibodies of the present disclosure include any antibody comprising a heavy chain variable domain and a light chain variable domain pair of SEQ ID NOs: 24 and 30; 25 and 31; 26 and 32; 27 and 33; 28 and 34; or 29 and 35.

[0252] Aspects of the present disclosure provide anti-myostatin / latent myostatin antibodies having heavy chain variable and / or light chain variable amino acid sequences homologous to any of the heavy chain variable and / or light chain variable amino acid sequences described herein. In some embodiments, the anti-myostatin / latent myostatin antibody comprises a heavy chain variable sequence or light chain variable sequence that is at least 75% (e.g., 80%, 85%, 90%, 95%, 98% or 99%) identical to the heavy chain variable sequence of any of SEQ ID NOs: 24-29 or the light chain variable sequence of any of SEQ ID NOs: 30-35. In some embodiments, the homologous heavy chain variable and / or light chain variable amino acid sequences are not altered within any of the CDR sequences provided herein. For example, in some embodiments, the degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) can be present in heavy chain variable and / or light chain variable sequences other than any of the CDR sequences provided herein.

[0253] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-68, 1990 (modified in Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-77, 1993). This algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol., 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the target protein molecule. In cases where gaps exist between the two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res., 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0254] In some embodiments, conservative mutations can be introduced into the CDR or framework sequences at positions where the residue is unlikely to be involved in the interaction with promyostatin / latent myostatin, as determined based on the crystal structure. As used herein, "conservative amino acid substitutions" refer to amino acid substitutions that do not alter the relative charge and size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods known to those skilled in the art for altering polypeptide sequences, as found in references compiling such methods, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FM Ausubel et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0255] In some embodiments, the antibodies provided herein include mutations that confer desired properties to the antibodies. For example, to avoid potential complications due to Fab-arm exchange (which is known to occur for the original IgG4 mAb), the antibodies provided herein may include a stable 'Adair' mutation (Angal S et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibodies", Mol Immunol 30, 105-108; 1993), wherein serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline, resulting in an IgG1-like (CPPCP (SEQ ID NO: 58)) hinge sequence. Therefore, any antibody may include a stable 'Adair' mutation or the amino acid sequence CPPCP (SEQ ID NO: 58).

[0256] The anti-promyostatin / latent myostatin binding agents of the present disclosure may optionally comprise an antibody constant region or a portion thereof. For example, V L The V domain can be linked at its C-terminus to a light chain constant domain such as CK or Cλ. H The domain or portion thereof can be linked to all or part of a heavy chain, such as IgA, IgD, IgE, IgG, and IgM, and any isotype subclass. The antibody may include a suitable constant region (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within the scope of the present disclosure may include a V domain combined with any suitable constant region. H and V L domain or antigen-binding portion thereof.

[0257] In certain embodiments, V H and / or V L The domains can be restored to germline sequences, for example, by mutating the FRs of these domains to match those produced by germline cells using conventional molecular biology techniques. H and / or V L The domains can be restored to the germline sequences of IgHV3-30 (SEQ ID NO: 36) and / or IgLV1-44 (SEQ ID NO: 37), respectively. H and / or V LThe domains can be reverted to any suitable germline sequence.In other embodiments, the FR sequences remain distinct from the consensus germline sequence.

[0258] IgHV3-30

[0259] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR(SEQ ID NO:36)

[0260] IgLV1-44

[0261] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNG(SEQ ID NO:37)

[0262] In some embodiments, the anti-promyostatin / latentmyostatin antibody or antigen-binding fragment may or may not include the framework regions of the antibodies set forth in SEQ ID NOs: 24 to 35. In some embodiments, the anti-promyostatin / latentmyostatin antibody is a murine antibody and includes murine framework region sequences.

[0263] In some embodiments, the anti-promyostatin / latent myostatin antibodies of the present disclosure can bind to promyostatin / latent myostatin with relatively high affinity, for example, with an affinity of less than 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 5 pM or less. For example, the anti-myostatin / latent myostatin antibody can bind to promyostatin / latent myostatin with an affinity between 5 pM and 500 nM, e.g., between 50 pM and 100 nM, e.g., between 500 pM and 50 nM. The present disclosure also includes antibodies or antigen-binding fragments that compete with any of the antibodies described herein for binding to promyostatin / latent myostatin and have an affinity of 50 nM or less (e.g., 20 nM or less, 10 nM or less, 500 pM or less, 50 pM or less, or 5 pM or less). The affinity and binding kinetics of the anti-myostatin / latent myostatin antibodies can be tested using any suitable method, including but not limited to biosensor technology (e.g., OCTET or BIACORE).

[0264] An antibody that "specifically binds" a target antigen binds the target antigen with greater affinity, avidity, more readily, and / or for a longer period of time than it binds to a non-target antigen. In some embodiments, disclosed herein are antibodies that specifically bind to promyostatin / latent myostatin. In some embodiments, any of the antibodies provided herein bind at or near the tolloid cleavage site or at or near the tolloid docking site of promyostatin / latent myostatin. In some embodiments, an antibody binds near or near the tolloid cleavage site if the antibody binds within 15 or fewer amino acid residues of the tolloid cleavage site or the tolloid docking site. In some embodiments, any of the antibodies provided herein bind within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the tolloid cleavage site or the tolloid docking site. In some embodiments, the antibody binds at or near the tolloid cleavage site of GDF8. For example, the antibody can bind to the amino acid sequence PKAPPLRELIDQYDVQRDDSSDGSLEDDDYHAT (SEQ ID NO: 62) set forth in SEQ ID NO: 62. In other embodiments, any of the antibodies provided herein bind at or near the proprotein convertase cleavage site of promyostatin / latent myostatin or at or near the proprotein convertase docking site. In some embodiments, an antibody binds near or near the proprotein convertase cleavage site if the antibody binds within 15 or fewer amino acid residues of the proprotein convertase cleavage site or the proprotein convertase docking site. In some embodiments, any of the antibodies provided herein bind within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the proprotein convertase cleavage site or the proprotein convertase docking site. In some embodiments, the antibody binds at or near the proprotein convertase cleavage site of GDF8. For example, the antibody can bind to the amino acid sequence GLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRC (SEQ ID NO: 63) set forth in SEQ ID NO: 63.

[0265] In one example, the anti-promyostatin / latent myostatin antibodies described herein specifically bind to promyostatin / latent myostatin as compared to other forms of myostatin and / or other members of the TGFβ family of growth factors. Members of the TGFβ family of growth factors include, but are not limited to, AMH, ARTN, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDNF, INHA, INHBA, INHBB, INHBC, INHBE, LEFTY1, LEFTY2, NODAL, NRTN, PSPN, TGFβ1, TGFβ2, and TGFβ3 proteins. Such antibodies can bind to promyostatin / latent myostatin with a much higher affinity (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1,000-fold higher) than other members of the TGFβ family of growth factors. In some embodiments, such antibodies can bind to promyostatin / latent myostatin with an affinity that is at least 1,000 higher than other members of the TGFβ family of growth factors. In some embodiments, the antibodies provided herein can bind to promyostatin / latent myostatin with a much higher affinity (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1,000-fold higher) than one or more forms of GDF11 or mature myostatin. In some embodiments, the antibodies provided herein can bind to promyostatin / latent myostatin with an affinity that is at least 1,000 higher than one or more forms of GDF11 (e.g., proGDF11, latent GDF11, or mature GDF11) or mature myostatin. Alternatively or additionally, the antibodies can exhibit much higher (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold higher) inhibitory activity against proteolytic cleavage of promyostatin / latent myostatin (e.g., by proprotein convertase or tolloid protease) than other members of the TGFβ family, such as pro / latent GDF11.

[0266] In some embodiments, the antibody binds to the antigen but cannot effectively eliminate the antigen from the plasma. Therefore, in some embodiments, the concentration of the antigen in the plasma can be increased by reducing the clearance of the antigen. However, in some embodiments, the antibodies provided herein (e.g., sweeping antibodies) have an affinity for the antigen that is sensitive to pH. Such pH-sensitive antibodies bind to the antigen in the plasma at neutral pH and dissociate from the antigen in the acidic endosome, thereby reducing antibody-mediated antigen accumulation and / or promoting antigen clearance from the plasma.

[0267] Aspects of the present disclosure relate to sweeping antibodies. As used herein, a "sweeping antibody" refers to an antibody that has both pH-sensitive antigen binding and at least a threshold level of binding to cell surface neonatal Fc receptors (FcRn) at neutral or physiological pH. In some embodiments, the sweeping antibody binds to the neonatal Fc receptor FcRn at neutral pH. For example, the sweeping antibody binds to FcRn at a pH in the range of 7.0-7.6. In some embodiments, the sweeping antibody can bind to the antigen at the antigen binding site and bind to the cellular FcRn through the Fc portion of the antibody. In some embodiments, the sweeping antibody can then neutralize, thereby releasing the antigen in acidic endosomes, which can be degraded. In some embodiments, the sweeping antibody, no longer bound to the antigen, can then be released by the cell (e.g., by exocytosis) back into the serum.

[0268] In some embodiments, FcRn in the vascular endothelium (e.g., of a subject) extends the half-life of the sweeping antibody. In some embodiments, the sweeping antibody, which in some embodiments binds to an antigen such as myostatin (e.g., promyostatin, latent myostatin, or primed myostatin), is neutralized by vascular endothelial cells. In some embodiments, the sweeping antibody is recirculated into the bloodstream. In some embodiments, the sweeping antibody has an increased half-life (e.g., in the serum of a subject) compared to its conventional counterpart. In some embodiments, the conventional counterpart of the sweeping antibody is an antibody from which the sweeping antibody is derived (e.g., before the Fc portion of the conventional antibody is engineered to bind with higher affinity at pH 7). In some embodiments, the sweeping antibody has a half-life in the serum of a subject that is at least 1%, 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 100%, 150%, 200%, or 250% longer than its conventional counterpart.

[0269] In some embodiments, the Fc portion of the sweeping antibody binds to FcRn. In some embodiments, the Fc portion of the sweeping antibody binds to FcRn at 10 -3 M-10 -8 In some embodiments, the sweeping antibody binds to FcRn at a pH of 10-3 M-10 -7 M, 10 -3 M-10 -6 M, 10 -3 M-10 -5 M, 10 -3 M-10 -4 M, 10 -4 M-10 -8 M, 10 -4 M-10 -7 M, 10 -4 M-10 -6 M, 10 -4 M-10 -5 M, 10 -5 M-10 -8 M, 10 -5 M-10 -7 M, 10 -5 M-10 -6 M, 10 -6 M-10 -8 M, 10 -6 M-10 -7 M or 10 -7 M-10 -8 In some embodiments, FcRn binds to FcRn with a Kd in the 5 μM range. In some embodiments, FcRn binds to the CH2-CH3 hinge region of the sweeping antibody. In some embodiments, FcRn binds to the same region as Protein A or Protein G. In some embodiments, FcRn binds to a different binding site than FcγR. In some embodiments, amino acid residue AA in the Fc region of the sweeping antibody is required for FcRn binding. In some embodiments, amino acid residue AA in the Fc region of the sweeping antibody influences FcRn binding.

[0270] In some embodiments, any of the antibodies provided herein are engineered to bind to FcRn with higher affinity. In some embodiments, any of the antibodies provided herein are engineered to bind to FcRn with higher affinity at pH 7.4. In some embodiments, the affinity of the sweeping antibodies for FcRn is improved to extend their pharmacokinetic (PK) properties compared to their conventional counterparts. For example, in some embodiments, the sweeping antibodies cause fewer side effects due to their efficacy at lower doses. In some embodiments, the sweeping antibodies are administered less frequently. In some embodiments, the transcytosis of the sweeping antibodies to certain tissue types is increased. In some embodiments, the sweeping antibodies enhance the efficiency of transplacental delivery. In some embodiments, the sweeping antibodies are produced at a lower cost.

[0271] In some embodiments, any of the antibodies provided herein are engineered to bind to FcRn with lower affinity. In some embodiments, any of the antibodies provided herein are engineered to bind to FcRn with lower affinity at pH 7.4. In some embodiments, the affinity of the sweeping antibodies for FcRn is reduced to shorten their pharmacokinetic (PK) properties compared to their conventional counterparts. For example, in some embodiments, the sweeping antibodies are cleared more quickly for imaging and / or radioimmunotherapy. In some embodiments, the sweeping antibodies promote the clearance of endogenous pathogenic antibodies as a treatment for autoimmune diseases. In some embodiments, the sweeping antibodies reduce the risk of adverse pregnancy outcomes (which can be caused by transplacental transport of maternal fetal-specific antibodies).

[0272] In some embodiments, the sweeping antibodies have a reduced affinity for the antigen at low pH compared to neutral or physiological pH (e.g., pH 7.4). In some embodiments, the sweeping antibodies have a reduced affinity for the antigen at acidic pH (e.g., pH in the range of 5.5-6.5) compared to physiological pH (e.g., pH 7.4). It should be understood that any antibody provided herein can be engineered to dissociate from the antigen according to changes in pH (e.g., pH sensitive antibodies). In some embodiments, the sweeping antibodies provided herein are engineered to bind to the antigen according to pH. In some embodiments, the sweeping antibodies provided herein are engineered to bind to FcRn according to pH. In some embodiments, the sweeping antibodies provided herein are internalized by endocytosis. In some embodiments, the sweeping antibodies provided herein are internalized by FcRn binding. In some embodiments, the endocytosed sweeping antibodies release the antigen in the endosome. In some embodiments, the sweeping antibodies recycle back to the cell surface. In some embodiments, the sweeping antibodies remain attached to the cell. In some embodiments, the endocytosed sweeping antibody is recirculated back into the plasma. It should be understood that the Fc portion of any of the antibodies provided herein can be engineered to have varying FcRn binding activity. In some embodiments, the FcRn binding activity affects the time it takes for the antigen to be cleared by the sweeping antibody. In some embodiments, the sweeping antibody can be a long-acting or fast-acting sweeping antibody.

[0273] In some embodiments, converting a conventional therapeutic antibody to a sweeping antibody reduces the effective dose. In some embodiments, converting a conventional therapeutic antibody to a sweeping antibody reduces the effective dose by at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, converting a conventional therapeutic antibody to a sweeping antibody reduces the effective dose by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 15-fold, 20-fold, 50-fold, or 100-fold.

[0274] In some embodiments, the selection of an appropriate dose of a sweeping antibody for treatment can be made empirically. In some embodiments, a high dose of a sweeping antibody can saturate FcRn, resulting in an antibody that stabilizes the antigen in the serum without internalization. In some embodiments, a low dose of a sweeping antibody may not be therapeutically effective. In some embodiments, the sweeping antibody is administered once a day, once a week, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every eight weeks, once every 10 weeks, once every 12 weeks, once every 16 weeks, once every 20 weeks, or once every 24 weeks.

[0275] In some embodiments, any of the antibodies provided herein can be modified or engineered into sweeping antibodies. In some embodiments, any of the antibodies provided herein can be converted into sweeping antibodies using any suitable method. For example, suitable methods for preparing sweeping antibodies have previously been described in Igawa et al. (2013) "Engineered Monoclonoal Antibody with Novel Antigen-Sweeping Activity In Vivo", PLoS ONE 8(5): e63236; and Igawa et al., "pH-dependent antigen-binding antibodies as a novel therapeutic modality", Biochimica et Biophysica Acta 1844 (2014) 1943-1950; the contents of each of which are hereby incorporated by reference. However, it should be understood that the methods for preparing the sweeping antibodies provided herein are not meant to be limiting. Therefore, additional methods for preparing sweeping antibodies are within the scope of the present disclosure.

[0276] Some aspects of the present disclosure are based on the recognition that the affinity (e.g., expressed as Kd) of any anti-promyostatin / latent myostatin antibody provided herein is sensitive to changes in pH. In some embodiments, the antibodies provided herein have an increased Kd for binding to promyostatin / latent myostatin at a relatively low pH (e.g., a pH in the range of 4.0-6.5) compared to a relatively high pH (e.g., a pH in the range of 7.0-7.4). In some embodiments, the antibodies provided herein have an increased Kd at a pH of 4.0-6.5. -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8In some embodiments, the antibodies provided herein have a Kd of 10 μM for binding to promyostatin / latent myostatin at pH 7.0-7.4. -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 In some embodiments, the antibodies provided herein have a Kd for binding to promyostatin / latent myostatin that is at least 2-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, or at least 10,000-fold higher at a pH of 4.0-6.5 compared to a pH of 7.0-7.4.

[0277] In some embodiments, provided herein are promyostatin / latentmyostatin antibodies that do not specifically bind to an epitope within the amino acid sequence set forth as SEQ ID NO: 64. In some embodiments, the promyostatin / latentmyostatin antibodies provided herein do not specifically bind to the same epitope as an antibody described in Table 2a, 11a, 11b, or 13 of International Patent Application Publication No. WO2016 / 098357, which was published on June 23, 2016 and is based on International Patent Application No. PCT / JP2015 / 006323, filed on December 18, 2015. In some embodiments, the promyostatin / latentmyostatin antibodies provided herein do not compete or cross-compete for binding to the same epitope as an antibody described in Table 2a, 11a, 11b, or 13 of International Patent Application Publication No. WO 2016 / 098357, which was published on June 23, 2016 and is based on International Patent Application No. PCT / JP2015 / 006323, filed on December 18, 2015. In some embodiments, the promyostatin / latentmyostatin antibodies provided herein do not specifically bind to the same epitope as an antibody comprising a VH and VL pair described in Table 2a, 11a, 11b, or 13 of International Patent Application Publication No. WO 2016 / 098357, which was published on June 23, 2016 and is based on International Patent Application No. PCT / JP2015 / 006323, filed on December 18, 2015. In some embodiments, the promyostatin / latentmyostatin antibodies provided herein do not compete or cross-compete for binding to the same epitope as an antibody comprising a VH and VL pair described in Table 2a, 11a, 11b, or 13 of International Patent Application Publication No. WO 2016 / 098357, which was published on June 23, 2016 and is based on International Patent Application No. PCT / JP2015 / 006323, filed on December 18, 2015.

[0278] peptides

[0279] Some aspects of the present disclosure relate to polypeptides having a sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29. In some embodiments, the polypeptide is a heavy chain variable domain. In some embodiments, the polypeptide is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29.

[0280] Some aspects of the present disclosure relate to polypeptides having a sequence selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35. In some embodiments, the polypeptide is a light chain variable domain. In some embodiments, the polypeptide is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of the amino acid sequences set forth in SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35.

[0281] Antibodies that compete with anti-promyostatin / latent myostatin antibodies

[0282] Aspects of the present disclosure relate to antibodies that compete or cross-compete with any of the antibodies provided herein. As used herein, the term "compete" with respect to an antibody means that a first antibody binds to an epitope of a protein (e.g., latent myostatin) in a manner sufficiently similar to the binding of a second antibody such that the result of binding of the first antibody to its epitope is detectably reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. An alternative approach in which the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody may be the case, but this is not necessarily the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its corresponding epitope. However, where each antibody detectably inhibits the binding of the other antibody to its epitope or ligand (whether to the same, greater, or lesser extent), the antibodies are said to "cross-compete" with each other for binding to their corresponding epitopes. Both competing and cross-competing antibodies are within the scope of the present disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational changes, or binding to a common epitope or portion thereof), one skilled in the art will understand that such competing and / or cross-competing antibodies are included and can be used in the methods and / or compositions provided herein.

[0283] Aspects of the present disclosure relate to antibodies that compete or cross-compete with any of the antibodies provided herein. In some embodiments, the antibody binds to or near the same epitope as any of the antibodies provided herein. In some embodiments, if the antibody binds to within 15 or less amino acid residues of the epitope, the antibody binds to the vicinity of the epitope. In some embodiments, any of the antibodies provided herein bind to within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the epitope to which any of the antibodies provided herein bind.

[0284] In another embodiment, the antibody is expressed at a concentration of less than 10 -6 The equilibrium dissociation constant Kd between the antibody and the protein of 10 M competes or cross-competes for binding to any antigen provided herein (e.g., promyostatin / latent myostatin). In other embodiments, the antibody is -11 M-10 -6 M range competes or cross-competes for binding to any of the antigens provided herein.

[0285] Aspects of the present disclosure relate to antibodies that compete with any of the antibodies provided herein for binding to promyostatin / latent myostatin. In some embodiments, the antibody binds to promyostatin / latent myostatin at the same epitope as any of the antibodies provided herein. For example, in some embodiments, any of the antibodies provided herein bind at or near the tolloid cleavage site or at or near the tolloid docking site of promyostatin / latent myostatin. In other embodiments, any of the antibodies provided herein bind at or near the proprotein convertase cleavage site or at or near the proprotein convertase docking site of promyostatin / latent myostatin. In another embodiment, the antibody binds to promyostatin / latent myostatin at a concentration of less than 10 -6 In other embodiments, the antibody that competes with any of the antibodies provided herein has an equilibrium dissociation constant, Kd, ​​between 10 and 10 M and promyostatin / latent myostatin. -11 M-10 -6 Binds promyostatin / latent myostatin with a Kd in the μM range.

[0286] Any antibody provided herein can be characterized using any suitable method. For example, one method is to identify the epitope or "epitope mapping" to which the antigen is bound. There are many suitable methods for locating and characterizing the epitope on the protein, including crystal structure analysis, competitive analysis, gene fragment expression analysis, and analysis based on synthetic peptides of the antibody-antigen complex, such as described in, for example, Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999, Chapter 11. In another example, epitope mapping can be used to determine the sequence to which the antibody is bound. The epitope can be a linear epitope, i.e., contained in a single amino acid extension chain, or a conformational epitope formed by the three-dimensional interaction of amino acids, which may not necessarily be contained in a single extension chain (primary structure linear sequence). Peptides of varying lengths (e.g., at least 4-6 amino acids long) can be separated or synthesized (e.g., recombinantly) and used for binding analysis with antibodies. In another example, the epitope to which the antibody is bound can be determined in a system screening by using overlapping peptides derived from the target antigen sequence and measuring the binding of the antibody. Based on gene fragment expression analysis, the open reading frame encoding the target antigen is fragmented randomly or by specific genetic reconstruction and the reactivity of the expressed antigen fragments with the antibody to be tested is determined. For example, gene fragments can be generated by PCR and then transcribed and translated into protein in vitro in the presence of radioactive amino acids. Binding of the antibody to the radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments can be assayed for binding to the test antibody in a simple binding assay. In another example, mutagenesis of the antigen-binding domain, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and / or essential for epitope binding. For example, domain swapping experiments can be performed using mutants of the target antigen in which various fragments of the promyostatin / latent myostatin polypeptide have been replaced (exchanged) with sequences from a closely related but antigenically different protein, such as another member of the TGFβ protein family (e.g., GDF11). By evaluating antibody binding to mutant promyostatin / latent myostatin, the importance of specific antigenic fragments for antibody binding can be assessed.

[0287] Alternatively, competition assays can be performed using other antibodies known to bind the same antigen to determine if the antibody binds to the same epitope as the other antibody.Competition assays are well known to those skilled in the art.

[0288] Any suitable method, such as the epitope mapping methods described herein, can be applied to determine whether an anti-myostatin / latent myostatin antibody binds to one or more specific residues / segments of promyostatin / latent myostatin as described herein. Furthermore, the interaction of the antibody with one or more of those defined residues in promyostatin / latent myostatin can be determined by conventional techniques. For example, a crystal structure can be determined, and the distances between the residues in promyostatin / latent myostatin and one or more residues in the antibody can be determined accordingly. Based on these distances, it can be determined whether a specific residue in promyostatin / latent myostatin interacts with one or more residues in the antibody. Furthermore, suitable methods, such as competition assays and targeted mutagenesis assays, can be applied to determine preferential binding of a candidate anti-myostatin / latent myostatin antibody to promyostatin / latent myostatin compared to another target, such as a mutant promyostatin / latent myostatin.

[0289] Generation of antibodies that bind promyostatin / latent myostatin

[0290] Various methods can be used to obtain antibodies or antigen-binding fragments thereof disclosed herein. For example, antibodies can be produced using recombinant DNA methods. Monoclonal antibodies can also be produced by generating hybridomas according to known methods (see, for example, Kohler and Milstein (1975) Nature, 256: 495-499). The hybridomas formed in this way are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (e.g., OCTET or BIACORE) analysis, to identify one or more hybridomas that produce antibodies that specifically bind to a given antigen. Any form of a given antigen can be used as an immunogen, for example, a recombinant antigen, a naturally occurring form, any variant or fragment thereof, and its antigenic peptide (e.g., described herein as a linear epitope or as any epitope in a skeleton as a conformational epitope). An exemplary method for preparing an antibody includes screening a protein expression library expressing an antibody or its fragment (e.g., scFv), for example, a phage or ribosome display library. Phage display is described in, for example, Ladner et al., US Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222:581-597; WO 92 / 18619; WO 91 / 17271; WO 92 / 20791; WO 92 / 15679; WO 93 / 01288; WO 92 / 01047; WO 92 / 09690 and WO 90 / 02809.

[0291] In addition to using display libraries, a given antigen (e.g., promyostatin) can be used to immunize a non-human animal, e.g., a rodent, e.g., a mouse, hamster, or rat. In one embodiment, the non-human animal is a mouse.

[0292] In another embodiment, the monoclonal antibody is obtained from a non-human animal and then modified, for example, chimerized, using appropriate recombinant DNA technology. Various approaches for preparing chimeric antibodies have been described. See, for example, Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985, Cabilly et al., US Pat. No. 4,816,567; Boss et al., US Pat. No. 4,816,397; Tanaguchi et al., European Patent Publication EP171496; European Patent Publication 0173494, British Patent GB 2177096B.

[0293] For additional antibody production techniques, see Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988. The present disclosure is not necessarily limited to any particular source, method of production, or other specific characteristics of the antibodies.

[0294] Some aspects of the present disclosure relate to host cells transformed with polynucleotides or vectors. The host cell can be prokaryotic or eukaryotic. The polynucleotide or vector present in the host cell can be integrated into the host cell's genome or can be maintained extrachromosomally. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial, insect, fungal, plant, animal, or human cells. In some embodiments, the fungal cell is, for example, that of the genus Saccharomyces, particularly that of the species Saccharomyces cerevisiae. The term "prokaryotic" includes all bacteria that can be transformed or transfected with DNA or RNA molecules for expression of antibodies or corresponding immunoglobulin chains. Prokaryotic hosts can include Gram-negative as well as Gram-positive bacteria such as, for example, Escherichia coli, Salmonella typhimurium, Serratia marcescens, and Bacillus subtilis. The term "eukaryotic" includes yeast, higher plants, insects, and vertebrate cells, for example, mammalian cells such as NSO and CHO cells. Depending on the host used in the recombinant production process, the antibody or immunoglobulin chain encoded by the polynucleotide may be glycosylated or non-glycosylated. The antibody or corresponding immunoglobulin chain may also include an initial methionine amino acid residue.

[0295] In some embodiments, once the vector has been incorporated into a suitable host, the host can be maintained under conditions suitable for high-level expression of the nucleotide sequence, and, as desired, immunoglobulin light chains, heavy chains, light / heavy chain dimers, or complete antibodies, antigen-binding fragments, or other immunoglobulin forms can be collected and purified; see Beychok, Cells of Immunoglobins Synthesis, Academic Press, NY, (1979). Thus, the polynucleotide or vector is introduced into cells, which then produce antibodies or antigen-binding fragments. In addition, transgenic animals, preferably mammals, comprising the aforementioned host cells can be used for large-scale production of antibodies or antibody fragments.

[0296] The transformed host cells can be grown in fermenters and cultured using any suitable technology to obtain optimal cell growth. Once expressed, full antibodies, their dimers, single light and heavy chains, other immunoglobulin forms or Fab can be purified according to the standard procedures of this area, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, etc.; Referring to Scopes, "Protein Purification", Springer Verlag, NY (1982). The antibody or Fab can then be partially separated from growth medium, cell lysates or cell membranes. For example, the separation and purification of bacterially expressed antibodies or Fab can be any conventional method, such as, for example, preparative chromatographic separations and immunoseparations, such as those involving the use of monoclonal or polyclonal antibodies for example the constant region of an antibody.

[0297] Aspects of the present disclosure relate to hybridomas, which provide a monoclonal antibody source of unlimited extension. As an alternative to obtaining immunoglobulin (Ig) directly from the culture of a hybridoma, immortalized hybridoma cells can be used as the source of heavy chain and light chain loci for subsequent expression and / or genetic manipulation. The antibody gene of the rearrangement can be reverse transcribed from suitable mRNA to produce cDNA. In some embodiments, the constant region of the heavy chain can be exchanged or eliminated together with the constant region of the heavy chain of different isotypes. The variable region can be connected to encode a single-chain Fv district. Multiple Fv districts can be connected to give the binding ability to more than a target or can adopt chimeric heavy chain and light chain combinations. Any suitable method can be used for the cloning of antibody variable regions and the generation of recombinant antibodies.

[0298] In some embodiments, suitable nucleic acids encoding the variable regions of the heavy and / or light chains are obtained and inserted into expression vectors that can be transfected into standard recombinant host cells. A variety of such host cells can be used. In some embodiments, mammalian host cells may be advantageous for efficient processing and production. Typical mammalian cell lines that can be used for this purpose include CHO cells, 293 cells, or NSO cells. The production of antibodies or antigen-binding fragments can be carried out by culturing the modified recombinant host under culture conditions suitable for the growth of the host cells and the expression of the coding sequences. The antibodies or antigen-binding fragments can be recovered by isolating them from the culture. The expression system can be designed to include a signal peptide so that the resulting antibody is secreted into the culture medium; however, intracellular production is also possible.

[0299] The present disclosure also includes polynucleotides encoding at least the variable region of the immunoglobulin chain of the antibodies described herein. In some embodiments, the variable region encoded by the polynucleotide comprises at least one complementarity determining region (CDR) of the VH and / or VL variable region of the antibody produced by any of the hybridomas described above.

[0300] The polynucleotide encoding the antibody or antigen-binding fragment can be, for example, DNA, cDNA, RNA or synthetically produced DNA or RNA, or a chimeric nucleic acid molecule comprising the recombinant production of any of these polynucleotides, either alone or in combination. In some embodiments, the polynucleotide is part of a vector. Such a vector can comprise further genes, such as marker genes, that allow the vector to be selected in a suitable host cell and under suitable conditions.

[0301] In some embodiments, the polynucleotide is operably connected to an expression control sequence and allowed to be expressed in a prokaryotic or eukaryotic cell. The expression of the polynucleotide includes that the polynucleotide is transcribed into translatable mRNA. Regulatory elements that ensure expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They can include regulatory sequences that promote transcription initiation and stable poly-A signals that promote transcription termination and transcripts. Other regulatory elements can include transcription and translation enhancers and / or naturally related or heterologous promoter regions. Possible regulatory elements that allow expression in prokaryotic host cells include, for example, the PL, Lac, Trp or Tac promoters in Escherichia coli, and examples of regulatory elements that allow expression in eukaryotic host cells are the AOX1 or GAL1 promoters in yeast or the CMV-promoter, SV40-promoter, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or globin introns in mammals and other animal cells.

[0302] In addition to the elements responsible for transcription initiation, these regulatory elements can also include transcription termination signals downstream of the polynucleotide, such as SV40-poly-A sites or tk-poly-A sites. In addition, depending on the expression system adopted, a leader sequence capable of directing polypeptide to the cell compartment or secreting it into the culture medium can be added to the coding sequence of the polynucleotide and has been described above. The leader sequence and translation, start and stop sequences, and preferably the leader sequence capable of directing the protein of translation or its part to be secreted into, for example, the extracellular medium are assembled with appropriate phases. Optionally, a heterologous polynucleotide sequence encoding a fusion protein comprising a C- or N-terminal identification peptide that imparts a desired characteristic (for example, stable or simplified purification of the recombinant product expressed) can be used.

[0303] In some embodiments, the polynucleotide encoding at least the variable domain of the light chain and / or heavy chain can encode the variable domain of two immunoglobulin chains or only one immunoglobulin chain. Similarly, the polynucleotides can be under the control of the same promoter or can be controlled separately for expression. In addition, some aspects relate to vectors, particularly plasmids, cosmids, viruses and phages conventionally used in genetic engineering, which contain polynucleotides encoding the variable domains of the immunoglobulin chain of an antibody or antigen-binding fragment; optionally in combination with a polynucleotide encoding the variable domain of another immunoglobulin chain of an antibody.

[0304] In some embodiments, expression control sequences are provided in the vector that can transform or transfect eukaryotic host cells as eukaryotic promoter system, but the control sequences for prokaryotic hosts can also be used. The expression vector that is derived from virus such as retrovirus, vaccinia virus, adeno-associated virus, herpes virus or bovine papilloma virus can be used for delivering polynucleotides or carrier to target cell colony (for example, to express antibody or Fab to cell engineering). A variety of suitable methods can be used for building recombinant viral vectors. In some embodiments, polynucleotides and carrier can be reconstructed into liposome for delivery to target cell. The carrier comprising polynucleotides (for example, the coding sequence and expression control sequence of the heavy chain of immunoglobulin chain and / or light chain variable domain) can be transferred to host cell by suitable method, and this method changes according to the type of cell host.

[0305] Modification

[0306] The antibodies or antigen-binding fragments of the present disclosure may be modified with detectable labels, including but not limited to enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, non-radioactive paramagnetic metal ions, and affinity labels for detecting and separating promyostatin / latent myostatin. The detectable material may be coupled or conjugated directly to the polypeptides of the present disclosure or indirectly through an intermediate (such as, for example, a linker) using suitable techniques. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, or acetylcholinesterase; non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; non-limiting examples of suitable fluorescent substances include biotin, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent substance includes luminol; non-limiting examples of bioluminescent substances include luciferase, luciferin, and aequorin; and examples of suitable radioactive substances include radioactive metal ions, for example, alpha emitters or other radioisotopes such as, for example, iodine ( 131 I, 125 I, 123 I,121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115 mIn, 113 mIn, 112 In, 111 In) and technetium ( 99 Tc, 99 mTc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), Palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F), 153 Sm、Lu、 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y. 47 Sc, 86 R. 188 Re、 142 Pr, 105 Rh, 97 Such as 68 Ge, 57 Co、 65 Zn, 85 Sr. 32 P. 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se and tin ( 113 Sn, 117 Sn). A detectable substance can be coupled or conjugated to the anti-promyostatin / latent myostatin antibodies of the present disclosure directly or indirectly through an intermediate (such as, for example, a linker) using suitable techniques. The anti-promyostatin / latent myostatin antibodies conjugated to a detectable substance can be used in diagnostic assays as described herein.

[0307] Pharmaceutical composition

[0308] One or more anti-myostatin / latent myostatin antibodies can be mixed with a pharmaceutically acceptable carrier (excipient) (including a buffer) to form a pharmaceutical composition for ameliorating a disease or disorder associated with a myopathy. "Acceptable" means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not harmful to the subject being treated. Examples of pharmaceutically acceptable excipients (carriers) (including buffers) will be clear to those skilled in the art and have been described previously. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover. In one example, the pharmaceutical compositions described herein comprise more than one anti-myostatin / latent myostatin antibody that recognizes different epitopes / residues of the target antigen.

[0309] The pharmaceutical composition used in the method of the present invention may contain a pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or an aqueous solution. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used, and may include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; and chloride); benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as Tween TM 、Pluronics TM or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0310] In some examples, the pharmaceutical compositions described herein comprise liposomes containing anti-promyostatin / latent myostatin antibodies, which can be prepared by any suitable method, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and US Pat. Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation time are disclosed in US Pat. No. 5,013,556. Particularly useful liposomes can be prepared by the reverse phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter with a defined pore size to produce liposomes with the desired diameter.

[0311] Anti-promyostatin / latent myostatin antibodies can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions. Exemplary techniques have been described previously, see, for example, Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0312] In other examples, the pharmaceutical compositions described herein can be formulated into sustained release forms. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies in the form of shaped particles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethyl vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT®, and the like. TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0313] Pharmaceutical compositions for in vivo administration must be sterile. This is easily achieved, for example, by filtration through a sterile filter membrane. Therapeutic antibody compositions are generally placed in a container with a sterile access port, for example, an intravenous solvent bag or vial with a stopper pierceable by a hypodermic needle.

[0314] The pharmaceutical compositions described herein may be in unit dosage form such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

[0315] To prepare solid compositions such as tablets, the active ingredient can be mixed with a pharmaceutical carrier, for example, conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or resins, and other pharmaceutical diluents, for example, water, to form a solid preformulation composition comprising a homogeneous mixture of a compound of the present disclosure or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is evenly dispersed throughout the composition so that the composition can be easily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the above type comprising 0.1 mg to about 500 mg of the active ingredient of the present disclosure. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form that provides an advantage of prolonged action. For example, a tablet or pill can comprise an inner dose and an outer dose component, the latter being in the form of a coating on the former. The two components can be separated by an enteric layer, which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, including a number of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0316] Suitable surface active agents include, in particular, non-ionic agents such as polyoxyethylene sorbitan (e.g., Tween TM 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span TM 20, 40, 60, 80 or 85). Compositions with surface active agents conveniently contain 0.05-5% surface active agent, and may be 0.1-2.5%. It will be appreciated that other ingredients, such as mannitol or other pharmaceutically acceptable media, may be added if desired.

[0317] Suitable emulsions include commercially available fat emulsions such as Intralipid TM , Liposyn TM Infonutrol TM 、LipofundinTM and Lipiphysan TM Preparation. The active ingredient can be dissolved in a premixed emulsion composition or alternatively it can be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil), and the emulsion is formed when mixed with a phospholipid (e.g., lecithin, soybean lecithin, or soy lecithin) and water. It should be understood that other ingredients, such as glycerol or glucose, can be added to adjust the tension of the emulsion. Suitable emulsions typically contain up to 20% oil, e.g., between 5 and 20%.

[0318] The emulsion composition can be prepared by mixing anti-promyostatin antibody with Intralipid TM or those prepared from its ingredients (soybean oil, lecithin, glycerin and water).

[0319] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as given above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0320] Compositions in preferably sterile pharmaceutically acceptable solvents can be nebulized using gases. Nebulized solutions can be breathed directly from the nebulizing device or the nebulizing device can be connected to a mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered from a device that delivers the formulation in an appropriate manner (preferably orally or nasally).

[0321] Use of anti-promyostatin / latent myostatin antibodies for treating diseases / disorders

[0322] The anti-promyostatin / latent myostatin antibodies described herein are effective in treating diseases or disorders associated with myopathies. As used herein, the term "myopathies" refers to muscle diseases in which muscle fibers do not function normally, typically resulting in muscle weakness. Myopathies include muscle diseases that are neuromuscular or skeletal in nature. In some embodiments, the myopathy is a hereditary myopathy. Hereditary myopathies include, but are not limited to, nutritional disorders, myotonia, congenital myopathies (e.g., nematode myopathy, multi / minicore myopathy, and centronuclear myopathy), mitochondrial myopathy, familial periodic myopathy, inflammatory myopathy, and metabolic myopathy (e.g., glycogen storage disease and lipid storage disorder). In some embodiments, the myopathy is an acquired myopathy. Acquired myopathies include, but are not limited to, exogenous substance-induced myopathies (e.g., drug-induced myopathies and glucocorticoid myopathies, alcoholic myopathies, and myopathies due to other toxic agents), myositis (e.g., dermatomyositis, polymyositis, and inclusion body myositis), myositis ossificans, rhabdomyolysis, and myoglobinuria, and disuse atrophy. In some embodiments, the myopathy is disuse atrophy, which can be caused by a fracture (e.g., a hip fracture) or by nerve damage (e.g., spinal cord injury (SCI)). In some embodiments, the myopathy is associated with a disease or disorder such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), cachexia syndrome due to renal failure, AIDS, heart disease, and / or cancer. In some embodiments, the myopathy is associated with aging.

[0323] Aspects of the present disclosure include methods for treating a subject suffering from a myopathy, the methods comprising administering to the subject an effective amount of the above-mentioned antibody. In some embodiments, the myopathy is a primary myopathy. In another embodiment, the primary myopathy comprises disuse atrophy. In other embodiments, disuse atrophy is associated with hip fracture, selective joint replacement, critical illness myopathy, spinal cord injury, or stroke. In some embodiments, the myopathy is a secondary myopathy, in which muscle loss is secondary to disease pathology. In other embodiments, the secondary myopathy comprises denervation, hereditary myasthenia, or cachexia. In another embodiment, the secondary myopathy is denervation associated with amyotrophic lateral sclerosis or spinal muscular atrophy. In some embodiments, the secondary myopathy is hereditary myasthenia associated with muscular dystrophy. In other embodiments, the secondary myopathy is cachexia associated with renal failure, AIDS, heart disease, cancer, or aging.

[0324] Another aspect of the present disclosure includes methods of treating a subject having a disease or condition associated with aging. Exemplary diseases or conditions associated with aging include, but are not limited to, sarcopenia (age-related muscle loss), frailty, and androgen deficiency.

[0325] Another aspect of the present disclosure includes a method of treating a subject having a disease or condition associated with disuse atrophy / trauma. Exemplary diseases or conditions associated with disuse atrophy / trauma include, but are not limited to, muscle weakness associated with time spent in an intensive care unit (ICU), hip / joint replacement, hip fracture, stroke, bed rest, SCI, rotator cuff injury, knee replacement, bone fracture, and burns.

[0326] Another aspect of the present disclosure includes methods of treating a subject having a neurodegenerative disease or condition.Exemplary neurodegenerative diseases or conditions include, but are not limited to, spinal muscular atrophy and amyotrophic lateral sclerosis (ALS).

[0327] Another aspect of the present disclosure includes methods of treating a subject having a disease or condition associated with cachexia. Exemplary diseases or conditions associated with cachexia include, but are not limited to, cancer, chronic heart failure, acquired immunodeficiency syndrome (AIDS), chronic obstructive pulmonary disease (COPD), and chronic kidney disease (CKD).

[0328] Another aspect of the present disclosure includes methods of treating a subject suffering from a disease or condition associated with a rare disease. Exemplary rare diseases and conditions include, but are not limited to, osteogenesis imperfecta, sporadic inclusion body myositis, and acute lymphoblastic leukemia.

[0329] Another aspect of the present disclosure includes treating a subject suffering from a disease or condition related to dysmetabolism and / or body composition. In some embodiments, the disease or condition is obesity (e.g., severe obesity), Prader-Willi, type II diabetes, or anorexia. However, other diseases or conditions related to dysmetabolism and / or body composition are well known to those skilled in the art and are within the scope of the present disclosure.

[0330] Another aspect of the present disclosure includes methods of treating a subject having a disease or condition associated with a congenital myopathy. Exemplary congenital myopathies include, but are not limited to, X-linked myotubular myopathy, autosomal dominant centronuclear myopathy, autosomal recessive centronuclear myopathy, nematode myopathy, and congenital fiber-type disproportionate myopathy.

[0331] Another aspect of the present disclosure includes methods of treating a subject having a disease or condition associated with a muscular dystrophy. Exemplary muscular dystrophy include, but are not limited to, Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy (FSH), and limb-girdle muscular dystrophy.

[0332] Another aspect of the present disclosure includes methods of treating a subject having a urogynecological-related disease or disorder, speech disorder (stenosis), extraocular myopathy, carpal tunnel syndrome, Guillain-Barré syndrome, or osteosarcoma.

[0333] To practice the methods disclosed herein, an effective amount of the pharmaceutical composition described above can be administered to a subject (e.g., a human) in need of treatment by a suitable route, such as intravenous administration, for example, by bolus injection or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intracapsular, oral, inhalation, or topical routes. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, can be used for administration. Liquid formulations can be directly nebulized and lyophilized powders can be nebulized after reconstitution. Alternatively, anti-promyostatin / latent myostatin antibodies can be nebulized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and ground powder.

[0334] The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to farm animals, sports animals, pets, primates, horses, dogs, cats, mice and rats. The human subject in need of treatment can be a human patient suffering from a disease / disorder associated with myopathy (such as those described above), at risk of suffering from the disease / disorder or suspected of suffering from the disease / disorder. Subjects with promyostatin / latent myostatin-related diseases or disorders can be identified by routine medical tests, for example, laboratory tests, organ function tests, CT scans or ultrasound. Subjects suspected of having any such disease / disorder can show one or more symptoms of the disease / disorder. A subject at risk of a disease / disorder can be a subject with one or more risk factors for the disease / disorder.

[0335] As used herein, "effective amount" refers to the amount of each active agent required for the treatment effect of a subject, either alone or in combination with one or more other active agents. As recognized by those skilled in the art, the effective amount varies according to the specific condition being treated, the severity of the condition, individual patient parameters (including age, physical condition, body shape, sex and weight), duration of treatment, the nature of simultaneous treatment (if any), specific route of administration and similar factors within the knowledge and skills of the health care provider. These factors are well known to those skilled in the art and can be solved using only routine experiments. It is generally preferred to use the maximum dose of a single component or a combination thereof, that is, the highest safe dose according to reasonable medical judgment. However, it will be understood by those skilled in the art that patients can adhere to lower doses or tolerable doses for medical reasons, physiological reasons or for any other reason in fact. In some embodiments, an effective amount refers to an antibody or its antigen-binding portion thereof that is sufficient to reduce or alleviate the severity and / or duration of the disorder or one or more symptoms thereof, prevent the progression of the disorder, cause the regression of the disorder, prevent the recurrence, development, onset or progression of one or more symptoms associated with the disorder, detect the disorder or enhance or improve the preventive or therapeutic effect of another treatment (e.g., a preventive or therapeutic agent).

[0336] In some embodiments, when a promyostatin / latent myostatin antibody is administered to a subject, an effective amount is an amount effective to increase the mass of the target muscle in the subject compared to a control muscle mass. In some embodiments, the increase in muscle mass is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 4-fold, at least 5-fold, or more compared to control muscle mass. In some embodiments, the increase in muscle mass is an increase in the range of 1-fold to 5-fold, 2-fold to 10-fold, 1-fold to 1.5-fold, 1-fold to 2-fold, etc., compared to control muscle mass.

[0337] As used herein, the term "control muscle mass" refers to a reference standard that can be used to assess the effect of a condition (e.g., treatment with a promyostatin / latent myostatin antibody) on the target muscle mass in a subject. In some embodiments, the control muscle mass is a predetermined value. In some embodiments, the control muscle mass is experimentally determined. In some embodiments, the control muscle mass is the mass of the target muscle in a subject that has not been administered promyostatin / latent myostatin antibody. In some embodiments, the control muscle mass is the mass (e.g., the average mass) of the target muscle in a population of subjects that have not been administered promyostatin / latent myostatin antibody. In some embodiments, the control muscle mass is the mass of the target muscle in a subject before (e.g., just before) administration of promyostatin / latent myostatin antibody. In some embodiments, the control muscle mass is the mass of the target muscle in a subject in which a normal antibody (e.g., an antibody of the same isotype as the promyostatin / latent myostatin antibody) obtained from an animal that has not been exposed to the antigen to which the promyostatin / latent myostatin antibody is directed is administered in place of the promyostatin / latent myostatin antibody. In some embodiments, the control muscle mass is the mass of the target muscle in a subject in which a vehicle (e.g., saline) is administered in place of the promyostatin / latent myostatin antibody.

[0338] In some embodiments, where a promyostatin / latent myostatin antibody is administered to a subject, an effective amount is an amount effective to increase the force generation capacity of a target muscle in the subject (e.g., maximal force generation as measured in vitro using a muscle lever system adapted for horizontal perfusion bath) compared to the force generation capacity of a control. In some embodiments, the increase in force generation capacity is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 4-fold, at least 5-fold, or more compared to the force generation capacity of a control. In some embodiments, the increase in force generation capacity is an increase in the range of 1-fold to 5-fold, 2-fold to 10-fold, 1-fold to 1.5-fold, 1-fold to 2-fold, etc., compared to the force generation capacity of a control.

[0339] As used herein, the term "control force capacity" refers to a reference standard that can be used to assess the effect of a condition (e.g., treatment with promyostatin / latent myostatin antibodies) on the force capacity of a muscle in a subject. In some embodiments, the control force capacity is a predetermined value. In some embodiments, the control force capacity is determined experimentally. In some embodiments, the control force capacity is the force capacity of a target muscle in a subject that has not been administered promyostatin / latent myostatin antibodies. In some embodiments, the control force capacity is the force capacity (e.g., the average force capacity) of a target muscle in a population of subjects that have not been administered promyostatin / latent myostatin antibodies. In some embodiments, the control force capacity is the force capacity of a target muscle in a subject before (e.g., immediately before) administration of promyostatin / latent myostatin antibodies. In some embodiments, the control force capacity is the force capacity of the target muscle in a subject who has been administered a normal antibody obtained from an animal that has not been exposed to the antigen to which the promyostatin / latent myostatin antibody is directed (e.g., an antibody of the same isotype as the promyostatin / latent myostatin antibody) in place of the promyostatin / latent myostatin antibody. In some embodiments, the control force capacity is the force capacity of the target muscle in a subject who has been administered a vehicle (e.g., saline) in place of the promyostatin / latent myostatin antibody.

[0340] Empirical considerations, such as half-life, generally aid in dosage determination. For example, antibodies compatible with the human immune system, such as humanized or fully human antibodies, can be used to extend the half-life of the antibody and protect the antibody from attack by the host immune system. The frequency of administration can be determined and adjusted during the course of treatment and is generally, but not necessarily, based on the treatment and / or inhibition and / or alleviation and / or delay of the disease / disorder associated with the myopathy. Alternatively, a sustained continuous release formulation of anti-promyostatin / latent myostatin may be appropriate. Various formulations and devices for achieving sustained release will be clear to those skilled in the art and are within the scope of this disclosure.

[0341] In one example, the dosage of an anti-promyostatin / latent myostatin antibody as described herein can be determined empirically in an individual who has been given one or more administrations of the antibody. The individual is given increasing doses of the antagonist. To assess the efficacy of the antagonist, indicators of the disease / disorder can be tracked.

[0342] Generally, for administration of any of the antibodies described herein, an initial candidate dose may be about 2 mg / kg. For the purposes of this disclosure, a typical daily dose range may be about any of 0.1 μg / kg to 3 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg, to 30 mg / kg to 100 mg / kg or higher, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the condition, treatment continues until the desired symptom suppression occurs or until sufficient therapeutic levels are achieved to alleviate the disease or disorder associated with promyostatin / latent myostatin or its symptoms. Exemplary dosing regimens include administration of an initial dose of about 2 mg / kg, followed by a maintenance dose of about 1 mg / kg of the antibody, or followed by a maintenance dose of about 1 mg / kg every other week. However, other dosing regimens may be useful, depending on the pharmacokinetic decay pattern that the practitioner desires to achieve. For example, administration 1-4 times a week is contemplated. In some embodiments, the administration of about 3 μg / mg-about 2 mg / kg (such as about 3 μg / mg, about 10 μg / mg, about 30 μg / mg, about 100 μg / mg, about 300 μg / mg, about 1 mg / kg and about 2 mg / kg) scope can be used. In some embodiments, the administration frequency is once a week, once every 2 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks or once every 10 weeks; or once a month, once every 2 months, or once every 3 months, once every 4 months, once every 5 months, once every 6 months, once every 8 months, once every 10 months, annually or longer. The progress of this treatment is easily monitored by conventional techniques and analysis. Dosage regimen (including the antibody used) can change over time.

[0343] In some embodiments, for an adult patient of normal weight, a dose in the range of about 0.3-5.00 mg / kg may be administered. The specific dosage regimen, such as dose, timing, and repetition, depends on the specific individual and the individual's medical history, as well as the properties of the individual agents (such as the half-life of the agent and other relevant considerations).

[0344] For the purposes of this disclosure, the appropriate dosage of an anti-myostatin / latent myostatin antibody depends on the specific antibody (or composition thereof) used, the type and severity of the disease / disorder, whether the antibody is being administered for prophylactic or therapeutic purposes, previous treatment, the patient's clinical history and response to antagonists, and the judgment of the attending physician. In some embodiments, the clinician administers an anti-myostatin / latent myostatin antibody until a dose is reached that achieves the desired result. Administration of an anti-myostatin / latent myostatin antibody can be continuous or intermittent, for example, depending on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to the skilled practitioner. Administration of an anti-myostatin / latent myostatin antibody can be substantially continuous over a preselected period of time or can be a series of spaced doses, for example, before, during, or after the onset of a disease or disorder associated with promyostatin / latent myostatin.

[0345] As used herein, the term "treat," ...

[0346] Alleviation of a disease / disorder associated with promyostatin / latent myostatin includes delaying the development or progression of the disease, or reducing the severity of the disease. Alleviation of a disease does not necessarily require a curative outcome. As used herein, "delaying" the development of a disease / disorder associated with promyostatin / latent myostatin means postponing, hindering, slowing, retarding, stabilizing and / or slowing the progression of the disease. Such a delay can be of varying lengths of time, depending on the medical history and / or the individual being treated. A method of "delaying" or alleviating the development of a disease or delaying the onset of a disease is a method that reduces the likelihood of one or more symptoms of the disease occurring within a given time period and / or reduces the extent of the symptoms within a given time period when compared to when the method was not used. Such comparisons are generally based on clinical studies using a sufficient number of subjects to give statistically significant results.

[0347] "Development" or "progression" of a disease means the first manifestation of the disease and / or subsequent progression. The progression of the disease may be detectable and assessed using standard clinical techniques. However, progression also refers to progression that may not be detectable. For the purposes of this disclosure, development or progression refers to the biological course of symptoms. "Development" includes onset, recurrence, and onset. As used herein, "onset" or "occurrence" of a disease / disorder associated with a myopathy includes both initial onset and / or recurrence.

[0348] In some embodiments, the anti-promyostatin / latent myostatin antibodies described herein are administered to a subject in need of treatment in an amount sufficient to inhibit the proteolytic activation of promyostatin / latent myostatin to active myostatin in vivo by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more). In other embodiments, the antibodies are administered in an amount effective to reduce the level of promyostatin / latent myostatin or latent myostatin by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more).

[0349] Conventional methods known to those skilled in the art of medicine can be used for administering pharmaceutical compositions to the subject, and it depends on disease type to be treated or disease site.This composition can also be administered via other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectum, nasally, oral cavity, vaginally or through implanted reservoir administration.Term " parenteral " as used herein comprises subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion technology.In addition, it can be administered to the subject via accumulating injection route of administration, as using 1-, 3- or 6-month accumulating injection or biodegradable materials and methods.

[0350] Injectable compositions can include various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies can be administered by drip infusion, thereby infusing a pharmaceutical preparation comprising the antibody and a pharmaceutically acceptable excipient. Physiologically acceptable excipients can include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. For intramuscular preparations, for example, sterile preparations of suitable antibody soluble salts can be dissolved and administered in a pharmaceutically acceptable excipient such as water for injection, 0.9% saline, or 5% glucose solution.

[0351] In one embodiment, the anti-promyostatin / latent myostatin antibody is administered by a site-specific or targeted local delivery technique. Examples of site-specific or targeted local delivery techniques include various implantable reservoirs or local delivery catheters of the anti-promyostatin / latent myostatin antibody, such as infusion catheters, indwelling catheters or intra-needle catheters, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site-specific carriers, direct injection or direct application. See, for example, PCT Publication No. WO 00 / 53211 and US Pat. No. 5,981,568.

[0352] Targeted delivery of therapeutic compositions comprising polynucleotides or expression vectors can also be used. Receptor-mediated DNA delivery technology is described in, for example, Findeis et al., Trends Biotechnol. (1993) 11: 202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (JA Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263: 621; Wu et al., J. Biol. Chem. (1994) 269: 542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87: 3655; Wu et al., J. Biol. Chem. (1991) 266: 338.

[0353] Therapeutic compositions comprising polynucleotides (e.g., those encoding the anti-promyostatin / latentmyostatin antibodies described herein) are administered in a gene therapy protocol in a range of about 100 ng to about 200 mg of DNA for local administration. In some embodiments, concentrations ranging from about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA, or higher, may also be used during a gene therapy protocol.

[0354] The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. Gene delivery vehicles can be viral or non-viral in origin (see generally Jolly, Cancer Gene Therapy (1994) 1: 51; Kimura, Human Gene Therapy (1994) 5: 845; Connelly, Human Gene Therapy (1995) 1: 185 and Kaplitt, Nature Genetics (1994) 6: 148). The expression of such coding sequences can be induced using endogenous mammalian promoters and / or enhancers or heterologous promoters and / or enhancers. The expression of coding sequences can be constitutive or regulated.

[0355] Suitable viral-based vectors for the delivery of desired polynucleotides (eg, encoding the antibodies disclosed herein) and expression in desired cells are within the scope of the present disclosure. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90 / 07936; WO 94 / 03622; WO 93 / 25698; WO 93 / 25234; WO 93 / 11230; WO 93 / 10218; WO 91 / 02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EP Patent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR-1251), and alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR-1251). 1249; ATCC VR-532)) and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655). Administration of DNA linked to inactivated adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can also be used.

[0356] Non-viral delivery vehicles and methods can also be used, including but not limited to, polycationic condensed DNA linked or unlinked to a separate inactivated adenovirus (see, e.g., Curiel, Hum. Gene Ther. (1992) 3: 147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264: 16985); eukaryotic cell delivery vehicles (see, e.g., US Pat. No. 5,814,482; PCT Publication Nos. WO 95 / 07994; WO 96 / 17072; WO 95 / 30763; and WO 97 / 42338) and nuclear charge neutralization or fusion with the cell membrane. Naked DNA can also be used. Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90 / 11092 and US Pat. No. 5,580,859. Liposomes useful as gene delivery vehicles are described in US Pat. No. 5,422,120; PCT Publication Nos. WO 95 / 13796; WO 94 / 23697; WO 91 / 14445; and EP Patent No. 0524968. Additional approaches are described in Philip, Mol. Cell. Biol. (1994) 14:2411 and Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0357] The specific dosage regimen, eg, dose, timing, and repetition, used in the methods described herein will depend on the particular subject and that subject's medical history.

[0358] In some embodiments, more than one anti-myostatin / latent myostatin antibody or a combination of an anti-myostatin / latent myostatin antibody and another suitable therapeutic agent may be administered to a subject in need of treatment. The antagonists may be of the same type or different from one another. The anti-myostatin / latent myostatin antibodies may also be used in conjunction with other agents to enhance and / or compensate for the efficacy of the drug.

[0359] The efficacy of the treatment of a disease / disorder associated with a myopathy can be evaluated using any suitable method. For example, the efficacy of the treatment of a disease / disorder associated with a myopathy can be evaluated by assessing muscle weakness (e.g., assessing the pattern and severity of muscle weakness), electromyography, assessing blood chemistry (e.g., assessing electrolytes, assessing endocrine causes, measuring creatinine kinase levels, determining erythrocyte sedimentation rate, and performing antinuclear antibody analysis), and biopsy assessment (e.g., by histology, histochemistry, electron microscopy, biochemistry, and genetic analysis).

[0360] Kit for ameliorating myopathy-related diseases / disorders

[0361] The present disclosure also provides kits for alleviating diseases / disorders associated with myopathy. Such kits can include one or more containers comprising an anti-promyostatin / latent myostatin antibody, for example, any of those described herein.

[0362] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include instructions for administering the anti-promyostatin / latent myostatin antibody to treat a target disease (such as those described herein), delay the onset of a target disease, or alleviate a target disease. The kit may further include instructions for selecting an individual suitable for treatment based on a determination of whether the individual has the target disease. In still other embodiments, the instructions include instructions for administering the antibody to an individual at risk for the target disease.

[0363] Instructions for use of the anti-promyostatin / latent myostatin antibodies generally include information on the dosage, dosing schedule, and route of administration for the intended treatment. The container can be a unit dose, a bulk package (e.g., a multi-dose package), or a subunit dose. The instructions provided in the kits of the present disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0364] The label or package insert indicates that the composition is used to treat, delay the onset of, and / or alleviate a disease or disorder associated with a myopathy.Instructions can be provided for practicing any of the methods described herein.

[0365] The kits of the present disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Packaging for use in conjunction with specific devices such as inhalers, nasal administration devices (e.g., nebulizers), or infusion devices such as miniature pumps is also contemplated. The kit can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). The container can also have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an anti-promyostatin / latent myostatin antibody such as those described herein.

[0366] The kit may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert on or associated with the container. In some embodiments, the present disclosure provides an article of manufacture comprising the contents of the kit described herein.

[0367] Assays for the detection of promyostatin / latent myostatin

[0368] In some embodiments, the methods and compositions provided herein relate to methods for detecting promyostatin / latent myostatin in a sample obtained from a subject. As used herein, "subject" refers to a single organism, e.g., a single mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, goat, cow, cat, or dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject can be of either sex and can be at any developmental stage. In some embodiments, the subject is a patient or a healthy volunteer.

[0369] In some embodiments, methods for detecting promyostatin / latent myostatin in a sample obtained from a subject comprise (a) contacting the sample with an anti-promyostatin / latent myostatin antibody under conditions suitable for binding of the antibody to the antigen (if the antigen is present in the sample), thereby forming a binding complex; and (b) measuring the level of the antibody or antigen-binding fragment bound to the antigen (e.g., measuring the level of the binding complex).

[0370] As used herein, a binding complex refers to a biomolecular complex of an antibody (including antigen binding fragments) that binds to an antigen (e.g., promyostatin / latent myostatin protein). A binding complex can comprise an antibody with a single specificity or two or more antibodies or antigen binding fragments with different specificities. In one embodiment, a binding complex comprises two or more antibodies that recognize different antigenic sites on the same antigen. In some cases, an antibody can bind to an antigen that is already bound to other biomolecules such as RNA, DNA, polysaccharides, or proteins. In one embodiment, a binding complex comprises two or more antibodies that recognize different antigens. In some embodiments, an antibody in a binding complex (e.g., an immobilized antibody that binds to an antigen) can itself act as an antigen to which an antibody (e.g., a detectably labeled antibody) binds. Thus, in some cases, a binding complex can comprise multiple antigens and multiple antibodies or antigen binding fragments.

[0371] In some embodiments, the antigen present in the binding complex may be in or not in its original original position conformation. In some embodiments, the binding complex is formed between an antibody and a purified protein antigen or an isolated protein comprising an antigen, wherein the antigen is not in its original original position conformation. In some embodiments, the binding complex is formed between an antibody and a purified protein antigen, wherein the antigen is not in its original original position conformation and is fixed on a solid support (e.g., a PVDF membrane). In some embodiments, the binding complex is formed with an antibody and a cell surface protein such as present in the original conformation (e.g., on the surface of a cell) in situ.

[0372] The antibody in the binding complex may be detectably labeled or unlabeled. In some embodiments, the binding complex comprises a detectably labeled antibody and an unlabeled antibody. In some embodiments, the binding complex comprises a detectably labeled antigen. In some embodiments, the antibody in the binding complex is fixed to one or more solid supports. In some embodiments, the antigen in the binding complex is fixed to one or more solid supports. Exemplary solid supports are disclosed herein and will be clear to those skilled in the art. The foregoing examples of binding complexes are not intended to be limiting. Other examples of binding complexes will be clear to those skilled in the art.

[0373] In any detection, diagnosis and monitoring method, the antibody (including Fab) or antigen can be coupled directly or indirectly to the solid support surface. The method for coupling to the solid support is standard and can be accomplished by covalent and non-covalent interactions. Non-limiting examples of coupling methods include: adsorption, cross-linking, protein A / G-antibody interaction and streptavidin-biotin interaction. Other coupling methods are readily apparent to those skilled in the art.

[0374] In some aspects, the detection, diagnosis and monitoring methods include comparing the level of an antibody (including antigen-binding fragments) that binds to an antigen (e.g., promyostatin / latent myostatin) to one or more reference standards. The reference standard can be, for example, the level of the corresponding promyostatin / latent myostatin in a subject with or without promyostatin / latent myostatin. In one embodiment, the reference standard is the level of promyostatin / latent myostatin detected in a sample that does not contain promyostatin / latent myostatin (e.g., background level). Alternatively, the background level can be determined from a sample containing a specific promyostatin / latent myostatin by contacting the sample with a nonspecific antibody (e.g., an antibody obtained from non-immune serum). Again, the reference standard can be the level of promyostatin / latent myostatin detected in a sample containing promyostatin / latent myostatin (e.g., a positive control). In some cases, the reference standard can be a range of levels that correlate with varying concentrations of promyostatin / latent myostatin in a sample and can be used to quantify the concentration of promyostatin / latent myostatin in a test sample. The foregoing examples of reference standards are not limiting and other suitable reference standards will be readily apparent to those skilled in the art. In some embodiments, the level of an antibody that binds to promyostatin / latent myostatin is compared to the level of mature myostatin. In some cases, the level of promyostatin / latent myostatin is compared to mature myostatin to determine the ratio of inactive to active myostatin in a sample.

[0375] The level of promyostatin / latent myostatin can be measured from a biological sample as provided herein. A biological sample refers to any biological material that can be obtained from a subject or cell. For example, a biological sample can be whole blood, plasma, serum, saliva, cerebrospinal fluid, urine, cells (or cell lysates) or tissue (e.g., normal tissue or tumor tissue). In some embodiments, the biological sample is a fluid sample. In some embodiments, the biological sample is a solid tissue sample. For example, a tissue sample can include, but is not limited to, skeletal muscle, myocardium, adipose tissue, and tissue from other organs. In some embodiments, the biological sample is a biopsy sample. In some embodiments, a solid tissue sample can be made into a fluid sample using conventional methods in the art.

[0376] Biological sample can also include one or more cells of cell line.In some embodiments, cell line includes human cell, primate cell (for example, vero cell), rat cell (for example, GH3 cell, OC23 cell) or mouse cell (for example, MC3T3 cell).There is multiple human cell line, include but not limited to human embryonic kidney (HEK) cell, HeLa cell, cancer cell (NCI60) of 60 cancer cell lines from National Cancer Institute, DU145 (prostate cancer) cell, Lncap (prostate cancer) cell, MCF-7 (breast cancer) cell, MDA-MB-438 (breast cancer) cell, PC3 (prostate cancer) cell, T47D (breast cancer) cell, THP-1 (acute myeloid leukemia) cell, U87 (glioblastoma) cell, SHSY5Y human neuroblastoma cell (from myeloma clone) and Saos-2 (bone cancer) cell.

[0377] Further embodiments relate to methods for monitoring a disease, condition, or any treatment thereof (e.g., a myopathy or myopathy treatment) in a subject having or at risk for a disease or condition, comprising: (a) obtaining a biological sample from the subject, (b) determining the level of promyostatin / latent myostatin in the biological sample using an antibody that detects promyostatin / latent myostatin; and (c) repeating steps (a) and (b) at one or more times. Myostatin has been used as a biomarker for muscle atrophy, however, currently available commercial methods and reagents (e.g., antibodies used in ELISA and Western blots) are either not specific for myostatin, or only detect mature myostatin or do not detect myostatin at all. Therefore, provided herein are methods and reagents (e.g., antibodies) for detecting promyostatin / latent myostatin for diagnostic purposes in the context of a disease and / or condition (e.g., muscle atrophy). As an example, the level of promyostatin / latent myostatin can be measured in a subject or in a biological sample therefrom to detect or monitor the progression of a disease or condition. As another example, the level of promyostatin / latent myostatin can be measured in a subject or in a biological sample therefrom to monitor the response to treatment for a disease or condition. It will be understood that the level of promyostatin / latent myostatin can be monitored over any suitable time period, which may vary depending on the disease or condition the subject is suffering from or any treatment regimen the subject may be undergoing.

[0378] Another embodiment relates to a diagnostic composition comprising any of the above-mentioned antibodies, Fabs, polynucleotides, carriers or cells and optionally a suitable method for detecting. For example, antibodies are suitable for use in immunoassays, wherein they can be used in liquid phase or incorporated into a solid phase carrier. Examples of immunoassays that can utilize the antibodies are competitive and non-competitive immunoassays in direct or indirect form. Examples of such immunoassays are enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIA), sandwich (immunoassay analysis), flow cytometry, Western blot analysis, immunoprecipitation analysis, immunohistochemistry, immunomicroscopy, lateral flow immunochromatographic analysis, and proteomics arrays. Antigens and antibodies can be incorporated into many different solid supports (e.g., carriers, membranes, columns, proteomics arrays, etc.). Examples of solid support materials include glass, polystyrene, polyvinyl chloride, polyvinylidene fluoride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses such as nitrocellulose, polyacrylamide, agarose, and magnetite. The nature of the support can be fixed or suspended in solution (e.g., beads).

[0379] According to a further embodiment, the antibodies (including antigen-binding fragments) provided herein can also be used in a method for assessing the expression of promyostatin / latent myostatin in a subject by obtaining a biological sample (which can be a tissue sample, blood sample or any other suitable body fluid sample) from the subject. The process can include contacting a blood sample (whole blood, serum, plasma), a tissue sample or a protein sample isolated therefrom with the antibody under conditions that allow the formation of a binding complex between the antibody and the antigen. The level of such a binding complex can then be determined by any suitable method. In some embodiments, the biological sample is contacted with the antibody under conditions suitable for binding of the antibody to the promyostatin / latent myostatin protein (if the antigen is present in the sample) and forming a binding complex consisting of the antibody bound to the antigen. This contacting step is typically performed in a reaction chamber such as a tube, a plate well, a membrane bath, a cell culture dish, or a microscope slide. In some embodiments, the antibody is immobilized on a solid support. In some embodiments, the antigen is immobilized on a solid support. In some embodiments, the solid support is the surface of the reaction chamber. In some embodiments, the solid support is a polymer membrane (eg, nitrocellulose strips, polyvinylidene fluoride (PVDF) membranes, etc.) Other suitable solid supports can be used.

[0380] In some embodiments, the antibody is immobilized on a solid support before contact with the antigen. In other embodiments, the antibody is immobilized after the binding complex is formed. In yet other embodiments, the antigen is immobilized on a solid support before the binding complex is formed. A detection reagent is added to the reaction chamber to detect the immobilized binding complex. In some embodiments, the detection reagent comprises a detectably labeled second antibody directed against the antigen. In some embodiments, the first antibody itself is detectably labeled and is thus a detection reagent.

[0381] In one aspect, the detection method includes the steps of immobilizing an antibody to a solid support; applying a sample (e.g., a biological sample or an isolated protein sample) to the solid support under conditions that allow binding of the antigen to the antibody (if present in the sample); removing excess sample from the solid support; applying a detectably labeled antibody under conditions that allow binding of the detectably labeled antibody to the immobilized antibody bound to the antigen; and washing the solid support and determining the presence of the label on the solid support.

[0382] In some embodiments, the antigen is fixed on a solid support such as a PVDF membrane in a reaction chamber (e.g., a membrane bath) before contact with the antibody. A detection reagent is added to the reaction chamber to detect the fixed binding complex. In some embodiments, the detection reagent comprises a detectably labeled second antibody for the antigen. In some embodiments, the detection reagent comprises a detectably labeled second antibody for the first antibody. As disclosed herein, detectable labeling can be, for example, a radioisotope, a fluorophore, a luminescent molecule, an enzyme, a biotin moiety, an epitope tag, or a dye molecule. In some embodiments, the first antibody itself is detectably labeled, and thus is a detection reagent. Suitable detectable labels are described herein and are readily apparent to those skilled in the art.

[0383] Thus, a diagnostic kit suitable for home or clinical use (field use) is provided, comprising (a) a detectably labeled and / or unlabeled antibody as an antigen binding reagent (e.g., a promyostatin / latent myostatin binding reagent); (b) a detection reagent; and optionally, (c) complete instructions for using the reagent to detect the antigen in a sample. In some embodiments, the diagnostic kit comprises antibodies and / or promyostatin / latent myostatin immobilized on a solid support. Any of the solid supports described herein is suitable for incorporation into the diagnostic kit. In a preferred embodiment, the solid support is the surface of a reaction chamber of a plate well. Typically, the plate well is in a multiwell plate having a plurality of wells selected from 6, 12, 24, 96, 384, and 1536, but is not limited thereto. In other embodiments, the diagnostic kit provides a detectably labeled antibody. The diagnostic kit is not limited to these embodiments, and other variations in the composition of the kit will be readily apparent to those skilled in the art.

[0384] The following detailed description is, therefore, to be construed as illustrative only, and the remainder of the disclosure is not limitative in any way.All publications cited herein are incorporated by reference for the purpose or subject matter referred to herein.

[0385] Example

[0386] Example 1: Antibody Generation and Selection

[0387] Antibody Summary

[0388] Ab2 is a fully human anti-promyostatin / latent myostatin monoclonal antibody of the IgG4 / λ isotype that binds to human promyostatin and latent myostatin with high affinity (Kd = 3420 pM, ForteBio BLI). The antibody is able to inhibit proteolytic activation of promyostatin / latent myostatin with an IC50 value in the 0.5 micromolar range, which is at or near the limit of the assay. The theoretical molecular weight of the polypeptide is 144,736 Da and its theoretical pI is 6.7. Affinity optimization was performed using antibody display to identify higher affinity variants Ab4 and Ab6. The affinity-optimized variants were similarly constructed on the human IgG4 / λ isotype framework.

[0389] Table 2: Biochemical properties of candidate anti-promyostatin / latent myostatin antibodies

[0390]

[0391] Platform and identification of parental antibodies

[0392] The parental Ab1 antibody was identified by selection from a primary phage display library using pro-myostatin and latent-myostatin as the primary antigens for selection. Phage selection and initial screening were performed using a library displaying conventional scFvs in a format similar to that described by McCafferty et al. (McCafferty et al., 1990). Each round of selection consisted of pre-clearing (to remove nonspecific phage antibodies), incubation with antigen, washing, elution, and amplification. Selection was performed over multiple rounds using both solid phase (biotinylated antigen coated on immunotubes) and solution phase (biotinylated antigen captured using streptavidin-coated beads) panning strategies.

[0393] A total of 10,000 individual scFv clones were screened for binding to promyostatin or latent-myostatin in two separate campaigns. The first campaign utilized promyostatin / latent-myostatin as the antigen, while the second campaign used latent-myostatin as the antigen. DNA for the target scFv clones was sequenced and 216 unique clones were identified. Positive binding scFv clones were counter-screened for binding to proGDF11 and to a panel of unrelated proteins to confirm specificity for promyostatin / latent-myostatin. From this set of unique scFv clones, 101 of the 134 GDF8-specific clones were converted to full-length IgG (IgG1 isotype) for additional characterization.

[0394] The full-length IgG antibodies were further characterized by ELISA for binding to the pro- and latent forms of human and mouse myostatin and GDF11. The antibodies were also screened for binding to the myostatin prodomain, proTGFβ (human and mouse), mature growth factor of myostatin, mature growth factor of GDF11, activin A growth factor, and proactivin A. Lead antibodies were selected based on their cross-reactivity with human and mouse promyostatin and latent myostatin without interaction with GDF11, activin, or TGFβ proteins.

[0395] Two forms of epitope panning were used. First, chimeric constructs were designed and generated that exchanged portions of the prodomains of myostatin and GDF11. These chimeric proteins were assayed for interaction with screening antibodies by ELISA. Epitope binning was performed using a ForteBio BLI instrument, where biotinylated promyostatin / latent myostatin antibodies were immobilized on a streptavidin-coated biosensor chip and cross-blocking of the antibodies was assessed by sensor reaction. These epitope binning experiments, along with data from ELISA binding experiments, allowed our functionally active lead antibodies (see below) to be separated into three distinct epitope groups (see Table 3).

[0396] Table 3: Ranking of five anti-promyostatin / latent myostatin IgG1 antibodies

[0397]

[0398] *Statistically significant by one-way ANOVA with Dunnett's test.

[0399] Ab8 did not bind to latent myostatin, only to promyostatin. The murine promyostatin / latent myostatin preparation had -40% latent material, which reduced apparent efficacy in the functional assay.

[0400] ND: Not determined.

[0401] To evaluate the ability of the antibodies to bind and inhibit the activation of promyostatin / latent myostatin, various biochemical and cellular assays were established. The binding kinetics to promyostatin and latent myostatin were measured using a ForteBio Octet, where biotinylated substrate proteins were immobilized on a streptavidin-coated sensor chip. The equilibrium dissociation constants for the candidates from the screen are shown in Table 3.

[0402] To measure the ability of IgG to inhibit myostatin signaling, a myostatin activation assay was developed. Conditioned medium from cells overexpressing mT112 (a tolloid protease required for myostatin activation) or furin (a proprotein convertase that cleaves mature growth factors from the prodomain) was generated. After pre-incubation with the test antibodies, promyostatin / latent myostatin or latent myostatin was incubated with a mixture of mT112 and furin-conditioned medium (promyostatin) or mT112-conditioned medium (latent myostatin). After an overnight proteolytic reaction, the release of mature growth factor was measured using a CAGA-based reporter assay in 293T cells. The antibodies were further validated by dose response in the same assay, the results of which are shown in Table 3.

[0403] Five parent antibodies (Table 3) consistently demonstrated strong selectivity and activity in all of the above assays and were also selected for further in vivo characterization (discussed in Example 2). For consistency, the binding and activity of these antibodies for promyostatin / latent myostatin are summarized, as Ab8 does not recognize latent myostatin.

[0404] To determine the mechanism of action of the antibody candidates, samples were analyzed by Western blot using a polyclonal antibody raised against the prodomain of myostatin, as shown in Figure 3. This allowed tracking of a fragment of the myostatin prodomain (boxed), which is produced after cleavage by mT112. A dose-dependent decrease in the production of this fragment was seen with increasing concentrations of Ab1. This experiment suggests that the antibodies in epitope bin 1 act by blocking the cleavage of pro-myostatin and latent-myostatin by the tolloid family of proteases.

[0405] Based on the in vitro and in vivo activities of the active anti-promyostatin / latentmyostatin antibodies, Abl was selected as a lead for further characterization, including affinity maturation, germlining, and manufacturability analysis.

[0406] Optimization of Ab1

[0407] The Ab1 antibody was selected for further characterization. Affinity for promyostatin / latent myostatin was optimized using yeast display. In addition, the sequence of Ab1 was germlined to reduce the potential immunogenicity of non-germline amino acid positions within the human variable region framework.

[0408] Affinity optimization of Ab1 by yeast display

[0409] The Ab1 parent antibody was optimized for binding to promyostatin / latent myostatin using a yeast-based scFv display approach. Briefly, three different scFv libraries were generated to introduce point mutations into selected CDR positions corresponding to the human framework utilized by Ab1 based on the amino acid frequencies observed in the natural human antibody repertoire using antibody deep sequencing. Each library contained scFvs based on the Ab1 sequence with a single point mutation introduced into each CDR, such that each variant of the resulting heavy or light chain had a total of three substitutions, one in each CDR. The three libraries were used for FACS-based sorting and selection to identify a pool of clones with higher affinity for promyostatin / latent myostatin (Figure 23). Direct binding of yeast-expressed scFv clones was used to select antibodies for conversion into full-length IgG expressed in mammalian cell culture.

[0410] Many of the higher affinity scFv clones identified in the yeast campaign contained substitutions at position 28 of the heavy chain. For some clones, the threonine to asparagine substitution resulted in the addition of an irregular N-glycosylation motif within CDRH1. Since N-glycosylation within the variable region of an antibody can be undesirable, any clones containing the glycosylation motif were further substituted to include an alanine at this position.

[0411] The binding kinetics to pro-myostatin and latent-myostatin were then evaluated by octet for each affinity-optimized construct and compared to the parental Ab1 (discussed in Example 2). All clones showed significantly improved binding affinity for myostatin, and two clones (Ab3 and Ab5) were selected based on their selective binding characteristics relative to GDF11.

[0412] Primary sequences and backbones of anti-promyostatin / latent myostatin antibodies

[0413] The sequence alignment of the variable regions of parent Abl and its affinity optimized variants is shown below. The complementary determining regions (CDRs) are defined using Kabat (underlined) and IMGT nomenclature (bold). Substitutions from parent Abl are shown in lowercase text (below and Figures 24A-24B ).

[0414] A. Heavy chain variable region

[0415]

[0416]

[0417] B. Light chain variable region

[0418]

[0419] Fundamentals of Antibody Engineering and Isotype Selection

[0420] In some embodiments, antibodies useful for myostatin blockade will lack effector function. Therefore, for the humanized construct, an IgG4-Fc region was selected. Antibodies of the IgG4 isotype bind poorly to complement C1q and therefore do not significantly activate complement. These antibodies also bind weakly to Fcγ receptors, resulting in insufficient or absent antibody-dependent cell-mediated cytotoxicity (ADCC).

[0421] To avoid potential complications due to Fab-arm exchange (which is known to occur in the original IgG4 mAb), Ab1 and its variants were engineered with a stabilizing "Adair" mutation (Angal, 1993), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline, resulting in an IgG1-like (CPPCP (SEQ ID NO: 58)) hinge sequence. This engineered Fc-sequence was used to produce the approved antibodies Keytruda, Mylotarg, and Tysabri, as well as a variety of current late-stage clinical candidate mAbs.

[0422] Germline and immunogenicity risk assessment

[0423] The Ab1 parent antibody and its variants are fully human IgG4 (S228P) lambda antibodies derived from phage display. The Fc portion of the antibody contains a single stabilizing mutation to prevent Fab arm exchange (as described above). This IgG4 Fc is not expected to have measurable binding to Fcγ receptors (see Example 2).

[0424] The variable framework region of Ab1 as isolated from fully human primary phage display contains five non-germline amino acids (see below and Figure 22 ). Complementarity determining regions (CDRs) are defined using Kabat nomenclature and are underlined. Non-germline residues are shown in lower case.

[0425] A. Heavy chain variable region

[0426]

[0427] B. Light chain variable region

[0428]

[0429] To mitigate the potential for immunogenicity, additional variants of the Abl molecule were generated that substituted non-germline framework residues with their corresponding germline amino acids. In some embodiments, substitutions involving Abl can be similarly applied to Ab3 and Ab4, or any antibody disclosed herein for which germlining is appropriate.

[0430] The sequence alignment of the variable region of Ab1 and its affinity-optimized variants is shown below: A.) heavy chain, B.) light chain. Complementarity determining regions (CDRs) are defined using Kabat (underlined) and IMGT nomenclature (boldface). Framework region substitutions present in the parent Ab1 are shown in lowercase.

[0431] A. Heavy chain variable region

[0432]

[0433] B. Light chain variable region

[0434]

[0435]

[0436] Three of the five substitutions were found to be far from the CDR regions and therefore had no effect on binding. The proline at position 2 of the light chain packs against CDRL3, and substitution to a germline serine actually improved binding to promyostatin / latent myostatin by stabilizing the CDR conformation.

[0437] Overall the antibodies were greater than 99% human (calculated as 100% minus % non-germline AAs, excluding CDRH3). No chemical conjugation was present. The heavy chain CDRH2 sequence contained a potential isomerization tendency (Asp-Gly) that is also present in the germline IgHV3-30 sequence.

[0438] Example 2: Pharmacological Characterization

[0439] In vitro pharmacology analysis

[0440] A total of 24 optimized Abl variants were expressed and purified as IgG4 and assayed for improved binding and functional activity. Changes to these molecules included germline mutations to the parental variable regions as well as mutations in the CDRs that conferred improved binding to promyostatin / latent myostatin in affinity maturation screens (see Example 1).

[0441] Ab1 variants were screened in several different ELISA-based assays, where binding to promyostatin and latent myostatin proteins (human, mouse, and cynomolgus monkey) was reassessed along with extensive screening of negative control proteins to verify that nonspecific binding was not introduced due to affinity maturation. Negative controls included GDF11 proteins (proGDF11, latent GDF11, and mature GDF11), TGFβ proteins, and activin proteins (proactivin). In addition, the antibodies were evaluated for multispecificity (which can result in rapid clearance) in a screen similar to that previously published (Hotzel et al., 2012). Any antibodies that had significant interactions with negative control proteins or with baculovirus particles in the multispecific screen were not further considered as candidates for the development program.

[0442] The 24 optimized Abl variants were also evaluated in a promyostatin activation assay to determine their functional potency, and the EC50 values ​​from the dose-response curves were compared to the parent Abl antibody. Most antibodies had equivalent or improved EC50 values, with a few showing reduced potency in this assay. Those with reduced potency in the activity assay were excluded from further analysis.

[0443] Three variants were identified that had improved binding to promyostatin and latent myostatin while maintaining specificity for promyostatin and latent myostatin. The binding and activity data for these three variants and the parent Abl molecule are summarized in Tables 4-7, and the sequences are shown in Example 1.

[0444] Table 4: Binding characteristics of antibodies against human / cynomolgus monkey / mouse promyostatin and parental Abl IgG4.

[0445]

[0446] Table 5: Binding characteristics of antibodies against human / cynomolgus / mouse promyostatin and Abl IgG4 with correct germline residues substituted with non-germlined residues (Ab2).

[0447]

[0448] Table 6: Binding characteristics of antibodies against human / cynomolgus / mouse promyostatin and Ab3 IgG4 (Ab4) containing corrected germline residues.

[0449]

[0450]

[0451] Table 7: Binding characteristics of antibodies against human / cynomolgus / mouse promyostatin and Ab5IgG4 (Ab6) containing corrected germline residues.

[0452]

[0453] Cell-based in vitro and in vivo bioactivity assays

[0454] In dose-response studies, Ab1 optimized variants were evaluated in GDF8 activation assays. In these experiments, 0.5 μM promyostatin was preincubated with increasing amounts of the test article. After this preincubation step, conditioned medium from HEK293 cells overexpressing mTll2 and furin was added to release the mature growth factor from promyostatin. After incubation overnight at 30°C, the substances were added to 293T cells carrying a SMAD-based luciferase reporter plasmid, and the activity of the released substances was recorded. Data from the screening are shown in Figure 4 middle.

[0455] Selectivity for myostatin relative to other TGFβ family members

[0456] The selectivity of the candidate antibodies was also evaluated by both binding and functional assays to verify the lack of cross-reactivity to other members of the TGFβ family. Human myostatin and GDF11 share 90% identity in the mature growth factor domain and 47% identity in the prodomain region. From epitope mapping studies, it was determined that the parental Ab1 molecule recognized an epitope on the prodomain of promyostatin and latent myostatin, as ELISA analysis had shown binding of this antibody to a construct consisting of the prodomain of myostatin. Although the prodomains of myostatin and GDF11 share less than 50% identity, and we did not expect significant cross-reactivity, the specificity of the lead antibody was carefully evaluated.

[0457] A sensitive assay was developed to detect the interaction between the target antibody and a negative control reagent. In this assay, biotinylated proGDF11 or biotinylated promyostatin was immobilized on a ForteBio BLI streptavidin-coated sensor tip, which was applied to a well containing 30 μg / mL of the antibody. The interaction of the analyte with the immobilized protein on the chip was measured by the reaction intensity of the biosensor chip. The biosensor response after 5 minutes of binding (saturation signal for proGDF8) was compared between the two antigens and expressed as a percentage of the response for GDF8 binding. Compared to the stable binding events measured for promyostatin, all antibodies had minimal interaction with proGDF11.

[0458] Table 8: Interaction with proGDF11 at high concentrations of candidate molecules

[0459] GDF11 response expressed as a percentage of GDF8 response Ab1 1.33％ Ab2 0.81％ Ab4 2.51％ Ab6 2.07％

[0460] Antibody candidates were also evaluated in a GDF11 activation assay. In this assay, 50 nM proGDF11 was preincubated with increasing concentrations of antibody. Following preincubation, conditioned medium from HEK293 cells overexpressing BMP-1 (a tolloid family protease) and PCSK5 (a furin family member specific for GDF11) was added to proteolytically activate proGDF11. After overnight incubation at 30°C, the reaction mixture was evaluated for GDF11 activity in a SMAD-based reporter cell line. As shown in Table 8, anti-myostatin antibodies did not inhibit proGDF11 activation, while the positive control antibody exerted dose-dependent inhibition of GDF11 activation.

[0461] The binding affinity of the antibody candidates was determined using the Forté BioOctet QKe dip-and-read label-free assay system utilizing biolayer interferometry. In each experiment, the antigen was immobilized on a biosensor (streptavidin-coated biosensors for proGDF8, proGDF11, and proactivin; direct amine coupling for all other antigens) and the antibody / construct was present in solution at a high concentration (50 μg / mL) to measure the binding interaction.

[0462] The binding affinity of the antibodies was determined using the FortéBio OctetQKe dip-and-read label-free assay system utilizing bio-layer interferometry. Human proGDF8, latent GDF8, proGDF11, and proactivin were biotinylated and immobilized on streptavidin-coated biosensors (FortéBio). Mature growth factors were immobilized on amine-reactive tips via direct amine coupling according to the manufacturer's instructions (FortéBio). In each experiment, the antibodies / constructs were present in solution at a single high concentration (50 μg / mL) to measure binding interactions. Growth factors were purchased from R&D systems, and biotinylated proteins were produced as described.

[0463] Table 9a: Comparison of antibodies for binding to different forms of several TGF[beta] family members.

[0464]

[0465] *Nonspecific binding.

[0466] The results from the antigen binding studies are summarized in Table 9a. Experiments with no detectable binding are indicated by a minus sign (-). There are some calculated Kd values ​​that are consistent with the data for poor binding responses, which are shown in the table as weak nonspecific binding (*).

[0467] Because the proGDF8 samples used in Table 9a contained approximately 10-15% latent GDF8, separate experiments were used to confirm that proGDF8 specifically binds to human and murine GDF8 antigens. Additionally, primed GDF8 (in which proGDF8 is proteolytically cleaved by both proprotein convertase and tolloid protease) was also evaluated for binding affinity to Ab2 and AbMyo. For these experiments, homologous preparations of human proGDF8 were purified from stably integrated 293 cells cultured in the presence of 30 μM decanoyl-RVKR-CMV. Primed human GDF8 was generated by in vitro cleavage of proGDF8 using conditioned medium from mTll2-overexpressing cells and purified furin. In these protein binding experiments, 150 nM of Ab2 or AbMyo was used to saturate the fixed sites on a human Fc capture tip (FortéBio), and the binding and dissociation of 150 nM analyte was assessed.

[0468] Binding affinity analysis for murine proteins was also evaluated and is reported in Table 9b. Murine proGDF8 protein was generated by negatively selecting against all mature and latent murine GDF8 from the sample using an antibody (AbMyo2) that tightly recognizes latent and mature GDF8. 50 nM of antibody was used to saturate an anti-human Fc capture tip (FortéBio). Initially, all antibodies were tested against a single 200 nM concentration of murine proGDF8, murine latent GDF8, and mature GDF8. If binding was observed, the Kd value was determined by immobilizing the antibody as previously described and titrating the analyte from 200 to 0.82 nM using a 3-fold dilution. Kd was determined using global fitting using FortéBio Data Analysis Software 8.2. For binding to mature myostatin, 5 ug / mL of growth factor (R&D systems) was coupled to an amine-reactive sensor tip (FortéBio) in acetate buffer at pH 5. All antibodies were initially tested for binding to this myostatin-coupled sensor at 333 nM. Antibodies that showed binding were then tested in a concentration range of 333-1.37 nM using 3-fold dilutions. Global fits were used to determine the Kd of the interaction using ForteBio Data Analysis 8.2.

[0469] Table 9b: Comparison of antibodies for binding to different forms of human and murine GDF8.

[0470]

[0471]

[0472] Results from the antigen binding studies are summarized in Table 9b. Experiments with no detectable binding are indicated by a minus (-) sign. Some values, labeled <1E-12, had very low off-rates, preventing quantification of high affinities. Surprisingly, AbMyo failed to recognize recombinant proGDF8, which differs from the results reported in Latres et al., 2015, in which the authors reported binding of AbMyo to proGDF8 in immunoprecipitation experiments with serum from mice dosed with the antibody, which could produce artifacts. Another surprising result was the interaction between Ab2 and stimulated GDF8 (GDF8 in complex with the tolloid-cleaved prodomain). This result was unexpected because Ab2 blocked tolloid cleavage of the prodomain and suggests that an intact tolloid cleavage site is not required for Ab2's interaction with proGDF8 and latent GDF8.

[0473] Evaluation of Fc-region functionality

[0474] In some embodiments, anti-myostatin / latent myostatin therapy involves binding to a soluble target (myostatin / latent myostatin) and preventing proteolytic activation. In some embodiments, antibody-dependent cell-mediated cytotoxicity and complement fixation are not involved in this process. Ab1 and its related variants are engineered to contain an IgG4-Fc region. It should be understood that IgG4 antibodies generally lack effector function due to their weak binding to complement component C1q and Fcγ receptors.

[0475] To demonstrate the ability to reduce effector function, Ab1 and related antibodies were tested for binding to CD64 (FcgRI) and C1q by ELISA. For comparison, an IgG1 variant of Ab1 was also prepared. In this analysis, all IgG4 antibodies showed significantly weaker binding to CD64 and C1q compared to IgG1 (10 to 20-fold). The relative binding values ​​at EC50 are listed in Table 10.

[0476] Table 10: Relative binding affinities of Ab2 and related antibodies for CD64.

[0477]

[0478] Not determined

[0479] The apparent binding affinities of Ab1 and its related variants to CD64 and C1q were similar to those of other IgG4 clinical candidate antibodies and significantly lower than those of antibodies of the IgG1 isotype. Based on the biology of IgG4 antibodies, it was concluded that anti-promyostatin / latent myostatin antibodies did not induce significant effector functions in vivo.

[0480] Efficacy in animal models

[0481] Based on in vitro characterization, four antibodies were selected for testing in an in vivo study (Ab7, Ab1, Ab8, and Ab9). The purpose of this study was to evaluate the ability of these four candidate antibodies to regulate muscle mass in mice. Five (5) groups of ten (10) female SCID mice received test article administration via intraperitoneal (IP) injection once a week on days 0, 7, 14, 21, 28, and 35. Prior to test article administration (day 0), all animals underwent a grip strength assessment. Grip strength assessment was also performed on the last day of the study (day 42). On day 0, blood was collected for evaluation of a complete blood count (CBC) by retro-ocular bleeding. Following administration, animals were assessed daily for body weight and overall health observations. On day 42, after grip strength evaluation, animals were sacrificed by CO2 overdose and blood was collected by cardiac puncture for CBC evaluation. Additional blood was collected for plasma preparation. Various tissues were separated and weighed. The muscles collected were: gastrocnemius, pectoralis, soleus, triceps, tibialis anterior, quadriceps (rectus femoris), and diaphragm. The organs collected were: heart, kidney, spleen, liver, and inguinal white adipose tissue. Except for the gastrocnemius, which was fixed in formalin (leg 1) and OCT (leg 2) for histological analysis, all tissues were weighed and snap-frozen.

[0482] Summarize

[0483] The mean daily percent weight change data for the animals in Study SCH-02 are shown in Figure 6 The animals in all five groups gained weight weekly. Figure 6 The animals shown in Figure 2 had the greatest increase in body weight (14.6%). Only the animals treated with Abl had a statistically significant increase in mean daily percent body weight change ( Figure 6 ).

[0484] The weights of the excised muscles are plotted in Figures 7 and 8. Animals treated with Abl had a gastrocnemius muscle ( Figure 7A ) and diaphragm ( Figure 8B) weights, 27.6% and 49.8%, respectively. Additional muscles from Ab1 treated animals showed increases in weight compared to the PBS control, but these differences were not statistically significant. For the mean tissue weights of heart, spleen, kidney, liver, and adipose tissue, there were no statistically significant differences between the treatment groups.

[0485] SCID dose-response studies

[0486] In the in vivo study (above), animals administered Ab1 at 25 mg / kg once a week for 6 weeks showed statistically significant increases in body weight and muscle weight (gastrocnemius and diaphragm) compared to animals given solvent (PBS). This muscle-enhancing activity of Ab1 was then studied in more detail in a dose-response study in SCID mice. In this study, it was examined whether the intensity of the effect on muscle mass could be increased by increasing the dosage of Ab1 to as high as 60 mg / kg / wk and whether the intensity of the effect on muscle mass could be reduced by reducing the dosage of Ab1 to as low as 2 mg / kg / wk. In this study, the activity of Ab1 was compared with two other antibodies (Ab8, which was originally tested in the above study, Ab10).

[0487] Ten (10) groups of ten (10) female SCID mice received test article administration via intraperitoneal (IP) injection (10 ml / kg) twice a week on days 0, 3, 7, 10, 14, 17, 21 and 24. The doses of the test articles were as follows: Abl (30 mg / kg, 10 mg / kg, 3 mg / kg and 1 mg / kg), Ab10 (10 mg / kg and 3 mg / kg) and Ab8 (10 mg / kg and 3 mg / kg). The control group was dosed with PBS and IgG-control (30 mg / kg). The treatment groups are described in Table 11. The animals were 10 weeks old at the beginning of the study. Body weight was measured on day -4 and twice a week (corresponding to the dosing days) throughout the study. Body mass composition parameters (fat mass, lean body mass and water content) were measured by Echo MRI (QNMR) on days -4, 7, 14, 21 and 28. Thirty (30) days after the first antibody dose, animals were sacrificed by CO2 overdose and blood was collected by cardiac puncture for CBC evaluation and plasma preparation. In addition, at the end of the study, various tissues were separated and weighed. The muscles collected were: gastrocnemius, soleus, tibialis anterior, quadriceps (rectus femoris) and diaphragm. Muscles were excised from both the right and left legs of the study mice. For analysis, the weights of the individual muscles from both legs were combined and the average muscle weight in grams was calculated. The other tissues collected were: heart, kidney, spleen, liver and adipose tissue. Except for the gastrocnemius muscle, which was fixed in formalin (left leg) and OCT (right leg) for histological analysis, all tissues were weighed and then snap-frozen.

[0488] Table 11: Study Design

[0489] Treatment group Test article dose Dosage # / week Total dose / week Animal ID 1 PBS control 2 0 1-10 2 IgG control (30 mg / kg) 2 60 mg / kg / week 11-20 3 Ab1 (30 mg / kg) 2 60 mg / kg / week 21-30 4 Ab1 (10 mg / kg) 2 20 mg / kg / week 31-40 5 Ab1 (3 mg / kg) 2 6mg / kg / week 41-50 6 Ab1 (1 mg / kg) 2 2mg / kg / week 51-60 7 Ab10 (10 mg / kg) 2 20 mg / kg / week 61-70 8 Ab10 (3 mg / kg) 2 6mg / kg / week 71-80 9 Ab8 (10 mg / kg) 2 20 mg / kg / week 81-90 10 Ab8 (3 mg / kg) 2 6mg / kg / week 91-100

[0490] The mean percent weight change and mean percent lean body mass change data from animals treated with vehicle (PBS), IgG control, and various doses of Abl are shown in Figure 9. Animals treated with 20 and 60 mg / kg / wk doses of Abl had significant weight gains on day 28 of the study, 15.3% and 14.4%, respectively, compared to animals treated with the IgG control. Figure 9A All four groups of animals treated with Ab1 (60, 20, 6, and 2 mg / kg / wk doses) had statistically significant increases in lean body mass at day 28 of the study compared to IgG control-treated animals, 14.1%, 12.4%, 17.1%, and 15.5%, respectively ( Figure 9B ).

[0491] The weights of four muscles (quadriceps, gastrocnemius, tibialis anterior and diaphragm) are plotted in Figure 10. The soleus muscle was also excised, but the small size of the muscle resulted in a highly variable data set. Animals treated with all doses of Ab1 had statistically significant increases in muscle weight compared to IgG control animals (Figure 10). The average percent change in muscle mass compared to the IgG control is shown above the corresponding bar on each muscle curve. The average percent weight change for the quadriceps ranged from 20.5% for the highest dose to 10.7% for the lowest dose (Figure 10). Figure 10A The mean percent weight change of the gastrocnemius ranged from 17.7% at the highest dose to 15.9% at the lowest dose ( Figure 10B The mean percentage weight change of the tibialis anterior muscle ranged from 24.0% at the highest dose to 18.0% at the lowest dose ( Figure 10C The mean percent weight change of the diaphragm was greater than 30% for all dose groups ( Figure 10D There were no statistically significant differences between treatment groups for mean tissue weights of heart, spleen, kidney, liver, and adipose tissue.

[0492] Ab1 treatment in the dexamethasone-induced muscle atrophy model

[0493] Given the known ability of the anti-myostatin antibody Abl to increase muscle mass in healthy SCID mice, it was determined whether Abl treatment could also protect animals against treatments that induce muscle atrophy. A model of corticosteroid-induced muscle atrophy was established by treating animals with dexamethasone in their drinking water for two weeks. The selected dose (2.5 mg / kg / day) was able to induce a significant decrease in lean body mass and the mass of individual hindlimb muscles. In the following experiments, animals were treated with different doses of Abl to determine whether it could protect animals from the effects of this dexamethasone-induced muscle atrophy.

[0494] In this study, eight (8) groups of ten (10) male mice (C57BL / 6) were enrolled at 13.5 weeks of age. Starting on day 0 of the study, mice were given either normal drinking water (groups 1-4) or water containing dexamethasone (groups 5-8). Test articles were administered by intraperitoneal (IP) injection (10 ml / kg) twice weekly on days 0, 3, 7, and 10. Test articles and doses were as follows: PBS (groups 1 and 5), 10 mg / kg IgG Ctl (groups 2 and 6), 10 mg / kg Ab1 (groups 3 and 7), and 1 mg / kg Ab1 (groups 4 and 8). Treatment groups are described in Table 12. Body weight was measured at least twice weekly throughout the study. Body mass composition parameters (fat mass, lean body mass, and water content) were measured by EchoMRI (QNMR) on days -1, 6, and 13. Fourteen (14) days after the first antibody dose, animals were sacrificed by CO2 overdose and blood was collected by cardiac puncture for plasma preparation. In addition, at the end of the study, various tissues were separated and weighed. The muscles collected were: gastrocnemius, soleus, tibialis anterior, quadriceps (rectus femoris) and diaphragm. Muscles were excised from both the right and left legs of the study mice. For analysis, the weights of the individual muscles from both legs were combined and the average muscle weight in grams was calculated. The other tissues collected were: heart, kidney, spleen, liver and adipose tissue. Except for the gastrocnemius muscle (which was fixed in formalin (left leg) and OCT (right leg) for histological analysis), all tissues were weighed and then snap-frozen.

[0495] Table 12: Treatment groups used in dexamethasone-induced atrophy model studies

[0496]

[0497] In this experiment, it was determined whether treatment of mice with the anti-myostatin antibody Ab1 could protect the animals from corticosteroid-induced muscle atrophy. During the study, body weight was measured twice weekly and lean body mass was measured by QNMR on days -1, 6, and 13. The mean percent weight change and mean percent lean body mass change data for animals in the non-disease control group (Group 1) and the dexamethasone-treated groups (Groups 5-8) are shown in Figure 11. There were no significant differences in mean percent weight change between any of the treatment groups on day 14 ( Figure 11A Treatment of mice with dexamethasone for two weeks resulted in a significant decrease in lean body mass (Groups 5 and 6) compared to the control group (Group 1) given normal drinking water ( Figure 11B However, mice treated with both dexamethasone and 20 mg / kg / wk of antibody Ab1 (Group 7) did not show a significant difference in percent change in lean body mass on day 14 of the study compared to the control group (Group 1). Animals treated with 20 mg / kg / wk of Ab1, but not 2 mg / kg / wk, showed a significant difference in percent change in lean body mass on day 14 when compared to either of the dexamethasone-treated control groups (Groups 5 and 6).

[0498] At the end of the two-week treatment with dexamethasone and the test article, individual muscles were excised and weighed. Weight data for both muscles (gastrocnemius and quadriceps) are available at Figures 12A-12B Graphed in . Animals that received dexamethasone through their drinking water and also received PBS or IgG control antibodies showed significant atrophy of the gastrocnemius and quadriceps compared to the non-disease control group (group 1) (groups 5 and 6). Animals (group 7) treated with both dexamethasone and 20 mg / kg / wk (rather than 2 mg / kg / wk) of Ab1 showed significant differences in muscle weight when compared to either of the dexamethasone-treated control groups (groups 5 and 6). In addition, mice (group 7) treated with both dexamethasone and 20 mg / kg / wk of antibody Ab1 did not show significant differences in gastrocnemius and quadriceps weight when compared to the non-disease control group (group 1). The average percentage difference in muscle weight of each group compared to the average muscle weight of the control group (group 1, PBS and water) is shown in Figures 12C-12D The percentage reductions in gastrocnemius muscle mass induced by dexamethasone treatment in the PBS and IgG Ctl groups were 16.5% and 18.9%, respectively. In contrast, animals treated with both dexamethasone and 20 mg / kg / wk of Ab1 had only a 4.0% reduction in gastrocnemius muscle mass, which was not significantly different from the non-disease control group (Group 1). Although animals treated with both dexamethasone and 2 mg / kg / wk of Ab1 (Group 8) had only a 10% reduction in gastrocnemius muscle mass, the reduction in muscle mass in this group was not significantly different from the reduction in the PBS and IgG control groups (Groups 5 and 6). Similar results were observed for the quadriceps ( Figure 12D ).

[0499] Ab1 treatment in the plaster-induced muscle atrophy model

[0500] Given the ability of the anti-myostatin antibody Ab1 to increase muscle mass in healthy SCID mice, we investigated whether Ab1 treatment could also protect animals against treatments that induce muscle atrophy. A model of disuse-induced atrophy was established by placing mice in a cast on their right leg for two weeks. Casting the right leg with the foot in a plantar flexed position for this period of time induced a significant decrease in muscle mass in a single hindlimb. In the following experiments, animals were treated with various doses of Ab1 to determine the extent to which it protected animals from this cast-induced muscle atrophy.

[0501] Table 13: Treatment groups used in the plaster-induced atrophy model study

[0502]

[0503] In this study, eight (8) groups of ten (10) male mice (C57BL / 6) were enrolled at 14.5 weeks of age. Starting on day 0 of the study, mice were placed under anesthesia and a cast was applied to the right hind limb with the foot in a plantar flexion position (Groups 5-8). The control group (Groups 1-4) was also placed under anesthesia but the cast was not placed on the hind limb. The test article was administered by intraperitoneal (IP) injection (10 ml / kg) twice a week on days 0, 3, 7, and 10. The test article and dose were as follows: PBS (Groups 1 and 5), 10 mg / kg IgGCtl (Groups 2 and 6), 10 mg / kg Abl (Groups 3 and 7), and 1 mg / kg Abl (Groups 4 and 8). The treatment groups are described in Table 13. Body weight was measured at least twice a week throughout the study. Body mass composition parameters (fat mass, lean body mass, and water content) were measured by EchoMRI (QNMR) on days -1, 7, and 14. Fourteen (14) days after the first antibody dose, animals were sacrificed by CO2 overdose and blood was collected by cardiac puncture for plasma preparation.

[0504] In addition, various tissues were separated and weighed. The muscles collected were: gastrocnemius, soleus, plantaris, tibialis anterior, and quadriceps (rectus femoris). For analysis, the weight of individual muscles from the right hind limb of the animal was collected. Other tissues collected were: heart, adipose tissue, and spleen. Except for the gastrocnemius muscle (which was fixed in formalin for histological analysis), all tissues were weighed and then snap-frozen.

[0505] Summarize

[0506] In this experiment, it was tested whether treatment of mice with the anti-myostatin antibody Ab1 could protect them from disuse muscle atrophy induced by right hind limb casting. During the study, body weight was measured twice a week and lean body mass was measured by QNMR on days -1, 7, and 14. The mean percent weight change and mean percent lean body mass change data for animals in the non-disease control group (Group 1) and the groups that were cast for two weeks (Groups 5-8) are shown in Figure 13. Right hind limb casting did not have any negative effect on weight gain ( Figure 13A ) and any differences in lean body mass between groups were not significant ( Figure 13B ).

[0507] At the end of the two-week study, individual muscles were excised and weighed. Weight data for both muscles (gastrocnemius and quadriceps) were obtained in Figures 14A-14B Graphed in . Animals with casts on their legs that also received PBS or IgG control antibody showed significant atrophy of the gastrocnemius and quadriceps muscles (Groups 5 and 6) compared to the uncast control group (Group 1). Animals that were cast and also administered Ab1 at 20 mg / kg / wk instead of 2 mg / kg / wk (Group 7) showed significant differences in muscle weight when compared to either of the cast control groups (Groups 5 and 6). In addition, cast-treated mice (Group 7) with 20 mg / kg / wk of antibody Ab1 did not show significant differences in gastrocnemius and quadriceps weight when compared to the uncast control group (Group 1). The average percentage difference in muscle weight for each group compared to the average muscle weight for the uncast control group (Group 1) is shown in Figures 14C-14D The percentage reduction in gastrocnemius muscle mass induced by casting was 22.8% and 23.5% in the PBS and IgG Ctl groups, respectively. In contrast, the cast mice treated with 20 mg / kg / wk of Ab1 had only a 10.0% reduction in gastrocnemius muscle mass. This difference was found to be statistically different from the cast control groups (Groups 5 and 6) that received PBS and IgG Ctl antibodies. The reduction in muscle mass in the cast mice treated with 2 mg / kg / wk Ab1 was not statistically different from the reduction in the PBS and IgG control groups (Groups 5 and 6). Similar results were observed for the quadriceps ( Figure 14D ).

[0508] The domain structures of promyostatin and latent myostatin (with protease cleavage sites indicated) are shown in Figure 16A An example of a portion of proprotein convertase-cleaved promyostatin run on an SDS PAGE gel is shown in Figure 16B Under reducing conditions, the protein band consists of promyostatin monomer (~50kD), prodomain (~37kD) and growth factor (12.5kD).

[0509] Ab1 specifically bound to promyostatin and latent myostatin, whereas no binding was observed to other members of the TGFβ superfamily, most notably the corresponding form of GDF11 ( Figure 17A Ab1 was applied at high concentration (50 ug / mL) to Forte-Bio BLI tips coated with the indicated antigens and the association and dissociation rates were measured to obtain approximate Kd values. The intensity of the biosensor response (indicating binding events) is graphically represented by the black bar, and the calculated Kd is indicated in orange. Furthermore, Ab1 blocked the activation of promyostatin, but not proGDF11 ( Figure 17B ).

[0510] SCID dose-response studies of Ab1, Ab2, Ab4, and Ab6

[0511] Previous in vivo studies of Ab1 have demonstrated that Ab1 can increase muscle mass in healthy animals and prevent muscle loss in a mouse model of muscle atrophy (dexamethasone and cast-induced atrophy). Antibody engineering work identified three antibodies with superior in vitro characteristics to Ab1. In this study, the in vivo activity of these antibodies was compared with the previously established activity of Ab1 at three different doses in SCID mice.

[0512] Fourteen (14) groups of eight (8) female SCID mice received test article administration twice weekly by intraperitoneal (IP) injection (10 ml / kg) on ​​days 0, 3, 7, 10, 14, 17, 21 and 24. The test article dosages were as follows: Abl, Ab2, Ab4 and Ab6 were administered at 3 different doses (10 mg / kg, 1 mg / kg and 0.25 mg / kg) and IgG-Ctl antibody was administered at 10 mg / kg. The treatment groups are described in Table 14. The animals were 10 weeks old at the start of the study. Body weight was measured twice weekly (corresponding to the dosing day) throughout the study. Body mass composition parameters (fat mass, lean body mass and water content) were measured by Echo MRI (QNMR) on days 0, 7, 14, 21 and 28. Twenty-eight (28) days after the first antibody dose, the animals were sacrificed by CO2 overdose and blood was collected by cardiac puncture for plasma preparation.

[0513] In addition, various tissues were separated and weighed. The muscles collected were: gastrocnemius, soleus, tibialis anterior, quadriceps (rectus femoris), extensor digitorum longus and diaphragm. Muscles were excised from both the right and left legs of the study mice. For analysis, the weights of the individual muscles from both legs were combined and the average muscle weight in grams was calculated. The other tissues collected were: heart, kidney, spleen, liver and adipose tissue. Except for the left gastrocnemius (which was fixed in formalin for histological analysis), all tissues were weighed and then snap-frozen.

[0514] Table 14: Treatment groups used in dose-response model studies

[0515] Treatment group Test article dose Dosage # / week Total dose / week Animal ID 1 PBS control 2 0 1-8 2 IgG control (10 mg / kg) 2 20 mg / kg / week 9-16 3 Ab1 (10 mg / kg) 2 20 mg / kg / week 17-24 4 Ab1 (1 mg / kg) 2 2mg / kg / week 25-32 5 Ab1 (0.25 mg / kg) 2 0.5mg / kg / week 33-40 6 Ab2 (10 mg / kg) 2 20 mg / kg / week 41-48 7 Ab2 (1 mg / kg) 2 2mg / kg / week 49-56 8 Ab2 (0.25 mg / kg) 2 0.5mg / kg / week 57-64 9 Ab4 (10 mg / kg) 2 20 mg / kg / week 65-72 10 Ab4 (1 mg / kg) 2 2mg / kg / week 73-80 11 Ab4 (0.25 mg / kg) 2 0.5mg / kg / week 81-88 12 Ab6 (10 mg / kg) 2 20 mg / kg / week 89-96 13 Ab6 (1 mg / kg) 2 2mg / kg / week 97-104 14 Ab6 (0.25 mg / kg) 2 0.5mg / kg / week 105-112

[0516] The mean percent lean body mass change (relative to day 0) of animals treated with vehicle (PBS), IgG control, and various doses of Ab1, Ab2, Ab4, and Ab6 is shown in Figure 2. Figure 15 Animals treated with the 20 mg / kg / wk dose level of Abl, Ab2, Ab4, and Ab6 had a significant increase in lean body mass on days 21 and 28 compared to the IgG control and vehicle (PBS) treated animals. Animals treated with the 2 mg / kg / wk dose level of Abl and Ab2 also had significant changes in lean body mass on days 21 and 28 of the study. For animals treated with the 0.5 mg / kg / wk dose level of Abl, Ab2, Ab4, and Ab6, there were no significant changes in lean body mass relative to the control group.

[0517] At the end of the study (day 28), muscles were collected and weighed. The weights of the quadriceps (rectus femoris) and gastrocnemius muscles were Figure 18A and 18B Graphed in. Animals treated with 20 mg / kg / wk dose levels of Abl, Ab2, Ab4 and Ab6 had a significant increase in gastrocnemius and quadriceps (rectus femoris) muscle weight compared to animals treated with solvent (PBS). Animals treated with 2 mg / kg / wk dose levels of Ab2 and Ab4 also had significant changes in gastrocnemius muscle weight. Animals treated with 2 mg / kg / wk dose levels of Ab2 also had significant changes in quadriceps (rectus femoris) muscle weight. For animals treated with 0.5 mg / kg / wk dose levels of Abl, Ab2, Ab4 and Ab6, there was no significant change in muscle mass relative to the control group. The percentage difference (when compared to the solvent control) in gastrocnemius and quadriceps (rectus femoris) muscle weight for animals treated with different doses of Abl, Ab2, Ab4 and Ab6 is shown in FIG. Figure 18C middle.

[0518] Duration of the Effect Study of Ab1 in SCID Mice

[0519] The ability of Ab1 to increase lean body mass after a single dose and after 3 weekly doses was tested in SCID mice. Seven (7) groups of eight (8) female SCID mice received test article administration by intraperitoneal (IP) injection (10 ml / kg) at day 0 (groups 1-4) or once a week (groups 5-7) on days 0, 7 and 14. See Table 15. Antibodies (IgG control, Ab1 and AbMyo) were administered at 10 mg / kg. Animals were 10 or 11 weeks old at the start of the study. Body weight was measured twice a week throughout the study (corresponding to the dosing day). Body mass composition parameters (fat mass, lean body mass and water content) were measured by Echo MRI (QNMR) on days 0, 7, 14 and 21.

[0520] Table 15: Treatment groups and dosing frequency.

[0521]

[0522]

[0523] The mean percent lean body mass change data for animals treated with vehicle (PBS), IgG control, Ab1, and AbMyo are shown in Figure 2. Figure 19 Data are presented as changes in lean body mass relative to day 0 of the study. At 21 days after a single dose of the test article, animals treated with Abl (Group 3) had a significant increase in lean body mass (compared to IgG control animals - Group 1), which was indistinguishable from the changes in lean body mass after 3 doses of Abl (Group 6). These changes in lean body mass were also comparable to those seen in animals treated with a single dose of AbMyo (Group 4) or animals treated with 3 weekly doses of AbMyo (Group 7).

[0524] Example 3: Chemistry / Pharmacology

[0525] Ab2 is a humanized monoclonal antibody of the IgG4 subtype with a proline replacing a serine at position 228. This results in an IgG1-like hinge sequence and minimizes incomplete formation of interchain disulfide bonds (a characteristic of IgG4). The complete amino acid sequences of the Ab2 heavy and light chains are shown below. The complementary determining regions (CDRs) are underlined. The NST sequence in bold: N-linked glycosylation consensus sequence site; the DP sequence in bold is a potential cleavage site; the NX sequence in bold, where X can be S, T or G, is a potential deamidation site; the DX sequence in bold, where X can be G, S, T or SDG, is a potential isomerization site; the methionine in bold is a potential methionine oxidation site; the Q in bold is the expected N-terminal pyroglutamate ( Figures 21A-21B ).

[0526] Molecular modeling of Ab1 identified several potential sites for post-translational modification. Two asparagines in the light chain and seven in the heavy chain are susceptible to deamidation. Two of these residues are located within the CDR region of the heavy chain.

[0527] Original IgG4 mAbs may have incomplete formation of inter-heavy chain disulfide bonds, in which two half molecules (each comprising one heavy chain and one light chain) are held in the intact antibody structure by non-covalent interactions. IgG4 molecules may be susceptible to exchange of half molecules in vitro and in vivo, and the level of half molecules must be consistent between manufacturing batches. The substitution of Ser to Pro in the backbone of the IgG4 structure results in an IgG1-like hinge sequence, thereby enabling the formation of inter-chain disulfide bonds and significantly stabilizing the antibody structure. The integrity and stability of the hinge region are monitored during development for extended characterization using analyses such as non-reducing capillary electrophoresis and quantification of free sulfhydryl groups. The potential for chain exchange can be monitored in vivo.

[0528] Summarize

[0529] Provided herein are promyostatin / latent myostatin-specific antibodies that block the activation of promyostatin and / or latent myostatin. Administration of this activation-blocking antibody to healthy mice increased lean body mass and muscle size, with only a single dose required to maintain muscle enhancement over a one-month period. Additionally, antibody administration protected healthy mice from muscle atrophy in two independent models of muscle atrophy. The data demonstrate that blocking myostatin activation promotes robust muscle growth and prevents muscle atrophy in vivo and represents an alternative mechanism for therapeutic intervention in muscle atrophy.

[0530] Example 4: Analysis of pro-myostatin and latent-myostatin in muscle atrophy

[0531] Western blot was performed to determine the presence of promyostatin and latent myostatin in muscle tissue and in the circulation during muscle atrophy and under normal conditions. A standard model of muscle atrophy involves treating mice with 2.5 mg / kg / week dexamethasone (administered in the drinking water) and collecting muscle and plasma after 2 weeks of treatment. This model often results in a 15-25% decrease in muscle mass during treatment. Control muscle and plasma were also collected from mice that were not treated with dexamethasone. The rectus femoris, tibialis anterior, and soleus muscles were excised, snap-frozen in liquid nitrogen, and stored at -80°C until needed. Muscle lysates were produced by grinding followed by dissolution in T-PER buffer supplemented with protease and phosphatase inhibitors. Plasma was collected by standard methods and stored at -80°C.

[0532] Multiple samples containing the same concentration of protein were separated by PAGE gel and Western blotting onto PVDF membranes. For muscle lysates, 10-50 ng of total protein were loaded onto the gel. Plasma was diluted 1:10 in PBS and 10 μl of each sample was loaded onto the gel. As a size standard, 0.1-1 ng of recombinant promyostatin and / or latent myostatin was also loaded onto the gel. Identification of myostatin protein was accomplished using an antibody that recognizes the prodomain of myostatin (AF1539, R&D Systems). This analysis showed that promyostatin is the predominant form in muscle, while latent myostatin is the major form in plasma ( Figure 25 ). Furthermore, it was demonstrated that in mice with dexamethasone-induced muscle atrophy, promyostatin was increased in muscle tissue, whereas latent myostatin was decreased in plasma.

[0533] To confirm these results, Western blots were performed using fluorescent labeling and detection in duplicate (Azure Biosystems). The relative levels of each myostatin form in plasma and in the rectus femoris and tibialis anterior muscles from normal and dexamethasone-treated mice were quantified. These data confirmed the results described above, showing a 2- to 2.5-fold increase in promyostatin in both muscles and a 2.3-fold decrease in latent myostatin in plasma ( Figure 26 ).

[0534] Based on these data, a model for myostatin "flux" in normal and diseased muscle was developed. As demonstrated, in normal muscle ( Figure 28A ), promyostatin is produced in muscle and converted to latent myostatin by cleavage by furin, which may be present intracellularly or extracellularly (Anderson et al., 2008). Latent myostatin in some fraction of the muscle is then released into the circulation, thereby forming a circulating pool of latent myostatin. In muscle atrophy, an increase in the production of active myostatin growth factor drives muscle atrophy. This increase is thought to be caused by upregulation of promyostatin levels in muscle and an increase in the conversion of latent myostatin to active growth factor ( Figure 28B The data outlined here directly support the first step of this model, demonstrating increased promyostatin in muscle. The data also support the second step, as decreased muscle mass was observed in dexamethasone-treated mice, suggesting increased production of mature myostatin without a concomitant increase in latent myostatin in muscle. Thus, decreased plasma myostatin levels suggest increased conversion to mature myostatin.

[0535] Example 5: Immunoprecipitation from mouse serum and muscle tissue

[0536] Immunoprecipitation was performed to determine the presence of circulating promyostatin and to investigate the binding of Ab2 and AbMyo to endogenous promyostatin in serum and muscle. Ab2 recognizes the major form of myostatin in muscle. Figure 27 The results shown in Figure 2 demonstrate that a pool of serum promyostatin is precipitated with Ab2, indicating the presence of extracellular promyostatin in vivo. Ab2 immunoprecipitated promyostatin from muscle extracts, in addition to binding to serum promyostatin, latent myostatin, and other partially processed forms of myostatin. In contrast, AbMyo efficiently bound to latent myostatin and partially processed precursors in serum without detectable interaction with promyostatin in muscle. This may provide a significant advantage in Ab2's mechanism of action, given that muscle is the site where myostatin signaling occurs.

[0537] Homogenized muscle lysates were prepared as follows: Frozen mouse quadriceps were ground using a CryoPrep grinder (Covaris, Woburn MA). The ground muscle was then lysed with 1x Halt TM The muscle samples were then incubated at 4°C for 30 minutes with end-to-end rotation. Finally, the samples were centrifuged at 16,100 g for 10 minutes to precipitate the insoluble fraction. The soluble fraction was aspirated and used in downstream experiments.

[0538] For immunoprecipitation, Ab2, IgG Ctl, or AbMyo antibodies were used using ThermoScientific Pierce TMCo-Immunoprecipitation test kit is covalently coupled to agarose beads according to the manufacturer's instructions. Each antibody of 75ug is coupled to the bead slurry of 50uL, and 30ug of antibody is used in each immunoprecipitation. Immunoprecipitation is carried out for 3mL of combined normal mouse serum (Bioreclamation) or 1.05mL of the homogenized soluble mouse quadriceps prepared as described above. The beads of antibody coupling and sample are incubated overnight with end-to-end rotation at 4°C. After incubation, beads are recovered using QIAvac 24Plus vacuum manifold (Qiagen) by making the entire sample volume pass through the spin filter included in the co-immunoprecipitation test kit. The beads are then washed 3x with 200uL of IP lysis / wash buffer and washed once with 100μL of 1x condition buffer according to the instructions of the test kit. Elution is carried out for five minutes with 50μL of elution buffer and then mixed with 5μL of 1M Tris, pH 9.5 in a collection tube.

[0539] Myostatin species pulled down by test antibodies using AF1539, (R&Dsystems) ab124721, (Abcam) Alexa 680AffiniPure donkey anti-sheep IgG (H+L), (Jackson ImmunoResearch) and Visualization by Western blotting was performed using 800CW donkey anti-rabbit IgG (H+L) (LI-COR Biosciences, Thermo Scientific). SEA BLOCK blocking buffer was used for blocking and primary antibody incubation.

[0540] Example 6: Increased muscle mass and altered myostatin protein expression in rats treated with Ab2

[0541] Study Design

[0542] Seven to eight week old female Sprague-Dawley rats were administered a single intravenous dose of Ab2 (10 mg / kg), a non-functional human IgG control antibody (10 mg / kg) or an equivalent volume of phosphate buffered saline (PBS). During the study, serum was collected from 3 rats / group at 4 hours, 48 ​​hours, 7 days, 14 days, 21 days and 28 days after administration. Collection was performed by standard methods and samples were stored at -80°C. Lean body mass was measured by quantitative nuclear magnetic resonance (qNMR) at baseline (before administration on day 0) and on days 7, 14, 21 and 28 (8 rats / group) and skeletal muscle (rectus femoris, tibialis anterior and soleus) was collected, weighed and snap-frozen in liquid nitrogen for storage at -80°C at the end of the study (day 28).

[0543] result

[0544] Drug exposure was measured in serum samples using an ELISA specific for human IgG with known amounts of each drug used as a reference standard. Figure 29 As shown in Figure 2, Ab2 and an IgG control antibody were detected in rat serum 4 hours after injection. As the study progressed, Ab2 demonstrated elevated circulating drug levels compared to the IgG control, with an average of 17.1 μg / ml of drug in the serum at the end of the study.

[0545] The pharmacodynamic effects of Ab2 treatment were evaluated by measuring lean body mass (by qNMR) during the study and by determining the weight of excised muscle at the end of the study. Figure 30A Lean body mass measurements are shown over the course of the study, with rats treated with Ab2 showing a significant increase in lean body mass compared to rats treated with PBS or with a human IgG control antibody. Muscle mass was measured by harvesting and weighing whole skeletal muscle at the end of the study (28 days), as Figure 30B As shown in , rats treated with Ab2 showed a 14% and 11% increase in rectus femoris and tibialis anterior muscle mass, respectively. Overall, these data indicate that treatment of rats with a single dose of Ab2 results in a sustained increase in muscle mass.

[0546] The relative levels of promyostatin and latent myostatin were determined by quantitative Western blotting of muscle lysates or serum samples. Muscle lysates were generated from snap-frozen muscle samples by grinding followed by solubilization in T-PER buffer supplemented with protease and phosphatase inhibitors. After solubilization, samples containing equal concentrations of protein were separated by PAGE gel and Western blotted onto low-fluorescence PVDF membranes. For muscle lysates, 10-50 ng of total protein were loaded onto the gel. Plasma was diluted 1:10 in PBS and 10 μl of each sample was loaded onto the gel. As a size standard, 0.1-1 ng of recombinant promyostatin and / or latent myostatin were also loaded onto the gel. Identification of myostatin protein was accomplished using an antibody that recognizes the prodomain of latent myostatin (AF1539, R&D Systems), followed by detection with a fluorescently labeled secondary antibody. For all Western blot analyses, a minimum of three samples / group were assayed.

[0547] Ab2 treatment increased latent myostatin levels in rat serum by ∼20-fold compared to IgG control-treated rats ( Figure 31A). These data are consistent with the effects observed for other antibody drugs, and thus reflect binding of the drug target in the circulation. In rat muscle (rectus femoris), Ab2 treatment resulted in a 1.9x increase in the latent form of myostatin (vs. IgG control treated rats). No statistically significant changes in promyostatin were observed. These data indicate that Ab2 binds to its target, promyostatin / latent myostatin, and alters myostatin processing in muscle as well as in the circulation. It was also observed that Ab2 treatment increased latent promyostatin in rat muscle, but did not increase promyostatin ( Figure 31B ).

[0548] Example 7: Increased muscle mass and changes in myostatin protein expression in mice treated with Ab2 and comparison with a comparative anti-myostatin antibody.

[0549] Study Design

[0550] Ten-week-old male SCID mice were administered a single intraperitoneal dose (5 mg / kg) of Ab2, a non-functional human IgG control antibody, or a comparative antibody (AbMyo) that blocks myostatin / receptor interactions. During the study, serum and skeletal muscle were collected at 1 hour, 4 hours, 48 ​​hours, 7 days, 14 days, 21 days, 28 days, and 56 days after dosing. Serum collection was performed by standard methods and samples were stored at -80°C. Skeletal muscle (rectus femoris, tibialis anterior, and soleus) was collected, weighed, and snap-frozen in liquid nitrogen for storage at -80°C. Lean body mass was measured by quantitative nuclear magnetic resonance (qNMR) at baseline (before dosing on Day 0) and weekly throughout the study.

[0551] result

[0552] The pharmacodynamic effects of Ab2 treatment were evaluated by measuring lean body mass (by qNMR) during the study. Figure 32 Lean body mass measurements during the study are shown, with mice treated with Ab2 showing a significant increase in lean body mass compared to mice treated with a human IgG control antibody. For the first three weeks of the study, mice treated with the comparison antibody (AbMyo) showed an increase in lean body mass equivalent to that of the Ab2 group. However, by 28 days post-dose, the AbMyo-treated mice did not maintain their increased lean body mass. In contrast, the mice in the Ab2-treated group maintained their increased lean body mass throughout the duration of the study (56 days). These data indicate that Ab2 has a longer duration of action than AbMyo.

[0553] Drug exposure was measured in serum samples using an ELISA specific for human IgG with known amounts of each drug used as a reference standard. Figure 33As shown in Figure 2, both Ab2 and the comparator antibody (AbMyo) were detected in serum as early as 1 hour after injection, and levels of both antibodies >1 μg / ml were detectable throughout the study. However, Ab2 exhibited a significantly longer half-life and extrapolated area under the curve (AUCINF) compared to AbMyo, indicating that at similar doses, Ab2 exhibited significantly greater exposure compared to AbMyo.

[0554] The relative levels of promyostatin and latent myostatin were determined by quantitative Western blotting of muscle lysates or serum samples. Muscle lysates were generated from snap-frozen muscle samples by grinding followed by solubilization in T-PER buffer supplemented with protease and phosphatase inhibitors. After solubilization, samples containing equal concentrations of protein were separated by PAGE gel and Western blotted onto low-fluorescence PVDF membranes. For muscle lysates, 10-50 ng of total protein were loaded onto the gel. Plasma was diluted 1:10 in PBS and 10 μl of each sample was loaded onto the gel. As a size standard, 0.1-1 ng of recombinant promyostatin and / or latent myostatin were also loaded onto the gel. Identification of myostatin protein was accomplished using an antibody that recognizes the prodomain of latent myostatin (AF1539, R&D Systems), followed by detection with a fluorescently labeled secondary antibody. For all Western blot analyses, a minimum of three samples / group were assayed.

[0555] Serum myostatin was measured in drug-treated mice and controls using fluorescent Western blotting. Despite increased Ab2 serum exposure, serum latent myostatin levels were similar in both Ab2- and AbMyo-treated mice ( Figure 34 These data suggest that circulating levels of free drug (not bound to target) are sufficiently above those of target that increased serum exposure of Ab2 does not translate into an increase in circulating latent myostatin greater than that observed for AbMyo.

[0556] Myostatin levels in muscle (rectus femoris) were also assessed by fluorescent Western blotting. The relative levels of latent myostatin and promyostatin were measured in mouse muscle lysates by fluorescent Western blotting. Latent myostatin was elevated in both Ab2- and AbMyo-treated muscles ( Figure 35A However, the elevation of latent myostatin in AbMyo-treated muscles returned to baseline by day 28, whereas the elevation of latent myostatin in Ab2-treated muscles remained elevated until at least this time (P < 0.003 vs. AbMyo-treated). Similar trends were observed for promyostatin ( Figure 35B), although the difference was not statistically significant (P=0.068). These data suggest that Ab2 has a longer duration of action at the site of drug action (skeletal muscle).

[0557] Example 8: Ab2 increases muscle force production.

[0558] In this example, the effect of Ab2 on muscle force production was evaluated. Briefly, male C57BL / 6J mice were administered IgG (20 mg / kg), Ab2 with a constant region of the mouse IgG1 isotype (20 mg / kg), or PBS intraperitoneally once weekly for 4 weeks (n=10 / group).

[0559] At the end of the study, the muscles were excised and weighed, and in vitro muscle performance of the extensor digitorum longus (EDL) muscle was measured in vitro using a 305C muscle lever system (Aurora Scientific Inc., Aurora, CAN) adapted for a transverse perfusion bath. The muscles were placed in ice-cold physiological buffer solution and silk sutures were tied to the proximal tendon. The muscles were placed in the transverse bath of the 305C muscle lever system and perfused with physiological buffer oxygenated with 95% O2 / 5% CO2 and maintained at 37°C.

[0560] The suture was tied to a fixed post on one side, and the lever arm was tied to the other side. A series of 1 Hz and 100 Hz field stimulation (0.2 ms pulse, 100 ms duration) at a frequency of 0.01 Hz was delivered through platinum electrodes on the flanks of the muscle to ensure that the suture was taut and the maximum force was stable. Once stable, direct muscle stimulation - force vs. frequency was measured. Platinum wire electrodes were placed proximally and distally at the muscle belly.

[0561] Twitch tension was monitored with 1 ms pulses and voltage was increased until maximal force was achieved. A series of stimulations (1 ms pulses, 250 ms training duration) was then performed at increasing stimulation frequencies: 1, 10, 20, 40, 60, 80, 100, 150 Hz, followed by a final stimulation of 1 Hz.

[0562] like Figure 36A As shown in , muscle mass and function were improved after 4 weeks of treatment with Ab 2. The average EDL weight increased by 33%, and the average gastrocnemius and quadriceps weights increased by 19%.

[0563] like Figure 36B As described in , maximal force production increased by 30% after 4 weekly doses of Ab2.

[0564] Although several embodiments of the present disclosure have been described and illustrated herein, it is readily apparent to those skilled in the art that a variety of other ways and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein may be envisioned, and such variations and / or improvements are each considered to be within the scope of the present disclosure. More generally, it is readily apparent to those skilled in the art that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that actual parameters, dimensions, materials, and / or configurations will depend on the specific application or applications to which the teachings of the present disclosure are applied. Those skilled in the art will recognize or be able to determine, using only routine experimentation, many equivalents to the specific embodiments of the present disclosure described herein. Therefore, it should be understood that the foregoing embodiments are given by way of example only, and within the scope of the appended claims and their equivalents, the present disclosure may be implemented in ways other than those specifically described and claimed. The present disclosure relates to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods is included within the scope of the present disclosure if such features, systems, articles, materials, and / or methods are not mutually inconsistent.

[0565] The indefinite articles "a" and "an" as used in this specification and claims, unless expressly indicated otherwise, should be understood to mean "at least one".

[0566] As used herein, the phrase "and / or" in the specification and claims should be understood to mean "either or both" of the elements so connected (i.e., elements presented conjunctively in some cases and separately in other cases). Unless expressly indicated to the contrary, additional elements other than those specifically designated by the "and / or" clause may optionally be present, whether related or unrelated to those specifically designated elements. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with an open-ended term such as "comprising," may, in one embodiment, refer to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); and so on.

[0567] As used herein, in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one of a plurality of or a series of elements, but also including more than one, and optionally including additional unlisted elements. Only explicitly indicating the opposite terms such as "only one of" or "exactly one of" or, when used in the claims, "consisting of" means including exactly one element of a plurality of or a series of elements. Generally, as used herein, the term "or" when preceded by an exclusive term such as "either," "one of," "only one of," or "exactly one of" should only be interpreted as indicating an exclusive optional manner (i.e., "one or the other but not both"). "Substantially consisting of" when used in the claims should have the ordinary meaning as used in the field of patent law.

[0568] As used herein, in the specification and claims, the phrase "at least one" with respect to a series of one or more elements should be understood to mean at least one element selected from any one or more elements in a list of elements, but does not necessarily include at least one of each and every element specifically listed in the list of elements and does not exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than the specifically named elements in the list of elements to which the phrase "at least one" refers, whether related or unrelated to those specifically named elements. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B," or equivalently, "at least one of A and / or B"), in one embodiment, may refer to at least one A, optionally including more than one A, without B (and optionally including elements other than B); in another embodiment, may refer to at least one B, optionally including more than one B, without A (and optionally including elements other than A); in yet another embodiment, may refer to at least one A, optionally including more than one A, and at least one B, optionally including more than one B, (and optionally including other elements); and so on.

[0569] In the claims and in the foregoing description, all conjunctions such as "comprising," "including," "with," "having," "containing," "involving," "accommodating," and the like are to be understood as open-ended, i.e., meaning including but not limited to. Only the conjunctions "consisting of" and "consisting essentially of" shall be closed or semi-closed conjunctions, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0570] The use of ordinal terms such as "first," "second," "third," etc. in the claims to modify claim elements does not in itself imply any priority, precedence, or order of one claim element relative to another element or the temporal order of the acts of performing the method, but serves merely as a marker to distinguish one claim element having a particular name from another element having the same name (for the use of ordinal terms only) to distinguish the claim elements.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds promyostatin / latent myostatin but not mature myostatin, wherein the antibody comprises a heavy chain variable region that is at least 98% identical to the amino acid sequence of SEQ ID NO: 25 and a light chain variable region that is at least 98% identical to the amino acid sequence of SEQ ID NO: 31, and wherein the antibody comprises 6 complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, wherein: a) CDRH1 consists of the amino acid sequence of SEQ ID NO: 1, CDRH2 consists of the amino acid sequence of SEQ ID NO:4, CDRH3 consists of the amino acid sequence of SEQ ID NO: 10, CDRL1 consists of the amino acid sequence of SEQ ID NO: 12, CDRL2 consists of the amino acid sequence of SEQ ID NO: 18, and CDRL3 consists of the amino acid sequence of SEQ ID NO:22, wherein the sequences of the CDRs are numbered according to the Kabat numbering system; or b) CDRH1 consists of the amino acid sequence of SEQ ID NO: 2, CDRH2 consists of the amino acid sequence of SEQ ID NO:5, CDRH3 consists of the amino acid sequence of SEQ ID NO: 10, CDRL1 consists of the amino acid sequence of SEQ ID NO: 13, CDRL2 consists of the amino acid sequence of SEQ ID NO: 19, and CDRL3 consists of the amino acid sequence of SEQ ID NO: 22, wherein the sequences of the CDRs are numbered according to the IMGT numbering system.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a fully human antibody or a humanized antibody.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is engineered to bind FcRn with higher affinity than a non-engineered corresponding antibody.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof has a reduced affinity for promyostatin / latent myostatin at acidic pH compared to physiological pH.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the acidic pH range is 4.0-6.5, and the physiological pH range is 7.0-7.

4.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 50, wherein the N-terminal Q is a pyroglutamic acid residue, and a light chain comprising the amino acid sequence of SEQ ID NO: 51, wherein the N-terminal Q is a pyroglutamic acid residue.

7. A method for preparing an antibody or an antigen-binding fragment thereof that specifically binds to promyostatin / latent myostatin but not to mature myostatin, comprising: (i) culturing a host cell comprising one or more polynucleotides encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; and (ii) purifying the antibody from the host cell.

8. Use of the antibody or antigen-binding fragment thereof according to claim 1 in the preparation of a medicament for treating myopathy.

9. The use according to claim 8, wherein the myopathy is spinal muscular atrophy (SMA).

10. The use according to claim 8, wherein the myopathy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, facioscapulohumeral muscular dystrophy (FSH), or disuse atrophy caused by bone fracture.

11. Use of the antibody or antigen-binding fragment thereof according to claim 1 in the preparation of a medicament for treating osteogenesis imperfecta.

12. Use of the antibody or antigen-binding fragment thereof according to claim 1 in the preparation of a medicament for treating amyotrophic lateral sclerosis (ALS).

13. The use according to any one of claims 8 to 12, wherein the medicament is formulated for subcutaneous administration of the antibody or antigen-binding fragment thereof.

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