Anti-pro / latent myostatin antibodies and uses thereof

Abstract

To provide antibodies that specifically bind to proMyostatin and / or latent Myostatin and uses thereof.SOLUTION: Antibodies herein can bind to Myostatin, preventing cleavage of Myostatin by a proprotein convertase and / or a tolloid protease. Preventing cleavage of proMyostatin or latent Myostatin can prevent Myostatin activation. The antibodies have an affinity to an antigen that can be sensitive to pH. Such pH sensitive antibodies are effective for clearing antigens from serum. The antibodies can be sweeping antibodies able to efficiently clear antigens (e.g., proMyostatin and / or latent Myostatin) from serum.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 219,094, filed September 15, 2015, entitled "ANTI-PRO / LATENT-MYOSTATIN ANTIBODIES AND USES THEREOF," the contents of which are incorporated herein by reference for all purposes.

[0002] Field of Disclosure Embodiments of the present disclosure may include modulators of growth factor activity. In some embodiments, such modulators may include antibodies, which are capable of modulating TGF-β family member activity and / or biology. [Background technology]

[0003] Background to the disclosure Myostatin is a secreted growth factor that negatively regulates muscle mass. Loss-of-function mutations in the myostatin gene that result in a hypermuscular phenotype have been described in cattle, sheep, fish, dogs, and humans. Myostatin expression is generally restricted to skeletal muscle, with low levels of expression reported in adipose and cardiac tissue. Inhibition of myostatin signaling leads to increased muscle size. Summary of the Invention [Means for solving the problem]

[0004] Summary of disclosure Aspects of the present disclosure relate to antibodies that, in some embodiments, 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 pro- and / or latent forms of myostatin, such as promyostatin and / or latent myostatin. In certain aspects, the present disclosure is based on the surprising discovery of antibodies provided herein that specifically bind to pure or substantially pure pro-GDF8 (also referred to as promyostatin). In some embodiments, the antibodies provided herein inhibit myostatin signaling. In some embodiments, inhibiting myostatin signaling is useful for increasing muscle mass or preventing muscle atrophy. In some embodiments, the antibodies provided herein bind to myostatin and prevent cleavage of myostatin by proprotein convertases and / or thrombin proteases. Preventing cleavage of promyostatin or latent myostatin, in some embodiments, prevents myostatin activation. A further aspect of the present disclosure relates to antibodies whose affinity for an antigen is sensitive to pH. In some embodiments, such pH-sensitive antibodies are effective in clearing antigens from serum. Further, in some embodiments, the antibodies provided herein are sweeping antibodies that can efficiently clear antigens (e.g., promyostatin and / or latent myostatin) from serum.

[0005] 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 a sequence set forth in any one of SEQ ID NOs: 10-11. In some embodiments, the antibody specifically binds to pro / latent myostatin. In some embodiments, the light chain variable domain comprises a complementarity determining region 3 (CDRH3) comprising a 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 set forth in any one of SEQ ID NOs: 1-3, CDRH2 comprises the sequence set forth in any one of SEQ ID NOs: 4-9, 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, CDRL2 comprises the sequence set forth in any one of SEQ ID NOs: 18-21, and CDRL3 comprises the sequence set forth in any one of SEQ ID NOs: 22-23.

[0006] In some embodiments, the CDRH1 comprises the sequence set forth in SEQ ID NO: 1 or 2, CDRH2 comprises the sequence set forth in SEQ ID NO: 4 or 5, CDRH3 comprises the sequence set forth in SEQ ID NO: 10, CDRL1 comprises the sequence set forth in SEQ ID NO: 12 or 13, CDRL2 comprises the sequence set forth in SEQ ID NO: 18 or 19, and CDRL3 comprises the sequence set forth in SEQ ID NO: 22.

[0007] In another embodiment, the CDRH1 comprises the sequence set forth in SEQ ID NO: 1 or 3, CDRH2 comprises the sequence set forth in SEQ ID NO: 6 or 7, CDRH3 comprises the sequence set forth in SEQ ID NO: 11, CDRL1 comprises the sequence set forth in SEQ ID NO: 14 or 15, 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.

[0008] 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.

[0009] In another embodiment, the antibody comprises a heavy chain variable domain sequence set forth in any one of SEQ ID NOs: 25 to 29. In some embodiments, the antibody comprises a light chain variable domain sequence set forth in any one of SEQ ID NOs: 30 to 35.

[0010] Other aspects of the present disclosure include antibodies that specifically bind to pro / 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 a sequence set forth in any one of SEQ ID NOs: 22-23. In some embodiments, the antibody comprises a light chain variable domain sequence of SEQ ID NO: 30.

[0011] Some aspects of the present disclosure relate to a polypeptide 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 variable heavy chain 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.

[0012] Some aspects of the present disclosure relate to a polypeptide 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 variable light chain 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.

[0013] Another aspect of the disclosure includes antibodies that compete with the antibodies described above for binding to pro / latent myostatin. In some embodiments, the antibodies bind to pro / latent myostatin at the same epitope as the antibodies described above. In another embodiment, the antibodies-6 In another embodiment, the antibody competes for binding to pro / latent myostatin with an equilibrium dissociation constant, Kd, ​​between the antibody and pro / latent myostatin that is less than 10 M. In another embodiment, the antibody has a Kd of 10 -11 M to 10 -6 It is in the range of M.

[0014] In some embodiments, the antibody is a humanized antibody, diabody, chimeric antibody, Fab fragment, F(ab')2 fragment, or 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 human germline sequences. In another embodiment, the antibody comprises a heavy chain constant domain selected from the group consisting of 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 creating an IgG1-like hinge and allowing interchain disulfide bond formation. In another embodiment, the antibody is conjugated to an agent selected from the group consisting of a fluorescent agent, a luminescent agent, an enzymatic agent, and a radioactive agent.

[0015] In another embodiment, the antibody specifically binds to pro / latent myostatin relative to mature myostatin. In some embodiments, the antibody specifically binds to pro / latent myostatin relative to another member of the transforming growth factor beta family. In another embodiment, the member is GDF11 or activin.

[0016] Further aspects of the present disclosure include antibodies that specifically bind to pro / latent myostatin and inhibit the formation of mature myostatin via proteolysis by thrombin proteases. In some embodiments, the antibodies inhibit the formation of mature myostatin via proteolysis by thrombin proteases with an IC50 of less than 1 μM. In some embodiments, the antibodies are cross-reactive with human and murine pro / latent myostatin. In other embodiments, the antibodies specifically bind to pro / latent myostatin relative to GDF11 or activin. In another embodiment, the antibodies specifically bind to pro / latent myostatin relative to mature myostatin.

[0017] Another aspect of the present disclosure includes a method of reducing myostatin receptor activation in cells present in a medium containing pro / latent myostatin, comprising delivering an antibody described above to the medium in an amount effective to inhibit proteolytic activation of pro / latent myostatin. In some embodiments, the medium further comprises a proprotein convertase. In other embodiments, the medium further comprises a thrombopoietin protease. In another embodiment, the antibody is delivered to the medium in an amount effective to inhibit proteolytic activation of pro / latent myostatin by the thrombopoietin protease. In some embodiments, the cells are in vitro. In other embodiments, the cells are in vivo.

[0018] Another aspect of the present disclosure includes a method of treating a subject having a myopathy, comprising administering to the subject an effective amount of an antibody described above. In some embodiments, the myopathy is a primary myopathy. In other embodiments, the primary myopathy comprises disuse atrophy. In other embodiments, the disuse atrophy is associated with hip fracture, elective joint replacement, critical care myopathy, spinal cord injury, or stroke. In some embodiments, the myopathy is a secondary myopathy in which muscle loss is secondary to a disease condition. In other embodiments, the secondary myopathy is a denervation myopathy. In another embodiment, the secondary myopathy is denervation associated with amyotrophic lateral sclerosis or spinal muscular atrophy. In some embodiments, the secondary myopathy is inherited muscle weakness associated with muscular dystrophy. In other embodiments, the secondary myopathy is cachexia associated with renal failure, AIDS, a heart condition, cancer, or aging.

[0019] Another aspect of the present disclosure includes a method of treating a subject with an age-related disease or condition. Exemplary age-related diseases and conditions include, but are not limited to, sarcopenia (age-related muscle loss), frailty, and androgen deficiency.

[0020] Another aspect of the present disclosure includes a method for treating a subject with a disease or condition associated with disuse atrophy / trauma.Exemplary diseases and 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 replacement surgery, hip fracture, stroke, bed rest, SCI, rotator cuff injury, knee replacement surgery, fracture and burn.

[0021] Another aspect of the present disclosure includes a method of treating a subject with a neurodegenerative disease or condition. Exemplary neurodegenerative diseases or conditions include, but are not limited to, spinal muscular atrophy and amyotrophic lateral sclerosis (ALS).

[0022] Another aspect of the present disclosure includes a method of treating a subject having a disease or condition associated with cachexia. Exemplary diseases and conditions associated with cachexia include, but are not limited to, cancer, chronic heart failure, acquired immune deficiency syndrome (AIDS), chronic obstructive pulmonary disease (COPD), and chronic kidney disease (CKD).

[0023] Another aspect of the present disclosure includes a method of treating a subject having 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.

[0024] Another aspect of the present disclosure includes a method of treating a subject having a disease or condition associated with metabolic disorders and / or body composition. In some embodiments, the disease or condition is obesity (e.g., severe obesity), Prader-Willi syndrome, type II diabetes, or anorexia. However, additional diseases or conditions associated with metabolic disorders and / or body composition are within the scope of the present disclosure.

[0025] Another aspect of the present disclosure includes a method 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, nemaline myopathy, and congenital fiber-type disproportion myopathy.

[0026] Another aspect of the present disclosure includes a method of treating a subject having a disease or condition associated with muscular dystrophy. Exemplary muscular dystrophies include, but are not limited to, Duchenne, Becker, facioscapulohumeral (FSH), and limb-girdle muscular dystrophy.

[0027] Another aspect of the present disclosure includes a method of treating a subject with a urogynecologic-related disease or condition, glottic disorder (stenosis), exophthalmic myopathy, carpel tunnel, Guillain-Barre, or osteosarcoma.

[0028] In some embodiments, the treatment results in improved muscle strength in the subject. In other embodiments, the treatment results in improved metabolic status in the subject.

[0029] In some embodiments, the antibody is administered at a dose ranging from 0.1 mg / kg to 100 mg / kg, and in other embodiments, the antibody is administered at a dose ranging from 0.3 mg / kg to 30 mg / kg.

[0030] In some embodiments, the antibody is administered to the subject intravenously. In other embodiments, the antibody is administered to the subject subcutaneously. In another embodiment, the antibody is administered to the subject on multiple occasions. In some embodiments, the multiple administrations are administered at least monthly. In another embodiment, the multiple administrations are administered at least weekly.

[0031] Further aspects of the present disclosure include compositions comprising any of the antibodies described above 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 other embodiments, 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 less than or equal to -65°C.

[0032] Other aspects of the disclosure include isolated nucleic acids encoding proteins 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.

[0033] Another aspect of the disclosure includes an isolated nucleic acid 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.

[0034] A further aspect of the present disclosure includes an isolated nucleic acid comprising the sequence set forth in any one of SEQ ID NOs: 38-49.

[0035] Another aspect of the present disclosure includes an isolated cell comprising the isolated nucleic acid described above.

[0036] In some aspects, the present disclosure includes methods for evaluating a biological sample obtained from a subject with a myopathy. In some embodiments, the method includes preparing an immunological reaction mixture comprising proteins from the biological sample obtained from the subject and an antibody that specifically binds to pro / latent myostatin; maintaining the immunological reaction mixture under conditions that allow for the formation of a binding complex between the antibody and pro / latent myostatin; and determining the extent of binding complex formation. In some embodiments, the method includes preparing an immunological reaction mixture comprising proteins from the biological sample obtained from the subject and an antibody that specifically binds to pro-myostatin; maintaining the immunological reaction mixture under conditions that allow for the formation of a binding complex between the antibody and pro-myostatin; and determining the extent of binding complex formation. In some embodiments, the method includes preparing an immunological reaction mixture comprising proteins from the biological sample obtained from the subject and an antibody that specifically binds to latent myostatin; maintaining the immunological reaction mixture under conditions that allow for the formation of a binding complex between the antibody and latent myostatin; and determining the extent of binding complex formation. In some embodiments, the method includes preparing an immunological reaction mixture comprising proteins of a biological sample obtained from a subject and an antibody that specifically binds to mature myostatin; maintaining the immunological reaction mixture under conditions that allow for the formation of a binding complex between the antibody and mature myostatin; and determining the extent of binding complex formation. This includes:

[0037] 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 aspect, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 51.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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, resulting in an IgG1-like hinge and allowing interchain disulfide bond formation.

[0045] In one embodiment, the antibody specifically binds to pro / latent myostatin. In one embodiment, the antibody specifically binds to promyostatin. In another embodiment, the antibody specifically binds to latent myostatin. In one embodiment, the antibody does not bind to mature myostatin.

[0046] In one embodiment, the antibody inhibits the formation of mature myostatin via proteolysis by throid proteases. In one embodiment, the antibody inhibits the formation of mature myostatin via proteolysis by throid proteases with an IC50 of less than 1 μM.

[0047] In one embodiment, the antibody is cross-reactive with human and murine pro / latent myostatin. In another embodiment, the antibody binds to pro / latent myostatin but does not bind to GDF11 or activin.

[0048] In one aspect, disclosed herein is a method of reducing myostatin receptor activation in cells present in a medium containing pro / latent myostatin, comprising delivering an antibody described herein to the medium in an amount effective to inhibit proteolytic activation of pro / latent myostatin. In one embodiment, the medium comprises a proprotein convertase. In another embodiment, the medium comprises a thrombin protease. In one embodiment, the cells are In another embodiment, the cell is in vivo.

[0049] In another aspect, disclosed herein is a method of treating a subject having a myopathy, comprising administering to the subject an effective amount of an antibody disclosed herein.

[0050] In one embodiment, the myopathy is primary myopathy. In another embodiment, the primary myopathy is disuse atrophy. In one embodiment, the disuse atrophy is associated with hip fracture, elective joint replacement, critical care myopathy, spinal cord injury, and / or stroke.

[0051] In another embodiment, the myopathy is a secondary myopathy in which muscle loss is secondary to a disease condition. In one embodiment, 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 yet another embodiment, the secondary myopathy is hereditary muscle weakness associated with muscular dystrophy. In one embodiment, the secondary myopathy is cachexia associated with renal failure, AIDS, a cardiac condition, cancer, or aging.

[0052] In one embodiment, administration results in improved muscle strength in the subject. In one embodiment, administration results in improved metabolic status in the subject.

[0053] In one embodiment, the antibody is administered at a dose ranging from 0.1 mg / kg to 100 mg / kg, in another embodiment, the antibody is administered at a dose ranging from 0.3 mg / kg to 30 mg / kg.

[0054] In one embodiment, the antibody is administered to the subject intravenously. In another embodiment, the antibody is administered to the subject subcutaneously.

[0055] In one embodiment, the antibody is administered to the subject on multiple occasions. In one embodiment, the multiple administrations are administered at least monthly. In another embodiment, the multiple administrations are administered at least weekly.

[0056] In another aspect, disclosed herein is a pharmaceutical composition 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 less than or equal to -65°C.

[0057] In another aspect, disclosed herein is a syringe containing the pharmaceutical composition described herein.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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: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.

[0063] 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.

[0064] 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.

[0065] In another aspect, disclosed herein is an isolated cell comprising an isolated nucleic acid described herein. [Brief explanation of the drawings]

[0066] [Figure 1] Figures 1A-1B show the domain structure and promyostatin assembly of myostatin. Figure 1A shows myostatin secreted as a proprotein, with an inhibitory prodomain followed by a C-terminal growth factor domain that exists as a disulfide-linked dimer. Figure 1B shows the assembled precursor protein in an inactive conformation, in which the prodomain (dark gray) encapsulates the growth factor (light gray) via a "straight jacket" assembly. This figure is an adaptation of the structure of latent TGFβ1 (Shi et al., Nature, 2011).

[0067] [Figure 2] Figure 2 shows that myostatin activation involves two distinct protease events, generating three major myostatin species. The biosynthetic precursor protein, promyostatin, is processed by two distinct 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 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 active growth factor are achieved after cleavage by additional proteases of 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 may be referred to as active or mature myostatin.

[0068] [Figure 3AB]Figures 3A-3C show that Ab1 blocks promyostatin cleavage by members of the tolloid family of proteases. Latent myostatin samples preincubated with increasing amounts of Ab1 were analyzed in a myostatin activation assay. After analysis of myostatin release by reporter assay (Figure 3A), samples were then run under reducing conditions and probed by Western blot with an antibody raised against the myostatin prodomain (Figure 3B). The approximately 18 kDa band (boxed), corresponding to the ARM portion of the prodomain generated after tolloid cleavage, decreased proportionally with increasing Ab1 doses. Latent and promyostatin standards (45 ng loaded) show that promyostatin migrated at approximately 50 kDa and the prodomain at approximately 37 kDa. Figure 3C shows that myostatin activation involves two distinct protease events, generating three major myostatin species. The biosynthetic precursor protein, promyostatin, is processed by two distinct 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 at a 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 illustrates the possible inhibition of the tolloid protease and blocks further cleavage of promyostatin. Activation and release of the active growth factor are achieved after cleavage by additional proteases of the BMP / tolloid family, such as TLL-2 (tolloid-like protein 2) or BMP1 (bone morphogenetic protein 1). [Figure 3C]Figures 3A-3C show that Ab1 blocks promyostatin cleavage by members of the tolloid family of proteases. Latent myostatin samples preincubated with increasing amounts of Ab1 were analyzed in a myostatin activation assay. After analysis of myostatin release by reporter assay (Figure 3A), samples were then run under reducing conditions and probed by Western blot with an antibody raised against the myostatin prodomain (Figure 3B). The approximately 18 kDa band (boxed), corresponding to the ARM portion of the prodomain generated after tolloid cleavage, decreased proportionally with increasing Ab1 doses. Latent and promyostatin standards (45 ng loaded) show that promyostatin migrated at approximately 50 kDa and the prodomain at approximately 37 kDa. Figure 3C shows that myostatin activation involves two distinct protease events, generating three major myostatin species. The biosynthetic precursor protein, promyostatin, is processed by two distinct 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 at a 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 illustrates the possible inhibition of the tolloid protease and blocks further cleavage of promyostatin. Activation and release of the active growth factor are achieved after cleavage by additional proteases of the BMP / tolloid family, such as TLL-2 (tolloid-like protein 2) or BMP1 (bone morphogenetic protein 1).

[0069] [Figure 4]Figure 4 shows the performance of the parent Ab1 antibody and other candidates in a cell-based reporter assay. After overnight proteolytic reactions with enzymes from both the proprotein convertase and thrombin protease families, the release of mature growth factors was measured by a CAGA-based reporter assay in 293T cells. Results were compared to control reactions, and the percentage of pro-myostatin or pro-GDF11 released during the assay was calculated. The standard deviations of the means of three replicates are shown; however, due to small values, most data points are not visible on the graph.

[0070] [Figure 5] FIG. 5 graphically shows that Ab1, Ab2, Ab4, and Ab6 antibodies do not inhibit pro-GDF11 activation.

[0071] [Figure 6] Figure 6 shows the results of an assay assessing mean percent weight change. Animals were weighed daily to calculate percent weight change from day 0. Data represent group mean ± SEM. Mean percent change data for each group on study day 42 were analyzed using one-way ANOVA followed by a Holm-Sidak post-hoc test in comparison to the PBS control group. **p<0.01.

[0072] [Figure 7] Figures 7A-7D show the results of assays assessing tissue weight. Figure 7A shows the mean gastrocnemius muscle weight. Figure 7B shows the mean pectoralis muscle weight. Figure 7C shows the mean soleus muscle weight. Figure 7D shows the mean triceps muscle weight. Statistical evaluation was performed using one-way ANOVA followed by a Holm-Sidak post-hoc test in comparison with the vehicle control group (Group 1). Data represent group means ± SEM. **p<0.01. Bar graphs show Groups 1-5 from left to right.

[0073] [Figure 8]Figures 8A-8C show the results of assays assessing tissue weight. Figure 8A shows the mean tibialis anterior muscle weight. Figure 8B shows the mean diaphragm weight. Figure 8C shows the mean quadriceps muscle weight. Statistical evaluation was performed using one-way ANOVA followed by a Holm-Sidak post-hoc test in comparison with the vehicle control group (Group 1). Data represent group means ± SEM. *p<0.05. Bar graphs show Groups 1-5 from left to right.

[0074] [Figure 9] Figures 9A-9B show the results of assays assessing mean percent body weight change and mean percent lean mass change. Figure 9A is a graph showing the calculated percent body weight change from day 0 for animals weighed twice weekly throughout the study. In Figure 9B, animals underwent EchoMRI (QNMR) to measure body composition on days -4, 7, 14, 21, and 28, and percent lean mass change from day 0 was calculated. Data represent group means ± SEM. For both body weight and lean mass, mean percent change data for each group at day 28 of the study were analyzed using one-way ANOVA followed by a Holm-Sidak post-hoc test in comparison to the IgG control group (Group 2). ***p<0.0005, **p<0.005, *p<0.05, ns (not significant).

[0075] [Figure 10AB] Figures 10A-10D are graphs showing the results of an assay assessing muscle weight. Figure 10A shows the mean quadriceps muscle weight, Figure 10B shows the mean gastrocnemius muscle weight, Figure 10C shows the mean tibialis anterior muscle weight, and Figure 10D shows the mean diaphragm weight. The percentage difference in mean muscle weight for the Ab1-treated group compared to the IgG control group is indicated above each bar. Statistical evaluation was performed using one-way ANOVA followed by a Holm-Sidak post-hoc test in comparison with 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). [Figure 10CD]Figures 10A-10D are graphs showing the results of an assay assessing muscle weight. Figure 10A shows the mean quadriceps muscle weight, Figure 10B shows the mean gastrocnemius muscle weight, Figure 10C shows the mean tibialis anterior muscle weight, and Figure 10D shows the mean diaphragm weight. The percentage difference in mean muscle weight for the Ab1-treated group compared to the IgG control group is indicated above each bar. Statistical evaluation was performed using one-way ANOVA followed by a Holm-Sidak post-hoc test in comparison with 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).

[0076] [Figure 11] Figures 11A-11B show the results of assays assessing mean percent body weight change and mean percent lean mass change. Figure 11A shows percent body weight change 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 percent lean mass change from day -1 was calculated. PBS = phosphate-buffered saline; Dex = dexamethasone; IgG(20) = IgG control antibody administered at 20 mg / kg / week; Ab1(20) = Ab1 antibody administered at 20 mg / kg / week; and Ab1(2) = Ab1 antibody administered at 2 mg / kg / week. Data represent group means ± SEM. The mean percent change data for each group on day 14 (for body weight) and day 13 (for lean mass) were analyzed using one-way ANOVA followed by Dunnett's multiple comparison test for Group 1 (****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05) and Group 5 (++++p<0.0001, +++p<0.0005, ++p<0.005, +p<0.05). ns (not significant).

[0077] [Figure 12AB]Figures 12A-12D are graphs showing the results of an assay assessing different muscle weights. Figure 12A shows the mean gastrocnemius muscle weight (grams), Figure 12B shows the mean quadriceps muscle weight (grams), Figure 12C shows the mean percent gastrocnemius muscle weight change compared to control animals treated with PBS (IP) and regular drinking water (Group 1), and Figure 12D shows the mean percent quadriceps muscle weight change compared to control animals treated with PBS (IP) and regular drinking water (Group 1). PBS = phosphate-buffered saline, Dex = dexamethasone, IgG(20) = IgG control antibody administered at 20 mg / kg / week, Ab1(20) = Ab1 antibody administered at 20 mg / kg / week, and Ab1(2) = Ab1 antibody administered at 2 mg / kg / week. For Figures 12A-12B, error bars represent standard deviation (SD). For Figures 12C-12D, error bars represent the standard error of the mean (SEM). Statistical evaluation was performed using one-way ANOVA followed by Dunnett's multiple comparison test for Group 1 (****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05) and Group 5 (++++p<0.0001, +++p<0.0005, ++p<0.005, +p<0.05). ns (not significant). Bars, from left to right, show PBS, water; PBS, dex; IgG control; Ab1(20); and Ab1(2). [Figure 12CD]Figures 12A-12D are graphs showing the results of an assay assessing different muscle weights. Figure 12A shows the mean gastrocnemius muscle weight (grams), Figure 12B shows the mean quadriceps muscle weight (grams), Figure 12C shows the mean percent gastrocnemius muscle weight change compared to control animals treated with PBS (IP) and regular drinking water (Group 1), and Figure 12D shows the mean percent quadriceps muscle weight change compared to control animals treated with PBS (IP) and regular drinking water (Group 1). PBS = phosphate-buffered saline, Dex = dexamethasone, IgG(20) = IgG control antibody administered at 20 mg / kg / week, Ab1(20) = Ab1 antibody administered at 20 mg / kg / week, and Ab1(2) = Ab1 antibody administered at 2 mg / kg / week. For Figures 12A-12B, error bars represent standard deviation (SD). For Figures 12C-12D, error bars represent the standard error of the mean (SEM). Statistical evaluation was performed using one-way ANOVA followed by Dunnett's multiple comparison test for Group 1 (****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05) and Group 5 (++++p<0.0001, +++p<0.0005, ++p<0.005, +p<0.05). ns (not significant). Bars, from left to right, show PBS, water; PBS, dex; IgG control; Ab1(20); and Ab1(2).

[0078] [Figure 13] Figures 13A-13B show the results of assays assessing mean percent body weight change and mean percent lean mass change. Figure 13A shows the percent body weight change from day 0 calculated for animals weighed twice weekly throughout the study. Figure 13B shows the percent lean mass change from day -1 calculated for animals that underwent EchoMRI (QNMR) to measure body composition on days -1, 7, and 14. PBS = phosphate-buffered saline, IgG(20) = IgG control antibody administered at 20 mg / kg / week, Ab1(20) = Ab1 antibody administered at 20 mg / kg / week, and Ab1(2) = Ab1 antibody administered at 2 mg / kg / week. Data represent group means ± SEM.

[0079] [Figure 14AB]Figures 14A-14D show the results of an assay assessing muscle weight. Figure 14A shows the average gastrocnemius muscle weight (grams) from the casted leg, Figure 14B shows the average quadriceps muscle weight (grams) from the casted leg, Figure 14C shows the average percent change in gastrocnemius muscle weight compared to non-casted control animals (Group 1) treated with PBS (IP), and Figure 14D shows the average percent change in quadriceps muscle weight compared to non-casted control animals (Group 1) treated with PBS (IP). PBS = phosphate-buffered saline; IgG(20) = IgG control antibody administered at 20 mg / kg / week; Ab1(20) = Ab1 antibody administered at 20 mg / kg / week; Ab1(2) = Ab1 antibody administered at 2 mg / kg / week. For Figures 14A-14B, error bars represent standard deviation (SD). For Figures 14C-14D, error bars represent the standard error of the mean (SEM). Statistical evaluation was performed using one-way ANOVA followed by Dunnett's multiple comparison test for Group 1 (****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05) and Group 5 (++++p<0.0001, +++p<0.0005, ++p<0.005, +p<0.05). ns (not significant). Bars from left to right: PBS, no immobilization; PBS, immobilization; IgG control (2), immobilization; Ab1 (20), immobilization; Ab1 (2), immobilization. [Figure 14CD]Figures 14A-14D show the results of an assay assessing muscle weight. Figure 14A shows the average gastrocnemius muscle weight (grams) from the casted leg, Figure 14B shows the average quadriceps muscle weight (grams) from the casted leg, Figure 14C shows the average percent change in gastrocnemius muscle weight compared to non-casted control animals (Group 1) treated with PBS (IP), and Figure 14D shows the average percent change in quadriceps muscle weight compared to non-casted control animals (Group 1) treated with PBS (IP). PBS = phosphate-buffered saline; IgG(20) = IgG control antibody administered at 20 mg / kg / week; Ab1(20) = Ab1 antibody administered at 20 mg / kg / week; Ab1(2) = Ab1 antibody administered at 2 mg / kg / week. For Figures 14A-14B, error bars represent standard deviation (SD). For Figures 14C-14D, error bars represent the standard error of the mean (SEM). Statistical evaluation was performed using one-way ANOVA followed by Dunnett's multiple comparison test for Group 1 (****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05) and Group 5 (++++p<0.0001, +++p<0.0005, ++p<0.005, +p<0.05). ns (not significant). Bars from left to right: PBS, no immobilization; PBS, immobilization; IgG control (2), immobilization; Ab1 (20), immobilization; Ab1 (2), immobilization.

[0080] [Figure 15]Figure 15 shows the results of an assay assessing lean mass change at day 21 (top right) and day 28 (top left). The figure also shows the percent lean mass change for the three different doses of antibody tested: 20 mg / kg / week (bottom left), 2 mg / kg / week (bottom center), and 0.5 mg / kg / week (bottom right), a PBS control, and an IgG control. Statistical evaluation was performed using one-way ANOVA followed by Dunnett's multiple comparison test for group 1 (****p<0.0001, ***p<0.005, **p<0.01, *p<0.05) and the IgG control. For the top two panels, the bar graphs are, from left to right, PBS; IgG control, 20 mg / kg / week; Ab1 20 mg / kg / week; Ab1 2 mg / kg / week; Ab1 0.5 mg / kg / week; Ab2 20 mg / kg / week; Ab2 2 mg / kg / week; Ab2 0.5 mg / kg / week; Ab4 20 mg / kg / week; Ab4 2 mg / kg / week; Ab4 0.5 mg / kg / week; Ab6 20 mg / kg / week; Ab6 2 mg / kg / week; and Ab6 0.5 mg / kg / week. For the bottom left panel (20 mg / kg / week), the data points correspond to 28 days after dosing, from top to bottom: Ab1, Ab4, Ab2, Ab6, IgG control, and PBS. For the lower middle panel (2 mg / kg / week), data points correspond to 28 days post-dose, from top to bottom: Ab2, Ab1, Ab6, Ab4, IgG control, and PBS. For the lower right panel (0.5 mg / kg / week), data points correspond to 28 days post-dose, from top to bottom: IgG control, Ab1, Ab2, PBS, Ab4, and Ab6.

[0081] [Figure 16] Figures 16A-16B show the domain structure and characterization of myostatin precursor forms. Figure 16A shows the domain structure of promyostatin and latent myostatin, indicating protease cleavage sites. Figure 16B shows partially proprotein convertase-cleaved promyostatin run on an SDS PAGE gel. Under reducing conditions, the protein bands consisted of the promyostatin monomer (approximately 50 kD), prodomain (approximately 37 kD), and growth factor (12.5 kD).

[0082] [Figure 17] Figures 17A-17B show that Ab1 is specific for myostatin. Figure 17A shows that Ab1 specifically binds to promyostatin and latent myostatin; no binding was observed to other members of the TGFB superfamily, particularly the corresponding forms of GDF11. Ab1 was administered at high concentrations (50 μg / mL) to Forte-Bio BLI chips coated with the indicated antigens, and on- and off-rates were measured to obtain approximate Kd values. The magnitude of the biosensor response, indicating binding events, is graphically displayed by black bars, with the calculated Kd shown in orange. Figure 17B shows that Ab1 blocks the activation of promyostatin, but not pro-GDF11. Following overnight proteolysis with enzymes from both the proprotein convertase and thrombin protease families, the release of mature growth factors was measured using a CAGA-based reporter assay in 293T cells. Results were compared to control reactions to calculate the percentage of promyostatin or proGDF11 released in the assay.

[0083] [Figure 18AB] Figures 18A-18C show SCID dose responses with candidate antibodies. Figure 18A shows gastrocnemius muscle weight, and Figure 18B shows quadriceps muscle weight. Figure 18C shows the mean percent muscle weight change compared to the PBS control. The bar graphs in Figures 18A-18B, from left to right, are PBS; IgG control 20 mg / kg / week; Ab1 20 mg / kg / week; Ab1 2 mg / kg / week; Ab1 0.5 mg / kg / week; Ab2 20 mg / kg / week; Ab2 2 mg / kg / week; Ab2 0.5 mg / kg / week; Ab4 20 mg / kg / week; Ab4 2 mg / kg / week; Ab4 0.5 mg / kg / week; Ab6 20 mg / kg / week; Ab6 2 mg / kg / week; and Ab6 0.5 mg / kg / week. [Figure 18C]Figures 18A-18C show SCID dose responses with candidate antibodies. Figure 18A shows gastrocnemius muscle weight, and Figure 18B shows quadriceps muscle weight. Figure 18C shows the mean percent muscle weight change compared to the PBS control. The bar graphs in Figures 18A-18B, from left to right, are PBS; IgG control 20 mg / kg / week; Ab1 20 mg / kg / week; Ab1 2 mg / kg / week; Ab1 0.5 mg / kg / week; Ab2 20 mg / kg / week; Ab2 2 mg / kg / week; Ab2 0.5 mg / kg / week; Ab4 20 mg / kg / week; Ab4 2 mg / kg / week; Ab4 0.5 mg / kg / week; Ab6 20 mg / kg / week; Ab6 2 mg / kg / week; and Ab6 0.5 mg / kg / week.

[0084] [Figure 19] Figure 19 shows the results of a duration of effect study comparing Ab1 with an existing myostatin antibody (AbMyo). PBS was used as a negative control. IgG was used as a positive control. Changes in lean mass were examined after 21 days following different dosing protocols.

[0085] [Figure 20] FIG. 20 is a schematic diagram illustrating an assay to reconstitute myostatin activation in vitro.

[0086] [Figure 21]Figures 21A-21B show the heavy chain (Figure 21A, SEQ ID NO: 50) and light chain (Figure 21B, SEQ ID NO: 51) of a humanized monoclonal antibody (Ab2) of the IgG4 subtype in which serine has been substituted with proline. This creates an IgG1-like hinge sequence and minimizes the incomplete formation of interchain disulfide bridges characteristic of IgG4. The complementarity-determining regions (CDRs) are underlined. The NST sequence, in bold, is an 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 a predicted N-terminal pyroglutamic acid.

[0087] [Figure 22] Figure 22 is a schematic diagram showing the reduction of immunogenicity risk by germlining. 24H4(WT) contains five non-germlined amino acids in the framework regions as shown in the schematic.

[0088] [Figure 23AB] Figures 23A-23C show the optimization of Ab1. Optimized candidates that specifically bind to promyostatin were selected, thereby yielding dozens of clones with increased affinity. FACS was performed to demonstrate increased binding of the yeast clones (Figure 23B) compared to Ab1 (Figure 23A). Figure 23C shows that the affinity-matured variants also have slower off-rates by octets. [Figure 23C] Figures 23A-23C show the optimization of Ab1. Optimized candidates that specifically bind to promyostatin were selected, thereby yielding dozens of clones with increased affinity. FACS was performed to demonstrate increased binding of the yeast clones (Figure 23B) compared to Ab1 (Figure 23A). Figure 23C shows that the affinity-matured variants also have slower off-rates by octets.

[0089] [Figure 24] Figures 24A-24B show sequence alignments of the variable heavy chain region (Figure 24A) and variable light chain region (Figure 24B) of parent Ab1 with affinity-optimized variants Ab3 and Ab5. Sequence identifiers, from top to bottom, correspond to SEQ ID NOS: 24, 26, and 28 (Figure 24A). Sequence identifiers, from top to bottom, correspond to SEQ ID NOS: 30, 32, and 34 (Figure 24B). Complementarity-determining regions (CDRs) are defined using Kabat (underlined) and IMGT nomenclature (bold). Substitutions from parent Ab1 are shown in light gray.

[0090] [Figure 25] Figure 25 shows the expression of promyostatin and latent myostatin in muscle and plasma from normal and atrophic mice.

[0091] [Figure 26] Figure 26 shows quantification of changes in promyostatin and latent myostatin in muscle and plasma. From left to right, the bar graphs show promyostatin, latent myostatin, promyostatin, latent myostatin, and latent myostatin.

[0092] [Figure 27] Figure 27 shows that Ab2 specifically recognizes promyostatin and latent myostatin and binds to the major forms of myostatin in both serum and muscle. Non-reducing Western blot for the prodomain (dark gray) and mature growth factor (light gray). Recombinant promyostatin (rpromyostatin) shows the migration of promyostatin and the myostatin prodomain (latent myostatin) on the gel, highlighted by arrows. In serum, both Ab2 and AbMyo bind to latent myostatin (prodomain band) and the multipartite processed precursor, but only Ab2 recognizes promyostatin (upper band). In muscle, Ab2 precipitates promyostatin, and there is no interaction between AbMyo and promyostatin in muscle tissue.

[0093] [Figure 28] Figures 28A-28B provide a model of myostatin flux in normal and atrophic muscle. In normal muscle (Figure 28A), promyostatin is produced in the muscle and converted to latent myostatin via cleavage by furin protease, which can occur either inside or outside the cell. A percentage of the latent myostatin in the 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 in the muscle and increased conversion of latent myostatin to active growth factor. As a result, the muscle pool of latent myostatin is redirected to form mature myostatin via mTLL2 cleavage, and circulating latent myostatin is reduced.

[0094] [Figure 29] Figure 29 shows the detection of Ab2 (top line) and IgG control (bottom line) antibodies in the serum of treated rats. Ab2 showed increased 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, with known amounts of each antibody used as a reference standard.

[0095] [Figure 30] Figures 30A-30B show the pharmacodynamic effects of Ab2 in treated rats. Figure 30A shows that rats treated with Ab2 exhibited increased lean mass compared to animals treated with PBS or IgG control. Ab2 and IgG were administered intravenously at a dose of 10 mg / kg on day 0. Lean mass was measured by qNMR (N=8 per group) 7, 14, 21, and 28 days after administration. Figure 30B shows that rectus femoris and tibialis anterior muscles were harvested from all groups (N=8 per group) at the end of the study and weighed to measure muscle mass. Rats treated with Ab2 exhibited a 14% and 11% increase in rectus femoris and tibialis anterior muscle mass, respectively.

[0096] [Figure 31]Figures 31A-31B show the levels of pro / latent myostatin in rats treated with Ab2. Figure 31A shows that treatment with Ab2 (top line) increases latent myostatin levels in rat serum by approximately 20-fold. Figure 31B shows that treatment with Ab2 increases the latent form of myostatin in rat muscle (rectus femoris) by 1.9-fold. The bars from left to right correspond to promyostatin, latent myostatin, promyostatin, and latent myostatin. No statistically significant changes in promyostatin are observed in rat muscle. These data are from quantitative Western analysis with n=3 samples per group.

[0097] [Figure 32] Figure 32 shows that treatment with Ab2 (Ab2) or comparator antibody (AbMyo) increases lean mass as early as 7 days after antibody administration. The increase in lean mass is comparable for Ab2 and AbMyo up to 21 days after administration. However, by 28 days after administration, the increase in lean mass is lost in the AbMyo-treated group, while the increase in Ab2-treated group is maintained throughout the course of the study. The top line corresponds to Ab2, the middle line corresponds to AbMyo, and the bottom line corresponds to the IgG control (5 mg / kg).

[0098] [Figure 33] Figure 33 shows that serum levels of drug were measured using an anti-human IgG ELISA after a single 5 mg / kg dose of Ab2 (top line) or comparator antibody (AbMyo; bottom line). Drug was detected in serum as early as 1 hour after dosing, and levels of greater than 1 μg / ml for both antibodies were detectable throughout the study. However, Ab2 exhibited a significantly longer half-life and estimated area under the curve (AUCINF) than AbMyo, suggesting that Ab2 provides significantly greater exposure than AbMyo at similar doses.

[0099] [Figure 34]Figure 34 shows that serum myostatin was measured using fluorescent Western blot in drug-treated mice and controls. Despite increased serum exposure of Ab2, serum latent myostatin levels were similar in both Ab2- and AbMyo-treated mice. These data suggest that circulating levels of free drug are sufficiently in excess of target levels that increased serum exposure of Ab2 does not result in a greater increase in circulating latent myostatin than observed in the AbMyo group. Data groups, from left to right, correspond to IgG, Ab2, AbMyo, IgG, Ab2, and AbMyo.

[0100] [Figure 35] Figures 35A-35B show the relative levels of latent and pro-myostatin measured by fluorescent Western blot in mouse muscle lysates. Figure 35A shows that latent myostatin is elevated in Ab2- and AbMyo-treated muscles. However, while the increase in latent myostatin in AbMyo-treated muscles returns to baseline by day 28, latent myostatin in Ab2-treated muscles remains elevated at least until this time (P<0.003 vs. AbMyo treatment). Figure 35B shows that a similar trend is observed for promyostatin, although the difference between the Ab2- and AbMyo-treated groups at day 28 is not statistically significant (P=0.068).

[0101] [Figure 36] Figure 36A shows the effect of Ab2 treatment on muscle mass and function in mice, and Figure 36B shows the effect of Ab2 treatment on maximal force production in mice. DETAILED DESCRIPTION OF THE INVENTION

[0102] Detailed Description 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 both initially expressed as inactive precursor polypeptides (termed promyostatin and proGDF11, respectively). The domain structure and nomenclature are shown in Figure 1A. Figure 1B illustrates a cartoon model of the overall structure of promyostatin, in which the mature growth factor is entrapped in a cage composed of two alpha helices connected by a loop called the "latency lasso."

[0103] Activation and release of mature growth factors is achieved by several separate protease cleavage events, outlined in Figure 2. The first cleavage step of promyostatin and proGDF11 is carried out by a proprotein convertase that cleaves at a 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 achieved after cleavage by additional proteases from the BMP / Tolloid family, such as mTLL-2 (Figure 2).

[0104] Exemplary pro-GDF8 sequences for human, rat, mouse, and cynomolgus monkey are provided below. In these pro-GDF8 sequences, the proprotein convertase cleavage site is shown in bold and the toroid protease site is underlined. In some embodiments, the proprotein convertase cleavage site comprises amino acid residues 240 to 243 of SEQ ID NOs: 52-55. In some embodiments, the toroid protease site comprises amino acid residues 74-75 of SEQ ID NOs: 52-55. It should be understood that the exemplary pro-GDF8 sequences provided herein are not intended to be limiting, and additional pro-GDF8 sequences from other species, including any isoforms thereof, are within the scope of the present disclosure.

[0105] [ka] [ka]

[0106] Myostatin and GDF11 share a relatively high degree of conservation between their mature growth factor domains, with 90 percent identity, but 2 in their prodomain regions. They are not well conserved, with less than 50 percent amino acid identity between them. Myostatin and GDF11 bind to and signal through the same receptor, consisting of a type I receptor (ALK4 / 5) associated with a type II receptor (ACTRIIA / B). Engagement of myostatin with both the type I and type II receptors initiates a signaling cascade that leads to SMAD phosphorylation and transcriptional activation of muscular atrophy genes. The relatively high degree of conservation among mature growth factors makes it difficult to identify reagents, such as monoclonal antibodies, that can distinguish between mature myostatin and GDF11.

[0107] In some embodiments, provided herein is a pro / latent myostatin antibody that specifically binds to a chimeric construct containing a growth factor domain and an N-terminal propeptide portion of GDF11 and a C-terminal portion of the propeptide of GDF8. This chimeric construct, described below, is referred to as GDF11Arm8. [ka]

[0108] The role of myostatin in myopathy Skeletal muscle accounts for approximately 40% of body weight and is a dynamic organ, turning over at a rate of 1–2% per day. Muscle atrophy is a highly regulated catabolic process that occurs during periods of inactivity (e.g., disuse atrophy) and / or in response to increased systemic inflammation (cachexia). In disuse atrophy, which can occur during prolonged periods of immobilization, such as bed rest, muscle loss occurs rapidly. For example, the average patient loses approximately 1.3 kg of muscle mass during a one-week hospital stay.

[0109] Muscle atrophy causes significant morbidity in a wide range of clinical conditions. In genetic disorders, including denervating diseases such as amyotrophic lateral sclerosis (ALS) or spinal muscular atrophy (SMA) and muscular dystrophies, loss of muscle strength and function is a highly disabling clinical manifestation without adequate treatment. In cachexia syndromes due to renal failure, AIDS, cardiac conditions, or cancer, muscle wasting often undermines successful treatment of the primary condition. Muscle loss also occurs as a natural progression of aging, and its most severe form is classified as sarcopenia, a widespread condition in the elderly that is increasingly recognized as a pathological condition requiring therapeutic intervention. Finally, disuse is a major factor in the progression of muscle atrophy. Immobilization causes rapid and significant muscle loss in a large group of conditions, including hip fracture, elective joint replacement, spinal cord injury, critical care myopathy, and stroke. Although their causes vary, these conditions share the characteristic of muscle weakness, which results in significant disability, long physical rehabilitation and recovery times, and reduced quality of life.

[0110] There is an unmet medical need for muscle wasting conditions. Accordingly, in some embodiments, methods are provided herein for treating muscle wasting conditions. In some embodiments, the methods provided herein relate to the treatment of primary myopathies. In embodiments, the methods provided herein relate to the treatment of secondary myopathies, such as denervation, inherited muscle weakness and cachexia diseases, conditions in which muscle loss is secondary to a disease pathology, etc. In some embodiments, the methods provided herein for the treatment of primary myopathies, such as disuse atrophy (e.g., associated with hip fracture or spinal cord injury (SCI)), result in increased muscle mass, strength, and function in a subject.

[0111] Myostatin pathway inhibition There are several myostatin pathway antagonists in various stages of clinical development for the treatment of muscle-related conditions. These pathway antagonists target either mature growth factors or their type II receptors, and most antagonize the signal transduction of multiple TGFβ family members. For example, some 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 may limit the patient population who can be safely treated due to unacceptable side effects.

[0112] Thus, provided herein are antibodies capable of binding to promyostatin and / or latent myostatin, thereby inhibiting myostatin activity, and their use to treat diseases and disorders involving myopathy. In some embodiments, provided herein are treatments that specifically target the more abundant and longer-present myostatin precursors, e.g., promyostatin and latent myostatin, rather than the mature growth factor, given the predominance of the latent complex in the circulation. While not wishing to be bound by any particular theory, the antibodies provided herein, for example, by binding to type I (ALK4 / 5) and type II (ACTRIIA / B) receptors, can prevent the activation of promyostatin and / or latent myostatin via proteolysis to mature myostatin, which is considered the "active" form of myostatin that can activate the myostatin pathway.

[0113] As used herein, the term "pro / latent myostatin" refers to promyostatin, latent myostatin, or both. In some embodiments, an anti-pro / latent myostatin antibody specifically binds to promyostatin. In some embodiments, an anti-pro / latent myostatin antibody specifically binds to latent myostatin. In some embodiments, an anti-pro / latent myostatin antibody specifically binds to both latent myostatin and promyostatin. It should be recognized that, as used herein, "latent myostatin" and "promyostatin" also refer to "latent GDF8" and "proGDF8," respectively.

[0114] 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 in vivo from its promyostatin form is achieved by several separate protease cleavage events. Initially, "promyostatin" is cleaved by a proprotein convertase to generate "latent myostatin," in which mature myostatin is blocked from binding to the receptor by a portion of the prodomain. Activation and release of mature myostatin is achieved after cleavage of latent myostatin by an additional protease from the BMP / tolloid family, such as mTLL-2. See, e.g., Figures 1A, 1B, and 2. As used herein, the term "mature myostatin" refers to both full-length mature myostatin and fragments that retain the biological activity of full-length mature myostatin. Exemplary mature myostatin sequences, variants thereof, and methods for producing mature myostatin are well known in the art and are described in more detail herein.

[0115] The term "promyostatin," also known as "pro-GDF8," refers to an inactive precursor of mature myostatin comprising a disulfide-linked homodimer, each molecule of which contains an amino-terminal prodomain covalently linked to a carboxyl-terminal mature myostatin domain. In one embodiment, "promyostatin" has not been cleaved by either proprotein convertases or BMP / thoroid family proteases. Exemplary promyostatin sequences, variants thereof, and methods for producing promyostatin are well known in the art and are described in more detail herein.

[0116] As used herein, the term "latent myostatin" refers to an inactive precursor of mature myostatin comprising a disulfide-linked homodimer, each molecule of which contains an amino-terminal prodomain noncovalently linked 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 BMP / tholoide family protease. In another embodiment, "latent myostatin" can be produced by combining the prodomain and the carboxy-terminal mature myostatin domain in vitro and allowing them to fold properly. See, e.g., Sengle et al., J. Biol. Chem., 286(7):5087-5099, 2011. Exemplary latent myostatin sequences, variants thereof, and methods for producing latent myostatin are well known in the art and are described in more detail herein.

[0117] As used herein, the term "pro / 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 promyostatin and latent myostatin.

[0118] As used herein, the terms "pure promyostatin" or "pure pro-GDF8" refer 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 pure promyostatin. In other words, such antibodies bind to promyostatin in a composition that lacks other forms of myostatin, latent myostatin, and mature myostatin.

[0119] As used herein, the term "proprotein convertase cleavage site" refers to the site at which promyostatin is cleaved by a proprotein convertase. In one embodiment, the proprotein convertase cleavage site is the conserved RXXR site between the prodomain and the biologically active domain, i.e., mature myostatin. See, e.g., Figures 1A, 1B, and 2.

[0120] As used herein, the term "BMP / thrombopoietin family cleavage site" refers to the site at which latent myostatin is cleaved by a BMP / thrombopoietin family member. In one embodiment, the BMP / thrombopoietin family member is mTLL-2. See, e.g., Figures 1A, 1B, and 2.

[0121] Antibodies that bind to pro / latent myostatin The present disclosure is based, at least in part, on the surprising discovery that certain pro / latent myostatin-specific antibodies (e.g., the antibody designated herein as Ab1) prevented the proteolytic activation of pro / latent myostatin to mature myostatin. Furthermore, inhibition of myostatin activation using such antibodies was effective in increasing muscle mass in both dexamethasone- and cast-induced muscle atrophy mouse models. Aspects of the Disclosure provides antibodies (e.g., antibodies and antigen-binding fragments) that bind to pro / latent myostatin and inhibit the proteolytic activation of pro / latent myostatin to mature myostatin.

[0122] Antibodies (used interchangeably with the plural) are immunoglobulin molecules capable of specifically binding 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 intact (e.g., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (Fab, Fab', F(ab')2, Fv, etc.), single chains (scFv), variants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configurations of immunoglobulin molecules containing an antigen recognition site of the desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies include antibodies of any class (or subclass thereof), such as IgD, IgE, IgG, IgA, or IgM, and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chain, immunoglobulins can be assigned to various classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known.

[0123] The term "isolated antibody," as used herein, 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 to pro / latent myostatin is substantially free of antibodies that specifically bind to antigens other than pro / latent myostatin). However, an isolated antibody that specifically binds to pro / latent myostatin may have cross-reactivity to other antigens, such as pro / latent myostatin molecules from other species. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0124] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences and fragments thereof. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences, for example in the CDRs, particularly CDR3 (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). 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, have been grafted onto human framework sequences.

[0125] The term "epitope" includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitopic 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 and / or charge characteristics. An epitope is the region of an antigen to which an antibody binds. 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.

[0126] The antibodies described herein are capable of binding to pro / latent myostatin, thereby inhibiting the proteolytic activation of pro / latent myostatin to mature myostatin. In some cases, the antibodies described herein can inhibit proteolytic activation of pro / latent myostatin by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some cases, the antibodies described herein can inhibit proteolytic cleavage of promyostatin by proprotein convertases (e.g., furin) by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some cases, the antibodies described herein can inhibit proteolytic cleavage of promyostatin or latent myostatin by tolloid proteases (e.g., mTLL2) by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. The inhibitory activity of anti-pro / latent myostatin antibodies can be measured by routine methods, such as Western blot analysis as described in Example 1 and Figure 3. However, it should be understood that additional methods can be used to measure the inhibitory activity of anti-pro / latent myostatin antibodies on proteolytic cleavage of pro / latent myostatin. In some embodiments, inhibition of pro / latent myostatin cleavage (e.g., by proprotein convertase and / or thrombocytopenic protease) provides a measure of inhibitor potency and can be reflected as an inhibition constant (Ki), which is the concentration of inhibitor (e.g., anti-pro / latent myostatin antibody) required to reduce the activity of a protease (e.g., of a proprotein convertase or thrombocytopenic protease) by half, and is independent of enzyme or substrate concentration.

[0127] In some embodiments, the proprotein convertase comprises (i) a catalytic domain that hydrolyzes the peptide bond of a protein containing a proprotein convertase cleavage site, and (ii) a binding pocket that binds to an rTGF having a proprotein convertase cleavage site. Examples of proprotein convertases for use according to the present disclosure include, but are not limited to, PCSK5 / 6, PACE4, PACE7, and PACE3 (e.g., furin). The proprotein convertase, in some embodiments, is obtained from any mammal, including, but not limited to, a human, monkey, or rodent (e.g., mouse, rat, hamster).

[0128] In some embodiments, the proprotein convertase is homologous to a proprotein convertase selected from the group consisting of PCSK5 / 6, PACE4, PACE7, and PACE3 (e.g., furin). For example, the proprotein convertase may 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).

[0129] A proprotein convertase cleavage site, in some embodiments, is an amino acid sequence that can be cleaved by a proprotein convertase (e.g., PCSK5 / 6, PACE4, PACE7, and PACE3). In some embodiments, a proprotein convertase cleavage site comprises the amino acid sequence RXXR, where R is arginine and X is any amino acid. In some embodiments, a proprotein convertase cleavage site comprises the amino acid sequence RX-(K / R)-R, where R is arginine, K is lysine, and X is any amino acid. In some embodiments, a proprotein convertase cleavage site comprises the amino acid sequence RVRR (SEQ ID NO: 57), where 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, a proprotein convertase cleavage site comprises the amino acid sequence RSRR (SEQ ID NO: 56).

[0130] In some embodiments, toroid proteases for use according to the present disclosure include, but are not limited to, BMP-1, mTLL-1, and mTLL-2. The toroid 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 toroid protease is homologous to a toroid protease selected from the group consisting of BMP-1, mTLL-1, and mTLL-2. For example, the toroid 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.

[0131] A tolloid protease cleavage site, in some embodiments, 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, a tolloid cleavage site comprises the amino acid sequence QR, where Q is glutamine and R is arginine.

[0132] In some embodiments, the antibodies described herein can bind to pro / latent myostatin, thereby inhibiting myostatin activity. In some cases, the antibodies described herein can inhibit myostatin signaling by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some embodiments, inhibition of myostatin signaling can be measured by routine methods, for example, using the myostatin activation assay described in Example 1. However, it should be understood that additional methods can be used to measure myostatin signaling activity.

[0133] It should be understood that the extent of proteolytic cleavage of myostatin, for example, by proprotein convertases and / or thrombin 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 factors (e.g., mature myostatin). As another example, antibodies that specifically bind to promyostatin, latent myostatin, and / or mature myostatin can be used in ELISA to measure the level of a particular form of myostatin (e.g., pro / latent / mature myostatin) and to quantitate 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, fluorescence anisotropy-based techniques, FRET assays, hydrogen-deuterium exchange mass spectrometry, and / or NMR spectroscopy.

[0134] 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, called lambda and kappa, can be found in antibodies. Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Each light chain contains an N-terminal variable (V) domain (V L ) and constant (C) domains (C L Each heavy chain typically comprises an N-terminal V domain (V H ), three or four C domains (C H 1-3) and a hinge region. H The most proximal C H The domain is CH It is named 1. V H and V L The domains are composed of relatively conserved regions called framework regions (FR1, FR2, FR3, and FR4) that form a scaffold for three regions of hypervariable sequence (complementarity determining regions, CDRs). The CDRs consist of four regions of sequence. The CDRs contain most of the residues involved in 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. The CDRs are listed in the Sequences of Proteins of Immunological The term typically refers to the Kabat CDRs described in "Interest, US Department of Health and Human Services" (1991), edited by Kabat et al. Another standard for characterizing antigen-binding sites refers to the hypervariable loops described by Chothia. See, for example, 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, for example, "Protein Sequence and Structure Analysis of Antibody Variable Domains" in Antibody Engineering Lab Manual (eds. Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). Embodiments described with respect to Kabat CDRs may alternatively be implemented using similarly described relationships with respect to Chothia hypervariable loops or with respect to AbM-defined loops, or with respect to any combination of these methods.

[0135] In some embodiments, the anti-pro / latent myostatin antibodies of the present disclosure and the nucleic acid molecules of the present disclosure encoding the antibodies comprise the CDR amino acid sequences shown in Table 1. [Table 1]

[0136] In Table 1, the single sequences for CDRH3 and CDRL3 reflect Kabat and IMGT.

[0137] In some embodiments, the anti-pro / latent myostatin binding agents (e.g., antibodies) of the present disclosure , any antibody (including antigen-binding fragment) comprising the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or combinations thereof, provided in any one of the antibodies shown in Table 1. In some embodiments, an anti-pro / latent myostatin binding agent comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of any one of the antibodies shown in Table 1. The present disclosure also includes any nucleic acid sequence encoding a molecule comprising the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3 of any one of the antibodies shown in Table 1. The antibody heavy and light chain CDR3 domains may play a particularly important role in the binding specificity / affinity of the antibody for the antigen. Thus, an anti-pro / latent myostatin binding agent or nucleic acid molecule thereof of the present disclosure may comprise at least the heavy and / or light chain CDR3 of an antibody shown in Table 1.

[0138] Embodiments of the present disclosure relate to a monoclonal antibody or antigen-binding fragment that binds to pro / latent myostatin protein and comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3.

[0139] 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.

[0140] In some embodiments (e.g., for the anti-pro / latent myostatin antibody Ab1 shown in Table 1), CDRH1 comprises the sequence set forth in SEQ ID NO: 1 or 2, CDRH2 comprises the sequence set forth in SEQ ID NO: 4 or 5, CDRH3 comprises the sequence set forth in SEQ ID NO: 10, CDRL1 comprises the sequence set forth in SEQ ID NO: 12 or 13, CDRL2 comprises the sequence set forth in SEQ ID NO: 18 or 19, and CDRL3 comprises the sequence set forth in SEQ ID NO: 22, and the antibody binds to pro / latent myostatin.

[0141] In some embodiments (e.g., for anti-pro / latent myostatin antibody Ab3 shown in Table 1), CDRH1 comprises the sequence set forth in SEQ ID NO: 1 or 3, CDRH2 comprises the sequence set forth in SEQ ID NO: 6 or 7, CDRH3 comprises the sequence set forth in SEQ ID NO: 11, CDRL1 comprises the sequence set forth in SEQ ID NO: 14 or 15, 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, and the antibody binds to pro / latent myostatin.

[0142] In some embodiments (e.g., for anti-pro / latent myostatin antibody Ab5 shown in Table 1), 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, and the antibody binds to pro / latent myostatin. In some examples, any of the anti-pro / latent myostatin binding agents (e.g., antibodies) of the disclosure include any antibody (including antigen-binding fragment) having one or more CDR (e.g., CDRH or CDRL) sequences substantially similar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3. For example, an antibody may comprise one or more CDR sequences set forth in Table 1 (SEQ ID NOs: 1-23) containing up to 5, 4, 3, 2, or 1 amino acid residue variation compared to the corresponding CDR region of any one of SEQ ID NOs: 1-23. The complete amino acid and nucleic acid sequences for the heavy and light chain variable regions of the antibodies listed in Table 1 are provided below. will be provided to. [ka] [ka] [ka] [ka] [ka] [ka]

[0143] In some embodiments, anti-pro / latent myostatin antibodies of the present disclosure include any antibody comprising a heavy chain variable domain of any one of SEQ ID NOs: 24-29 or a light chain variable domain of any one of SEQ ID NOs: 30-35. In some embodiments, anti-pro / latent myostatin antibodies of the present disclosure include any antibody comprising a heavy chain variable and light chain variable pair of SEQ ID NOs: 24 and 30; 25 and 31; 26 and 32; 27 and 33; 28 and 34; or 29 and 35).

[0144] Aspects of the present disclosure provide anti-pro / latent myostatin antibodies having heavy and / or light chain variable amino acid sequences homologous to any of those described herein. In some embodiments, the anti-pro / latent myostatin antibodies comprise a heavy chain variable sequence or a 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 one of SEQ ID NOs: 30-35. In some embodiments, the homologous heavy chain variable and / or light chain variable amino acid sequence is unchanged within any of the CDR sequences provided herein. For example, in some embodiments, a degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) may occur within the heavy and / or light chain variable sequences to the exclusion of any of the CDR sequences provided herein.

[0145] 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 as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such algorithms are described in Altschul et al., J. Mol. Biol. 215:403-10, 1990, incorporated into the NBLAST and XBLAST programs (version 2.0). BLAST protein searches can be performed with the XBLAST program, score=50, word length=3, to obtain amino acid sequences homologous to the protein molecule of interest. When gaps exist between the two sequences, gapped BLAST can be performed as described by 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 (eg, XBLAST and NBLAST) can be used.

[0146] In some embodiments, conservative mutations may be introduced into CDR or framework sequences at positions where the residue is unlikely to be involved in interactions with pro / latent myostatin as determined based on the crystal structure. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants may be made in accordance with references summarizing such methods, such as Molecular Cloning: A Laboratory Manual, eds. J. Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or may be prepared according to methods for altering polypeptide sequences known to those of skill in the art, such as those found in Current Protocols in Molecular Biology, edited by F. M. Ausubel et al., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made between amino acids within 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.

[0147] In some embodiments, the antibodies provided herein contain mutations that confer desirable properties to the antibody. For example, to avoid potential difficulties with Fab arm exchange, which is known to occur in natural IgG4 mAbs, the antibodies provided herein may contain a stabilizing "Adair" mutation in which serine 228 (EU numbering, Kabat numbering residue 241) is converted to proline to create an IgG1-like (CPPCP (SEQ ID NO: 58)) hinge sequence (Angal S. et al., "A single amino acid substitution abolishes the heterogeneity of "Chimeri mouse / human (IgG4) antibody," Mol Immunol 30:105-108; 1993). Thus, any of the antibodies may contain the stabilizing "Adair" mutation or the amino acid sequence CPPCP (SEQ ID NO:58).

[0148] The anti-pro / latent myostatin binding agents of the present disclosure may optionally comprise an antibody constant region or portion thereof. L The domain may be attached at its C-terminus to a light chain constant domain such as Cκ or Cλ. H The domain or a portion thereof can be attached to all or part of a heavy chain, such as IgA, IgD, IgE, IgG, and IgM, and any isotype subclass. The antibody can include a suitable constant region (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within this scope can include a V domain combined with any suitable constant region. H and V L domain, or an antigen-binding portion thereof.

[0149] In certain embodiments, V H and / or V LDomains may be reverted to germline sequences, e.g., the FRs of these domains are mutated using conventional molecular biology techniques to match those produced by germline cells. H and / or V L The domains may be reverted to the germline sequences of IgHV3-30 (SEQ ID NO: 36) and / or IgLV1-44 (SEQ ID NO: 37), respectively. H and / or V L It should be understood that any of the domains may be reverted to any suitable germline sequence. In other embodiments, the FR sequences remain deviated from the consensus germline sequence. [ka]

[0150] In some embodiments, the anti-pro / latent myostatin antibody or antigen-binding fragment comprises a sequence It may or may not include the framework regions of the antibodies shown in columns 24 to 35. In some embodiments, the anti-pro / latent myostatin antibody is a murine antibody and includes murine framework region sequences.

[0151] In some embodiments, the anti-pro / latent myostatin antibodies of the present disclosure are capable of binding with relatively high affinity, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11The anti-pro / latent myostatin antibody can bind to pro / latent myostatin with a Kd of less than 100 nM or less. For example, an anti-pro / latent myostatin antibody can bind to pro / latent myostatin with an affinity of 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 pro / 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 anti-pro / latent myostatin antibodies may be tested using any suitable method, including, but not limited to, biosensor technology (e.g., OCTET or BIACORE).

[0152] An antibody that "specifically binds" to a target antigen binds to the target antigen with greater affinity, avidity, more readily, and / or with longer duration than it binds to a non-target antigen. In some embodiments, antibodies that specifically bind to pro / latent myostatin are disclosed herein. In some embodiments, any of the antibodies provided herein binds to or near the toroid cleavage site or toroid docking site of pro / latent myostatin. In some embodiments, an antibody binds near the toroid cleavage site or toroid docking site if it binds within 15 or fewer amino acid residues of the toroid cleavage site or toroid 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 toroid cleavage site or toroid docking site. In some embodiments, an antibody binds to or near the toroid cleavage site of GDF8. For example, the antibody can bind to the amino acid sequence set forth in SEQ ID NO: 62 PKAPPLRELIDQYDVQRDDSSDGSLEDDDYHAT (SEQ ID NO: 62). In other embodiments, any of the antibodies provided herein bind to or near the proprotein convertase cleavage site or proprotein convertase docking site of pro / latent myostatin. In some embodiments, the antibody binds near the proprotein convertase cleavage site or proprotein convertase docking site if it binds within 15 or fewer amino acid residues of the proprotein convertase cleavage site or 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 proprotein convertase docking site. In some embodiments, the antibody binds to or near the proprotein convertase cleavage site of GDF8. For example, the antibody can bind to the amino acid sequence set forth in SEQ ID NO: 63. GLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRC (SEQ ID NO: 63).

[0153] In one example, the anti-pro / latent myostatin antibodies described herein specifically bind to pro / latent myostatin relative to other forms of myostatin and / or other members of the TGFβ family of growth factors, including, but 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, GDF8A, GDF8B, GDF9A, GDF10B, GDF11C, GDF11D, GDF11E, GDF11F, GDF11G, GDF11H ... Examples of antibodies that can bind to pro / latent myostatin include DF8, GDF9, GDNF, INHA, INHBA, INHBB, INHBC, INHBE, LEFTY1, LEFTY2, NODAL, NRTN, PSPN, TGFβ1, TGFβ2, and TGFβ3 proteins. Such antibodies can bind to pro / latent myostatin with 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 pro / latent myostatin with at least 1,000-fold higher affinity than other members of the TGFβ family of growth factors. In some embodiments, the antibodies provided herein can bind to pro / latent myostatin with 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 pro / latent myostatin with at least 1,000-fold higher affinity than one or more forms of GDF11 (e.g., proGDF11, latent GDF11, or mature GDF11) or mature myostatin. Alternatively or additionally, the antibody may exhibit much greater inhibitory activity (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold greater) against proteolytic cleavage of pro / latent myostatin (e.g., by proprotein convertases or thrombin proteases) compared to other members of the TGFβ family, such as pro / latent GDF11.

[0154] In some embodiments, antibodies bind to antigens but cannot effectively remove antigens from plasma. Thus, in some embodiments, the concentration of antigens in plasma can be increased by reducing antigen clearance. However, in some embodiments, the antibodies provided herein (e.g., sweeping antibodies) have pH-sensitive affinity for antigens. Such pH-sensitive antibodies can bind to antigens in plasma at neutral pH and dissociate from the antigen in acidic endosomes, thereby reducing antibody-mediated antigen accumulation and / or promoting antigen clearance from plasma.

[0155] Aspects of the present disclosure relate to sweeping antibodies. As used herein, "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 receptor (FcRn) at neutral or physiological pH. In some embodiments, the sweeping antibody binds to neonatal Fc receptor FcRn at neutral pH. For example, the sweeping antibody can bind to FcRn at a pH ranging from 7.0 to 7.6. In some embodiments, the sweeping antibody can bind to an antigen at the antigen-binding site and to cellular FcRn via the Fc portion of the antibody. In some embodiments, the sweeping antibody can then release the antigen into acidic endosomes, where it may be internalized and degraded. In some embodiments, the sweeping antibody no longer binds to the antigen and can then be released back into the serum by cells (e.g., by exocytosis).

[0156] In some embodiments, FcRn in the vascular endothelium (e.g., of a subject) increases the half-life of the sweeping antibody. In some embodiments, vascular endothelial cells internalize the sweeping antibody, which binds to an antigen such as myostatin (e.g., promyostatin, latent myostatin, or primed myostatin). In some embodiments, the sweeping antibody is recycled back 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 refers to the antibody from which the sweeping antibody is derived (e.g., before engineering the Fc portion of the conventional antibody to bind FcRn with greater affinity at pH 7). In some embodiments, the sweeping antibody has at least 1%, 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 100%, 150%, 150%, 250%, 350%, 500%, 15 ... have a half-life that is 150%, 200% or 250% longer.

[0157] 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 a pH of 7.4. -3 M to 10 -8 In some embodiments, the sweeping antibody binds to FcRn with a Kd in the range of 10 M at a pH of 7.4. -3 M to 10 -7 M, 10 -3 M to 10 -6 M, 10 -3 M to 10 -5 M, 10 -3 M to 10 -4 M, 10 -4 M to 10 -8 M, 10 -4 M to 10 -7 M, 10 -4 M to 10 -6 M, 10 -4 M to 10 -5 M, 10 -5 M to 10 -8 M, 10 -5M to 10 -7 M, 10 -5 M to 10 -6 M, 10 -6 M to 10 -8 M, 10 -6 M to 10 -7 M or 10 -7 M to 10 -8 In some embodiments, the sweeping antibody Fc region binds with a Kd in the range of M. In some embodiments, the FcRn binds to the CH2-CH3 hinge region of the sweeping antibody. In some embodiments, the FcRn binds to the same region as Protein A or Protein G. In some embodiments, the FcRn binds to a binding site different from that of FcγR. In some embodiments, amino acid residue AA of the sweeping antibody Fc region is required for binding to FcRn. In some embodiments, amino acid residue AA of the sweeping antibody Fc region affects binding to FcRn.

[0158] 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 to FcRn is increased to enhance their pharmacokinetic (PK) properties compared to their conventional counterparts. For example, in some embodiments, the sweeping antibodies induce fewer adverse reactions due to efficacy at lower doses. In some embodiments, the sweeping antibodies are administered less frequently. In some embodiments, the transcytosis of the sweeping antibodies into certain tissue types is increased. In some embodiments, the sweeping antibodies enhance the efficiency of transplacental delivery. In some embodiments, the sweeping antibodies are less expensive to produce.

[0159] In some embodiments, any of the antibodies provided herein are engineered to bind to FcRn with low affinity. In some embodiments, any of the antibodies provided herein are engineered to bind to FcRn with low affinity at pH 7.4. In some embodiments, the affinity of the sweeping antibodies to FcRn is reduced to shorten their pharmacokinetic (PK) properties compared to their conventional counterparts. For example, in some embodiments, the sweeping antibodies are more rapidly cleared 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 that may result from transplacental transport of material fetal-specific antibodies.

[0160] In some embodiments, the sweeping antibody has decreased affinity for the antigen at low pH compared to neutral or physiological pH (e.g., pH 7.4). In some embodiments, the sweeping antibody has decreased affinity for the antigen at acidic pH (e.g., a pH ranging from 5.5 to 6.5) compared to physiological pH (e.g., pH 7.4). It should be understood that any of the antibodies provided herein may be engineered to dissociate from the antigen in response to a change in pH (e.g., a pH-sensitive antibody). In some embodiments, the sweeping antibody provided herein is engineered to bind to the antigen in response to pH. In some embodiments, the sweeping antibody provided herein is engineered to bind to FcRn in response to pH. In some embodiments, the sweeping antibody provided herein is internalized by endocytosis. In some embodiments, the sweeping antibody provided herein is internalized by FcRn binding. In some embodiments, the sweeping antibody provided herein is internalized by endocytosis. The endocytosed sweeping antibody releases the antigen into the endosome. In some embodiments, the sweeping antibody is recycled back to the cell surface. In some embodiments, the sweeping antibody remains bound to the cell. In some embodiments, the endocytosed sweeping antibody is recycled back to the plasma. It should be understood that the Fc portion of any of the antibodies provided herein may be engineered to have different FcRn binding activities. In some embodiments, the FcRn binding activity affects the clearance time of the antigen by the sweeping antibody. In some embodiments, the sweeping antibody may be a long-acting or fast-acting sweeping antibody.

[0161] 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-, 2-, 3-, 4-, 5-, 6-, 8-, 10-, 15-, 20-, 50-, or 100-fold.

[0162] In some embodiments, selecting an appropriate dose of the sweeping antibody for treatment can be performed empirically. In some embodiments, a high dose of the sweeping antibody may saturate FcRn, resulting in an antibody that stabilizes the antigen in serum without internalizing it. In some embodiments, a low dose of the 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 ten weeks, once every 12 weeks, once every 16 weeks, once every 20 weeks, or once every 24 weeks.

[0163] In some embodiments, any of the antibodies provided herein may be modified or engineered to become a sweeping antibody. In some embodiments, any of the antibodies provided herein may be converted into a sweeping antibody using any suitable method. For example, suitable methods for generating sweeping antibodies have been previously described in Igawa et al. (2013) "Engineered Monoclonal Antibody with Novel Antigen-Sweeping Activity In Vivo," PLoS ONE, Vol. 8(5):e63236; and Igawa et al., "pH-dependent antigen-binding antibodies as a novel therapeutic modality," Biochimica et Biophysica Acta, Vol. 1844 (2014) pp. 1943-1950, the contents of each of which are incorporated herein by reference. However, it should be understood that the methods for generating sweeping antibodies provided herein are not meant to be limiting. Accordingly, additional methods for generating sweeping antibodies are within the scope of this disclosure.

[0164] Some aspects of the present disclosure are based on the recognition that the affinity (e.g., expressed as Kd) of any of the anti-pro / latent myostatin antibodies provided herein is sensitive to changes in pH. In some embodiments, the antibodies provided herein have an increased Kd for binding to pro / latent myostatin at relatively low pH (e.g., a pH in the range of 4.0 to 6.5) compared to relatively high pH (e.g., a pH in the range of 7.0 to 7.4). In some embodiments, the antibodies provided herein have an increased Kd for binding to pro / latent myostatin at a pH between 4.0 and 6.5, with an increased Kd of 10 or more. -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 In some embodiments, the antibodies provided herein have a Kd for binding to pro / latent myostatin in the range of 10 M when the pH is between 7.0 and 7.4. -6M, 10 -7 M , 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M. In some embodiments, the antibodies provided herein have a Kd for binding to pro / 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 10000-fold greater between pH 4.0 and 6.5 compared to between pH 7.0 and 7.4.

[0165] In some embodiments, provided herein are pro / latent myostatin antibodies that do not specifically bind to an epitope within the amino acid sequence set forth in (SEQ ID NO: 64). In some embodiments, the pro / latent myostatin antibodies provided herein do not specifically bind to the same epitope as an antibody set forth in Tables 2a, 11a, 11b, or 13 of International Patent Application Publication No. WO2016 / 098357, published June 23, 2016, based on International Patent Application No. PCT / JP2015 / 006323, filed December 18, 2015. In some embodiments, the pro / latent myostatin antibodies provided herein do not compete or cross-compete for binding to the same epitope as an antibody described in Tables 2a, 11a, 11b, or 13 of International Patent Application Publication No. WO2016 / 098357, published on June 23, 2016, based on International Patent Application No. PCT / JP2015 / 006323, filed on December 18, 2015. In some embodiments, the pro / latent myostatin antibodies provided herein do not specifically bind to the same epitope as an antibody comprising a VH and VL pair described in Tables 2a, 11a, 11b, or 13 of International Patent Application Publication No. WO2016 / 098357, published on June 23, 2016, based on International Patent Application No. PCT / JP2015 / 006323, filed on December 18, 2015. In some embodiments, the pro / latent myostatin antibodies provided herein do not compete or cross-compete for binding to the same epitope as antibodies comprising a VH and VL pair set forth in Tables 2a, 11a, 11b, or 13 of International Patent Application Publication No. WO2016 / 098357, published June 23, 2016, based on International Patent Application No. PCT / JP2015 / 006323, filed December 18, 2015.

[0166] Polypeptides Some aspects of the present disclosure relate to a polypeptide 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 variable heavy chain 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.

[0167] Some aspects of the present disclosure relate to a polypeptide 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 variable light chain 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.

[0168] Antibodies that compete with anti-pro / latent myostatin antibodies Aspects of the present disclosure relate to antibodies that compete or cross-compete with any of the antibodies provided herein. As used herein with respect to antibodies, the term "compete" 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 that the result of the binding of the first antibody to that 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. The binding of the second antibody to that epitope is also detectably reduced in the presence of the first antibody. Although this is possible, it is not necessary that other antibodies be competitive. 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 respective epitope. However, if each antibody detectably inhibits the binding of the other antibody to its epitope or ligand, whether to the same extent, a greater extent, or a lesser extent, the antibodies are said to "cross-compete" with each other for binding to their respective epitope(s). 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 change, or binding to a common epitope or portion thereof), those skilled in the art will understand that such competing and / or cross-competing antibodies are encompassed and may be useful in the methods and / or compositions provided herein.

[0169] 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 at or near the same epitope as any of the antibodies provided herein. In some embodiments, an antibody binds near an epitope if it binds within 15 or fewer amino acid residues of the epitope. In some embodiments, any of the antibodies provided herein binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the epitope bound by any of the antibodies provided herein.

[0170] In another embodiment, the antibody is -6 In other embodiments, the antibody competes or cross-competes for binding to any of the antigens provided herein (e.g., pro / latent myostatin) with an equilibrium dissociation constant, Kd, ​​between the antibody and the protein of less than 10 M. -11 M to 10 -6 Compete or cross-compete for binding to any of the antigens provided herein with a Kd in the range of M.

[0171] Aspects of the present disclosure relate to antibodies that compete for binding to pro / latent myostatin with any of the antibodies provided herein. In some embodiments, the antibody binds to pro / latent myostatin at the same epitope as any of the antibodies provided herein. For example, in some embodiments, any of the antibodies provided herein binds to or near the tolloid cleavage site or tolloid docking site of pro / latent myostatin. In other embodiments, any of the antibodies provided herein bind to or near the proprotein convertase cleavage site or proprotein convertase docking site of pro / latent myostatin. In another embodiment, the antibody binds to or near the proprotein convertase cleavage site or proprotein convertase docking site of pro / latent myostatin. -6 In other embodiments, an antibody that competes with any of the antibodies provided herein competes for binding to pro / latent myostatin with an equilibrium dissociation constant Kd between the antibody and pro / latent myostatin of less than 10 M. -11 M to 10 -6 It binds to pro / latent myostatin with a Kd in the range of M.

[0172] Any of the antibodies provided herein may be characterized using any suitable method. For example, one method is to identify the epitope to which the antigen binds, i.e., "epitope mapping." There are numerous suitable methods for mapping and characterizing the location of epitopes on proteins, including analyzing the crystal structure of an antibody-antigen complex, competitive assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, "Using Antibodies," a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In a further example, epitope mapping can be used to determine the sequence to which an antibody binds. The epitope may be a linear epitope, i.e., a conformation formed by three-dimensional interactions of amino acids that may not necessarily be contained in a single stretch (primary structure linear sequence) of amino acids. The epitope may be a conformational epitope. Peptides of various lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used in antibody binding assays. In another example, the epitope to which an antibody binds can be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and determining antibody binding. In a gene fragment expression assay, the open reading frame encoding the target antigen is fragmented, either randomly or by specific genetic construction, and the reactivity of the expressed antigen fragments with the antibody being tested is determined. The gene fragments can be generated, for example, 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 fragments is then determined by immunoprecipitation and gel electrophoresis. Specific epitopes can also be identified using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, defined libraries of overlapping peptide fragments can be tested for binding to a test antibody in a simple binding assay. In additional examples, mutagenesis of the antigen-binding domain, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and / or necessary for epitope binding. For example, domain swapping experiments can be performed using mutants of the target antigen in which various fragments of the pro / latent myostatin polypeptide are replaced (swapped) with sequences from a closely related but antigenically distinct protein, such as another member of the TGFβ protein family (e.g., GDF11). By assessing the binding of the antibody to mutant pro / latent myostatin, the importance of specific antigen fragments to antibody binding can be assessed.

[0173] Alternatively, a competition assay may be performed using other antibodies known to bind to the same antigen to determine whether the antibody binds to the same epitope as the other antibody. Competition assays are well known to those of skill in the art.

[0174] Any suitable method, such as the epitope mapping methods described herein, can be applied to determine whether an anti-pro / latent myostatin antibody binds to one or more of the specific residues / segments in pro / latent myostatin described herein. Furthermore, the interaction of the antibody with one or more of these defined residues in pro / latent myostatin can be determined by routine techniques. For example, a crystal structure can be determined, and the distance between a residue in pro / latent myostatin and one or more residues in the antibody can be determined accordingly. Based on such distance, it can be determined whether a particular residue in pro / 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 the preferential binding of a candidate anti-pro / latent myostatin antibody to pro / latent myostatin compared to another target, such as mutant pro / latent myostatin.

[0175] Production of antibodies that bind to pro- and latent myostatin Numerous methods can be used to obtain the antibodies or antigen-binding fragments thereof of the present disclosure. For example, antibodies can be produced using recombinant DNA methods. Monoclonal antibodies can also be produced by generating hybridomas using known methods (see, e.g., Kohler and Milstein (1975) Nature, 256:495-499). Hybridomas formed in this manner 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 particular antigen. Any form of a particular antigen can be used as an immunogen, including recombinant antigens, naturally occurring forms, any variants or fragments thereof, and antigenic peptides thereof (e.g., any of the epitopes described herein within a scaffold, either as a linear epitope or as a conformational epitope). Antibodies can be generated using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (e.g., OCTET or BIACORE) analysis, to identify hybridomas that produce antibodies that specifically bind to a particular antigen. Any form of a particular antigen can be used as an immunogen, including recombinant antigens, naturally occurring forms, any variants or fragments thereof, and antigenic peptides thereof (e.g., any of the epitopes described herein within a scaffold, either as a linear epitope or as a conformational epitope). One exemplary method involves screening a protein expression library, such as a phage or ribosome display library, that expresses antibodies or fragments thereof (e.g., scFvs). Phage display is described, for example, in Ladner et al., U.S. Patent 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; WO92 / 18619; WO91 / 17271; WO92 / 20791; WO92 / 15679; WO93 / 01288; WO92 / 01047; WO92 / 09690; and WO90 / 02809.

[0176] In addition to using display libraries, a particular antigen (e.g., promyostatin) may be used to immunize a non-human animal, such as a rodent, such as a mouse, hamster, or rat. In one embodiment, the non-human animal is a mouse.

[0177] In another embodiment, monoclonal antibodies are obtained from non-human animals and then modified (e.g., chimeric) using suitable recombinant DNA techniques. Various approaches for producing 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., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., European Patent Publication Nos. EP 171496, 0173494, and GB 2177096B.

[0178] 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 particular characteristics of the antibodies.

[0179] Some aspects of the present disclosure relate to host cells transformed with a polynucleotide or vector. The host cell may be a prokaryotic or eukaryotic cell. The polynucleotide or vector present in the host cell may be integrated into the host cell's genome or maintained extrachromosomally. The host cell may be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal, or human cell. In some embodiments, the fungal cell is, for example, of the genus Saccharomyces, specifically the species S. cerevisiae. The term "prokaryote" includes all bacteria that can be transformed or transfected with DNA or RNA molecules for expression of antibodies or corresponding immunoglobulin chains. Prokaryotic hosts may include gram-negative and gram-positive bacteria, such as E. coli, S. typhimurium, Serratia marcescens, and Bacillus subtilis. The term "eukaryote" includes yeast, higher plants, insects, and vertebrate cells, such as mammalian cells, such as NS0 and CHO cells. Depending on the host employed in a recombinant production procedure, the antibody or immunoglobulin chain encoded by the polynucleotide may be glycosylated or may be non-glycosylated. The antibody or the corresponding immunoglobulin chain may also include an initial methionine amino acid residue.

[0180] In some embodiments, once the vector has been incorporated into a suitable host, the host may be maintained under conditions suitable for high level expression of the nucleotide sequence, followed, if desired, by recovery and purification of immunoglobulin light chains, heavy chains, light / heavy chain dimers or intact antibodies, antigen-binding fragments, or other immunoglobulin forms; see Beychok, Cells of Immunoglobulin Synthesis, Academic Press, NY, (1979). Thus, the polynucleotide or vector may be The host cells may then be introduced into cells that produce antibodies or antigen-binding fragments. Furthermore, transgenic animals, preferably mammals, containing the aforementioned host cells may be used for the large-scale production of antibodies or antibody fragments.

[0181] Transformed host cells may be grown in fermentors and cultured using any suitable technique to achieve optimal cell growth. Once expressed, whole antibodies, their dimers, individual light and heavy chains, other immunoglobulin forms, or antigen-binding fragments may be purified by standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like; see Scopes, "Protein Purification," Springer Verlag, NY (1982). The antibodies or antigen-binding fragments may then be isolated from the growth medium, cell lysates, or cell membrane fractions. Isolation and purification of antibodies or antigen-binding fragments expressed, for example, in microorganisms, may be by any conventional means, including, for example, preparative chromatographic separations and immunological separations, including, for example, those involving the use of monoclonal or polyclonal antibodies directed against the constant regions of the antibodies.

[0182] Aspects of the present disclosure relate to hybridomas that provide an indefinitely sustainable source of monoclonal antibodies. As an alternative to obtaining immunoglobulins directly from hybridoma cultures, immortalized hybridoma cells may be used as a source of rearranged heavy and light chain loci for subsequent expression and / or genetic manipulation. Rearranged antibody genes may be reverse transcribed from the appropriate mRNA to produce cDNA. In some embodiments, the heavy chain constant region may be replaced with one of a different isotype or removed entirely. Variable regions may be linked to encode a single-chain Fv region. Multiple Fv regions may be linked to confer binding ability to more than one target, or chimeric heavy and light chain combinations may be used. Any suitable method may be used for cloning antibody variable regions and generating recombinant antibodies.

[0183] In some embodiments, appropriate nucleic acids encoding the heavy and / or light chain variable regions are obtained and inserted into expression vectors that can be transfected into standard recombinant host cells. A variety of such host cells may be used. In some embodiments, mammalian host cells may be advantageous for efficient processing and production. Exemplary mammalian cell lines useful for this purpose include CHO cells, 293 cells, or NSO cells. Production of antibodies or antigen-binding fragments may be carried out by culturing the modified recombinant host under culture conditions appropriate for host cell growth and expression of the coding sequences. Antibodies or antigen-binding fragments may be recovered by isolating them from the culture. Expression systems may be designed to include a signal peptide so that the resulting antibody is secreted into the medium; however, intracellular production is also possible.

[0184] The present disclosure also includes polynucleotides encoding at least the variable regions of the immunoglobulin chains of the antibodies described herein. In some embodiments, the variable regions encoded by the polynucleotides comprise at least one complementarity-determining region (CDR) of the VH and / or VL variable regions of the antibodies produced by any one of the hybridomas described above.

[0185] The polynucleotide encoding the antibody or antigen-binding fragment may be, for example, DNA, cDNA, RNA, or synthetically produced DNA or RNA, or a recombinantly produced chimeric nucleic acid molecule comprising any of these polynucleotides, alone or in combination. In some embodiments, the polynucleotide is part of a vector. Such vectors may contain additional genes, such as marker genes, that allow for the selection of the vector in a suitable host cell and under suitable conditions.

[0186] In some embodiments, the polynucleotide is operably linked to an expression control sequence that allows expression in prokaryotic or eukaryotic cells. Expression of the polynucleotide includes transcription of the polynucleotide into translatable mRNA. Control elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They may include a control sequence that promotes transcription initiation and, optionally, a polyA signal that promotes transcription termination and transcript stabilization. Additional control elements may include transcriptional and translational enhancers and / or naturally associated or heterologous promoter regions. Possible control elements that allow expression in prokaryotic host cells include, for example, the PL, Lac, Trp, or Tac promoters in E. coli. Examples of control elements that allow expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast, or the CMV promoter, SV40 promoter, RSV promoter (Rous sarcoma virus), CMV enhancer, SV40 enhancer, or globin intron in mammalian and other animal cells.

[0187] Such control elements, in addition to those involved in the initiation of transcription, may also include transcription termination signals downstream of the polynucleotide, such as the SV40 polyA site or the tk polyA site. Furthermore, depending on the expression system used, a leader sequence capable of directing the polypeptide into a cellular compartment or secreting it into the medium may be added to the coding sequence of the polynucleotide, as previously described. The leader sequence(s) is assembled in appropriate phase with the translation, initiation, and termination sequences, and preferably, the leader sequence is capable of directing the secretion of the translated protein or portion thereof, for example, into the extracellular medium. Heterologous polynucleotide sequences encoding fusion proteins containing C- or N-terminal identification peptides that confer desirable characteristics, such as stabilization or simplified purification of the expressed recombinant product, may also optionally be used.

[0188] In some embodiments, the polynucleotides encoding at least the variable domains of the light and / or heavy chains may encode both immunoglobulin chains or only one variable domain. Similarly, the polynucleotides may be under the control of the same promoter or may be separately regulated for expression. Furthermore, some aspects relate to vectors conventionally used in genetic engineering, particularly plasmids, cosmids, viruses, and bacteriophages, that contain polynucleotides encoding the variable domains of an immunoglobulin chain of an antibody or antigen-binding fragment, optionally in combination with polynucleotides encoding the variable domains of other immunoglobulin chains of the antibody.

[0189] In some embodiments, the expression control sequences are provided as eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, although control sequences for prokaryotic hosts may also be used. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses may be used to deliver polynucleotides or vectors to targeted cell populations (e.g., to engineer cells to express antibodies or antigen-binding fragments). Various suitable methods may be used to construct recombinant viral vectors. In some embodiments, polynucleotides and vectors may be reconstituted into liposomes for delivery to target cells. Vectors containing polynucleotides (e.g., immunoglobulin chain heavy and / or light chain variable domain(s) encoding sequences and expression control sequences) may be transferred into host cells by suitable methods, which vary depending on the type of cellular host.

[0190] qualification The antibodies or antigen-binding fragments of the present disclosure can be used with, but are 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 the detection and isolation of pro / latent myostatin. The polypeptides of the present disclosure may be modified with a detectable label, including, but not limited to, a detectable substance. Detectable substances may be coupled or conjugated to the polypeptides of the present disclosure either directly or indirectly through an intermediate (e.g., 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 materials include biotin, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; non-limiting examples of bioluminescent materials include luciferase, luciferin, and aequorin; and an example of a suitable radioactive material is, 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 Ru,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 Detectable substances include radioactive metal ions such as Sn), e.g., alpha emitters or other radioisotopes. Detectable substances may be coupled or conjugated to the anti-pro / latent myostatin antibodies of the present disclosure either directly or indirectly through an intermediate (e.g., a linker) using suitable techniques. Anti-pro / latent myostatin antibodies conjugated to a detectable substance can be used for the diagnostic assays described herein.

[0191] Pharmaceutical Composition One or more anti-pro / latent myostatin antibodies may be mixed with pharmaceutically acceptable carriers (excipients), including buffering agents, to form a pharmaceutical composition for use in alleviating diseases or disorders associated with myopathy. By "acceptable," it is meant that the carrier must be compatible with (and preferably be able to stabilize) the active ingredients of the composition and not deleterious to the subject being treated. Examples of pharmaceutically acceptable excipients (carriers), including buffering agents, will be apparent to those skilled in the art and have been previously described. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, eds., K. E. Hoover. In one example, the pharmaceutical compositions described herein contain more than one anti-pro / latent myostatin antibody that recognizes different epitopes / residues of the target antigen.

[0192] The pharmaceutical compositions used in the present methods may contain pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, eds., K. E. Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, or other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (fewer than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; glycine, glutamine, aspartic acid, hydroxybenzoates ... The excipients may include amino acids such as arginine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0193] In some examples, the pharmaceutical compositions described herein include liposomes containing anti-pro / latent myostatin antibodies, which can be prepared by any suitable method, such as those 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 U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be produced by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to obtain liposomes with the desired diameter.

[0194] Anti-pro / latent myostatin antibodies can also be entrapped in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacylate) microcapsules, respectively, prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions. Exemplary techniques have been previously described, see, for example, Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing (2000).

[0195] In other examples, the pharmaceutical compositions described herein may be formulated in a sustained-release format. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, the matrices being in the form of shaped articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide lactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0196] Pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0197] 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 for administration by inhalation or insufflation.

[0198] To prepare solid compositions such as tablets, the main active ingredient may be mixed with a pharmaceutical carrier, e.g., corn starch. The compound of the present disclosure may be mixed with conventional tableting ingredients, such as starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gums, and other pharmaceutical diluents, such as water, or non-toxic pharmaceutically acceptable salts thereof, to form a solid preformulation composition containing a homogeneous mixture of the compound of the present disclosure. When these preformulation compositions are referred to as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition, allowing the composition to be easily divided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid preformulation composition is then divided into unit dosage forms of the type described above containing from 0.1 mg to about 500 mg of the active ingredient of the present disclosure. The tablets or pills of the novel composition may be coated or otherwise compounded to provide a dosage form offering the advantage of prolonged action. For example, the tablet or pill may comprise an inner dosage and an outer dosage component, the latter in the form of an envelope surrounding the former. The two components may be separated by an enteric layer which serves to resist disintegration in the stomach and permits 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, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0199] Suitable surfactants include, in particular, non-ionic agents such as polyoxyethylene sorbitan (e.g., Tween™ 20, 40, 60, 80, or 85) and other sorbitan (e.g., Span™ 20, 40, 60, 80, or 85). Compositions that include a surfactant conveniently contain 0.05 to 5% surfactant, and may be 0.1 to 2.5%. It will be appreciated that other ingredients, such as mannitol or other pharmaceutically acceptable vehicles, may be added as required.

[0200] Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™, and Lipiphysan™. The active ingredient can be dissolved in a premixed emulsion composition, or alternatively, in an emulsion formed by mixing oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and phospholipids (e.g., egg phospholipids, soybean phospholipids, or soybean lecithin) with water. It is understood that other ingredients, such as glycerol or glucose, can be added to adjust the tonicity of the emulsion. Suitable emulsions typically contain up to 20% oil, for example, 5 to 20%.

[0201] The emulsion composition may be prepared by mixing an anti-promyostatin antibody with Intralipid™ and its components (soybean oil, egg phospholipids, glycerol, and water).

[0202] 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 described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0203] Compositions, preferably in sterile, pharmaceutically acceptable solvents, may be nebulized by the use of gases. Nebulized solutions may be breathed directly from the nebulizing device, or the nebulizing device may be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered from devices that deliver the formulation in an appropriate manner, preferably orally or nasally.

[0204] Use of anti-pro / latent myostatin antibodies to treat diseases / disorders The anti-pro / latent myostatin antibodies described herein are effective in treating diseases or disorders associated with myopathy. As used herein, the term "myopathies" refers to muscle diseases in which muscle fibers do not function properly, typically resulting in muscle weakness. Myopathy includes muscle diseases that are neuromuscular or musculoskeletal in nature. In some embodiments, the myopathy is an inherited myopathy. Inherited myopathies include, but are not limited to, dystrophies, myotonias, congenital myopathies (e.g., nemaline myopathy, multi / minicore myopathy, and centronuclear myopathy), mitochondrial myopathies, familial periodic myopathies, inflammatory myopathies, and metabolic myopathies (e.g., glycogen storage diseases and lipid storage disorders). 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, polymositis, and inclusion body myositis), myositis ossificans, rhabdomyolysis, and myoglobinuria, and disuse atrophy. In some embodiments, the myopathy is disuse atrophy and may result from a fracture (e.g., a hip fracture) or from nerve injury (e.g., a 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, a cardiac condition, and / or cancer. In some embodiments, the myopathy is associated with aging.

[0205] Aspects of the present disclosure include methods of treating a subject with a myopathy, comprising administering to the subject an effective amount of an antibody described above. In some embodiments, the myopathy is a primary myopathy. In another embodiment, the primary myopathy comprises disuse atrophy. In other embodiments, the disuse atrophy is associated with hip fracture, elective joint replacement, critical care myopathy, spinal cord injury, or stroke. In some embodiments, the myopathy is a secondary myopathy in which muscle loss is secondary to a disease condition. In other embodiments, the secondary myopathy comprises denervation, inherited 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 inherited muscle weakness associated with muscular dystrophy. In other embodiments, the secondary myopathy is cachexia associated with renal failure, AIDS, a cardiac condition, cancer, or aging.

[0206] Another aspect of the present disclosure includes a method of treating a subject with an age-related disease or condition. Exemplary age-related diseases and conditions include, but are not limited to, sarcopenia (age-related muscle loss), frailty, and androgen deficiency.

[0207] Another aspect of the present disclosure includes a method for treating a subject with a disease or condition associated with disuse atrophy / trauma.Exemplary diseases and 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 replacement surgery, hip fracture, stroke, bed rest, SCI, rotator cuff injury, knee replacement surgery, fracture and burn.

[0208] Another aspect of the present disclosure includes a method of treating a subject with a neurodegenerative disease or condition. Exemplary neurodegenerative diseases or conditions include, but are not limited to, spinal muscular atrophy and amyotrophic lateral sclerosis (ALS).

[0209] Another aspect of the present disclosure includes a method of treating a subject having a disease or condition associated with cachexia. Exemplary diseases and conditions associated with cachexia include, but are not limited to, cancer, chronic heart failure, acquired immune deficiency syndrome (AIDS), chronic obstructive pulmonary disease (COPD), and chronic kidney disease (CKD).

[0210] Another aspect of the present disclosure includes a method of treating a subject having 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.

[0211] Another aspect of the present disclosure includes a method of treating a subject having a disease or condition associated with metabolic disorders and / or body composition. In some embodiments, the disease or condition is obesity (e.g., severe obesity), Prader-Willi syndrome, type II diabetes, or anorexia. However, additional diseases or conditions associated with metabolic disorders and / or body composition will be apparent to those skilled in the art and are within the scope of the present disclosure.

[0212] Another aspect of the present disclosure includes a method 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, nemaline myopathy, and congenital fiber imbalance myopathy.

[0213] Another aspect of the present disclosure includes a method of treating a subject having a disease or condition associated with muscular dystrophy. Exemplary muscular dystrophies include, but are not limited to, Duchenne, Becker, facioscapulohumeral (FSH), and limb-girdle muscular dystrophy.

[0214] Another aspect of the present disclosure includes a method of treating a subject with a urogynecologic-related disease or condition, glottic disorder (stenosis), exophthalmic myopathy, carpal tunnel, Guillain-Barre, or osteosarcoma.

[0215] To practice the methods described herein, an effective amount of the pharmaceutical composition described above can be administered to a subject (e.g., a human) in need of treatment via a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation, or topical. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be directly nebulized, or lyophilized powders can be nebulized after reconstitution. Alternatively, anti-pro / latent myostatin antibodies can be aerosolized using a fluorocarbon formulation and a metered-dose inhaler, or inhaled as a lyophilized, milled powder.

[0216] The subject treated by the methods described herein may be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats. A human subject in need of treatment may be a human patient having, at risk of, or suspected of having a disease / disorder involving myopathy, such as those described above. A subject with a pro / latent myostatin-related disease or disorder may be identified by routine medical tests, such as laboratory tests, organ function tests, CT scans, or ultrasound. A subject suspected of having any of such diseases / disorders may exhibit one or more symptoms of the disease / disorder. A subject at risk for a disease / disorder may be a subject with one or more risk factors for the disease / disorder.

[0217] As used herein, "effective amount" refers to the amount of each active agent, either alone or in combination with one or more other active agents, required to provide a therapeutic effect to a subject. As will be recognized by those skilled in the art, the effective amount will vary depending on the specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex and weight, duration of treatment, the nature of concurrent treatment (if any), the specific route of administration, and similar factors within the knowledge and professional opinion of medical personnel. These factors are well known to those skilled in the art. and can be addressed with no more than routine experimentation. It is generally preferred that the maximum dose of each component or combination thereof, i.e., the highest safe dose according to sound medical judgment, be used. However, it will be understood by those of skill in the art that a patient may insist on a lower or tolerable dose for medical reasons, psychological reasons, or virtually any other reason. In some embodiments, an effective amount refers to the amount of an antibody or antigen-binding portion thereof sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof, prevent progression of a disorder, cause regression of a disorder, prevent the recurrence, onset, onset, or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., a prophylactic or therapeutic agent).

[0218] In some embodiments, in terms of administering a pro / latent myostatin antibody to a subject, an effective amount is an amount effective to increase target muscle mass in the subject relative 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, increase compared to the control muscle mass. In some embodiments, the increase in muscle mass is 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., increase compared to the control muscle mass.

[0219] As used herein, the term "control muscle mass" refers to a reference standard useful for evaluating the effect of a condition (e.g., treatment with a pro / latent myostatin antibody) on a 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 a target muscle mass in a subject who has not been administered a pro / latent myostatin antibody. In some embodiments, the control muscle mass is a target muscle mass (e.g., average mass) in a population of subjects who have not been administered a pro / latent myostatin antibody. In some embodiments, the control muscle mass is a target muscle mass in a subject before (e.g., immediately before) being administered a pro / latent myostatin antibody. In some embodiments, the control muscle mass is a target muscle mass in a subject who has been administered a normal antibody (e.g., of the same isotype as the pro / latent myostatin antibody) obtained from an animal that has not been exposed to the antigen against which the pro / latent myostatin antibody is directed instead of the pro / latent myostatin antibody. In some embodiments, the control muscle mass is the target muscle mass in a subject administered a vehicle, e.g., saline, instead of a pro / latent myostatin antibody.

[0220] In some embodiments, in terms of administering a pro / latent myostatin antibody to a subject, an effective amount is an amount effective to increase the force-generating capacity of a target muscle in the subject (e.g., maximal force production determined in vitro using a muscle lever system adapted for horizontal perfusion bathing) compared to a control force-generating capacity. In some embodiments, the increase in force-generating 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 control force-generating capacity. In some embodiments, the increase in force-generating capacity is 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 control force-generating capacity.

[0221] As used herein, the term "control force-generating capacity" refers to a reference standard useful for comparing the effect of a condition (e.g., treatment with a pro / latent myostatin antibody) on the force-generating capacity of a muscle in a subject. In some embodiments, the control force-generating capacity is a predetermined value. The control force-generating capacity is experimentally determined. In some embodiments, the control force-generating capacity is the force-generating capacity of a target muscle in a subject who has not been administered a pro / latent myostatin antibody. In some embodiments, the control force-generating capacity is the force-generating capacity (e.g., average force-generating capacity) of the target muscle in a population of subjects who have not been administered a pro / latent myostatin antibody. In some embodiments, the control force-generating capacity is the force-generating capacity of the target muscle in a subject prior to (e.g., immediately before) being administered a pro / latent myostatin antibody. In some embodiments, the control muscle force-generating capacity is the force-generating capacity of the target muscle in a subject who has been administered a normal antibody (e.g., of the same isotype as the pro / latent myostatin antibody) obtained from an animal that has not been exposed to the antigen to which the pro / latent myostatin antibody is directed, instead of the pro / latent myostatin antibody. In some embodiments, the control force-generating capacity is the force-generating capacity of the target muscle in a subject who has been administered a vehicle, e.g., saline, instead of the pro / latent myostatin antibody.

[0222] Empirical considerations, such as half-life, generally contribute to determining dosage. Antibodies that are compatible with the human immune system, such as humanized or fully human antibodies, may be used to extend the half-life of the antibody and prevent it from being attacked by the host's immune system. The frequency of administration may be determined and adjusted over the course of treatment and is generally, but not necessarily, based on the treatment and / or suppression and / or reversal and / or delay of the disease / disorder associated with myopathy. Alternatively, sustained release of anti-pro / latent myostatin may be used. A variety of formulations and devices for achieving sustained release will be apparent to those skilled in the art and are within the scope of the present disclosure.

[0223] In one example, dosages for the anti-pro / latent myostatin antibodies described herein can be empirically determined in individuals given one or more administrations of the antibody. Individuals are given increasing dosages of the antagonist. Disease / disorder indicators can be followed to assess the efficacy of the antagonist.

[0224] Generally, for administration of any of the antibodies described herein, the initial candidate dose may be about 2 mg / kg. For purposes of this disclosure, a typical daily dose may range anywhere from about 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 more, depending on the factors discussed above. For repeated administration over several days or longer, treatment is continued until a desired suppression of symptoms occurs or until a sufficient therapeutic level is achieved to alleviate a disease or disorder associated with pro- or latent myostatin or its symptoms, depending on the condition. An exemplary dosing regimen involves an initial dose of about 2 mg / kg, followed by weekly maintenance doses of about 1 mg / kg of antibody, or followed by maintenance doses of about 1 mg / kg every other week. However, other dosing regimens are useful depending on the pattern of pharmacodynamic decay the clinician wishes to achieve. For example, dosing one to four times per week is contemplated. In some embodiments, about 3 μg / mg to about 2 mg / kg (e.g., 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) are used. In some embodiments, the administration frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks, or once every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 8 months, every 10 months, every year, or longer. The progress of this treatment can be easily monitored by conventional techniques and assays. The administration regimen (including the antibody used) may be modified over time.

[0225] In some embodiments, for a normal weight adult patient, a dose ranging from about 0.3 to 5.00 mg / kg may be administered. The particular dosing regimen, e.g., dose, timing, and repetition, will depend on the particular individual and their medical history and the characteristics of the individual drug (such as the drug's half-life and other relevant considerations).

[0226] For purposes of this disclosure, an appropriate dosage of an anti-pro / latent myostatin antibody is used. The administration of an anti-pro / latent myostatin antibody, whether for therapeutic or prophylactic purposes, can be continuous or intermittent, depending, for example, on the physiological condition of the recipient and other factors known to a skilled physician. Administration of an anti-pro / latent myostatin antibody, whether for therapeutic or prophylactic purposes, can be essentially continuous or intermittent, depending, for example, on the physiological condition of the recipient and other factors known to a skilled physician. Administration of an anti-pro / latent myostatin antibody can be essentially continuous over a preselected period of time, or in a series of spaced doses, for example, either before, during, or after the onset of a disease or disorder associated with pro / latent myostatin.

[0227] As used herein, the term "treating" refers to the application or administration of a composition containing one or more active agents to a subject having a disease / disorder, symptom of a disease / disorder, or predisposition to a disease / disorder involving myopathy to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, symptom of the disease, or predisposition to the disease / disorder.

[0228] Alleviating a disease / disorder associated with pro / latent myostatin includes delaying the development or progression of the disease or reducing the severity of the disease. Alleviating a disease does not necessarily require a curative outcome. As used herein, "delaying" the onset of a disease / disorder associated with pro / latent myostatin means deferring, hindering, slowing, retarding, stabilizing, and / or postponing the progression of the disease. This delay may be for various lengths of time depending on the history of the disease and / or the individual being treated. A method of "delaying" or alleviating the onset of a disease or delaying the onset of a disease is a method that reduces the likelihood of the onset of one or more symptoms of the disease in a given time frame and / or reduces the severity of the symptoms in a given time frame compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to produce statistically significant results.

[0229] "Onset" or "progression" of a disease refers to the initial manifestation of the disease and / or the subsequent progression of the disease. Disease onset may be detectable and can be assessed using standard clinical techniques. However, onset also refers to progression, which may be undetectable. For the purposes of this disclosure, onset or progression refers to the biological course of symptoms. "Onset" includes occurrence, recurrence, and onset. As used herein, "onset" or "onset" of a disease / disorder involving myopathy includes initial onset and / or recurrence.

[0230] In some embodiments, the anti-pro / latent myostatin antibodies described herein are administered to a subject in need of treatment in an amount sufficient to inhibit proteolytic activation of pro / 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 pro / latent myostatin or latent myostatin levels by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more).

[0231] Conventional methods known to those skilled in the art of medicine may be used to administer pharmaceutical compositions to a subject depending on the type of disease or site of disease being treated. The compositions may also be administered via other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. As used herein, the term "parenteral" refers to subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, thoracic, or rectal administration. These include intraosseous, intrathecal, intralesional, and intracranial injection or infusion techniques. They may also be administered to a subject via an injectable depot route of administration, such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.

[0232] Injectable compositions may contain a variety of 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 may be administered by infusion, in which a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. For intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the antibody may be dissolved in a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% glucose solution and administered.

[0233] In one embodiment, the anti-pro / latent myostatin antibody is administered via a site-specific or targeted local delivery technique. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of anti-pro / latent myostatin antibody or local delivery catheters such as infusion catheters, indwelling catheters, or needle catheters, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site-specific carriers, direct injection, or direct application. See, e.g., PCT Publication No. WO 00 / 53211 and U.S. Patent No. 5,981,568.

[0234] Targeted delivery of therapeutic composition containing polynucleotide or expression vector 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 (edited by JA Wolff) (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.

[0235] Therapeutic compositions containing polynucleotides (e.g., those encoding anti-pro / latent myostatin antibodies described herein) are administered in the range of about 100 ng to about 200 mg of DNA for local administration in gene therapy protocols. In some embodiments, concentration ranges of 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 or more of DNA may also be used during gene therapy protocols.

[0236] The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. Gene delivery vehicles can be of viral or non-viral 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). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and / or enhancers. Expression of the coding sequences can be either constitutive or regulated.

[0237] Viral-based vectors suitable for delivery and expression of a desired polynucleotide (eg, encoding an antibody disclosed herein) in a desired cell are within the scope of this disclosure. Exemplary virus-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO90 / 07936, WO94 / 03622, WO93 / 25698, WO93 / 25234, WO93 / 11230, WO93 / 10218, WO91 / 02805, U.S. Patent Nos. 5,219,740 and 4,777,127; British Patent No. 2,200,651 and European Patent No. 0345242), 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-1247). VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532) and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94 / 12649, WO93 / 03769, WO93 / 19191, WO94 / 28938, WO95 / 11984, and WO95 / 00655). Administration of DNA linked to inactivated adenovirus, as described in Curiel, Hum. Gene Ther. (1992) 3:147, can also be used.

[0238] Non-viral delivery vehicles and methods may also be used, including, but not limited to, polycationic condensed DNA (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147), with or without linkage to inactivated adenovirus alone; ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicle cells (see, e.g., U.S. Patent No. 5,814,482, PCT Publication Nos. WO95 / 07994, WO96 / 17072, WO95 / 30763, and WO97 / 42338), and nuclear charge neutralization or fusion with cell membranes. Naked DNA may also be used. Exemplary naked DNA transfer methods are described in PCT Publication No. WO 90 / 11092 and U.S. Patent No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120, PCT Publication Nos. WO 95 / 13796, WO 94 / 23697, WO 91 / 14445, and European Patent No. 0524968. Additional approaches are described in Philip, Mol. Cell. Biol. (1994) 14:2411, and Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0239] The particular administration regimen used in the methods described herein, eg, dosage, timing, and repetition, will depend on the particular subject and that subject's medical history.

[0240] In some embodiments, more than one anti-pro / latent myostatin antibody or a combination of an anti-pro / 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 each other. Anti-pro / latent myostatin antibodies may also be used in conjunction with other agents that serve to enhance and / or complement the effectiveness of the agents.

[0241] The efficacy of a treatment for a disease / disorder associated with myopathy can be evaluated using any suitable method. For example, the efficacy of a treatment for a disease / disorder associated with myopathy can be evaluated by assessing muscle weakness (e.g., assessing the pattern and severity of weakness), electromyography, assessing blood chemistry (e.g., assessing electrolytes, assessing endocrine causes, measuring creatine kinase levels, determining the erythrocyte sedimentation rate, and performing an antinuclear antibody assay). The present invention can be evaluated by administering biopsies (e.g., by histological, histochemical, electron microscopic, biochemical, and genetic analysis), as well as by evaluating biopsies.

[0242] Kits for use in alleviating diseases / disorders associated with myopathy The present disclosure also provides kits for use in alleviating diseases / disorders associated with myopathy. Such kits may include one or more containers containing an anti-pro / latent myostatin antibody, such as any of those described herein.

[0243] In some embodiments, the kit may include instructions for use in accordance with any of the methods described herein. The included instructions may include instructions for administering an anti-pro / latent myostatin antibody to treat, delay the onset of, or alleviate a target disease described herein. The kit may further include instructions for selecting an individual suitable for treatment based on identifying whether the individual has the target disease. In yet other embodiments, the instructions include instructions for administering the antibody to an individual at risk for the target disease.

[0244] Instructions for use of anti-pro / latent myostatin antibodies generally include information about dosage, administration schedule, and route of administration for the intended treatment. Containers may be unit dose, bulk packages (e.g., multi-dose packages), or partial unit doses. Instructions provided in kits of the present disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions written on a magnetic or optical storage disk) are also acceptable.

[0245] The label or package insert indicates that the composition is used for treating, delaying the onset of, and / or ameliorating a disease or disorder associated with myopathy. Instructions may be provided for practicing any of the methods described herein.

[0246] 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 combination with specific devices, such as inhalers, nasal administration devices (e.g., atomizers), or injection devices such as minipumps, is also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an anti-pro / latent myostatin antibody described herein.

[0247] Optionally, the kit can provide additional components such as buffers and interpretive information. Typically, the kit includes a container and a label or package insert(s) on or associated with the container. In some embodiments, the disclosure provides an article of manufacture including the contents of the kit described above.

[0248] Assays for detecting pro / latent myostatin In some embodiments, the methods and compositions provided herein relate to methods for detecting pro / latent myostatin in a sample obtained from a subject. As used herein, "subject" refers to an individual organism, for example, an individual 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, amphibian, reptile, fish, insect, or the like. In some embodiments, the subject is a laboratory animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development. In some embodiments, the subject is a patient or a healthy volunteer.

[0249] In some embodiments, a method for detecting pro / latent myostatin in a sample obtained from a subject includes (a) contacting the sample with an anti-pro / 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 bound complex, and (b) determining the level of antibody or antigen-binding fragment bound to the antigen (e.g., determining the level of the bound complex).

[0250] As used herein, a bound complex refers to a biomolecular complex of an antibody (including an antigen-binding fragment) bound to an antigen (e.g., pro / latent myostatin protein). A bound complex may contain an antibody with a single specificity or two or more antibodies or antigen-binding fragments with different specificities. In one embodiment, a bound complex contains 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 bound to another biomolecule, such as RNA, DNA, polysaccharide, or protein. In one embodiment, a bound complex contains two or more antibodies that recognize different antigens. In some embodiments, an antibody in a bound complex (e.g., an immobilized antibody bound to an antigen) can itself bind to an antibody (e.g., a detectably labeled antibody) as an antigen. Thus, a bound complex may, in some cases, contain multiple antigens and multiple antibodies or antigen-binding fragments.

[0251] Antigens present in the binding complex may or may not be in their native conformation in situ. In some embodiments, the binding complex is formed between an antibody and a purified protein antigen or an isolated protein comprising the antigen, in which the antigen is not in its native conformation in situ. In some embodiments, the binding complex is formed between an antibody and a purified protein antigen, in which the antigen is not in its native conformation in situ. The antibody is not in its native conformation in situ but is immobilized on a solid support (e.g., a PVDF membrane). In some embodiments, the binding complex is formed between the antibody and a cell surface protein that is present in situ in its native conformation (e.g., on the cell surface).

[0252] The antibody in the binding complex may or may not be detectably labeled. 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 immobilized to one or more solid supports. In some embodiments, the antigen in the binding complex is immobilized to one or more solid supports. Exemplary solid supports are disclosed herein and will be apparent to those of skill in the art. The foregoing examples of binding complexes are not intended to be limiting. Other examples of binding complexes will be apparent to those of skill in the art.

[0253] In any of the detection, diagnosis, and monitoring methods, antibodies (including antigen-binding fragments) or antigens can be directly or indirectly conjugated to the surface of a solid support.Methods for conjugation to a solid support are standard and can be achieved through covalent and non-covalent interactions.Non-limiting examples of conjugation methods include adsorption, cross-linking, protein A / G antibody interaction, and streptavidin-biotin interaction.Other methods of conjugation will be readily apparent to those skilled in the art.

[0254] In some embodiments, the detection, diagnosis, and monitoring methods involve measuring the level of antibodies (including antigen-binding fragments) bound to an antigen (e.g., pro / latent myostatin) using one or more This includes comparing the level of pro / latent myostatin to a reference standard. The reference standard may be, for example, the level of corresponding pro / latent myostatin in a subject with or without pro / latent myostatin. In one embodiment, the reference standard is the level of pro / latent myostatin detected in a sample that does not contain pro / latent myostatin (e.g., background level). Alternatively, the background level may be determined from a sample containing a specific pro / latent myostatin by contacting the sample with a non-specific antibody (e.g., an antibody obtained from non-immune serum). Again, the reference standard may be the level of pro / latent myostatin detected in a sample containing pro / latent myostatin (e.g., a positive control). In some cases, the reference standard may be a range of levels that correlate with variations in the concentration of pro / latent myostatin in a sample and may be useful for quantifying the concentration of pro / 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 one of skill in the art. In some embodiments, the level of an antibody that binds to pro / latent myostatin is compared to the level of mature myostatin. In some cases, the level of pro / latent myostatin is compared to mature myostatin to determine the ratio of inactive to active myostatin in the sample.

[0255] The level of pro / latent myostatin can be measured as provided herein from a biological sample. A biological sample refers to any biological material that may be obtained from a subject or a cell. For example, the biological sample may be whole blood, plasma, serum, saliva, cerebrospinal fluid, urine, a cell (or cell lysate), or a tissue (e.g., normal 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, tissue samples may include, but are not limited to, skeletal muscle, cardiac muscle, adipose tissue, and tissue from other organs. In some embodiments, the biological sample is a biopsy sample. In some embodiments, a solid tissue sample may be converted into a fluid sample using routine methods in the art.

[0256] The biological sample may also contain one or more cells of a cell line. In some embodiments, the cell line comprises human cells, primate cells (e.g., Vero cells), rat cells (e.g., GH3 cells, OC23 cells), or mouse cells (e.g., MC3T3 cells). Various human cell lines include, but are not limited to, human embryonic kidney (HEK) cells, HeLa cells, cancer cells from the National Cancer Institute's 60 cancer cell lines (NCI60), DU145 (prostate cancer) cells, Lncap (prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-438 (breast cancer) cells, PC3 (prostate cancer) cells, T47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, SHSY5Y human neuroblastoma cells (cloned from a myeloma), and Saos-2 (bone cancer) cells.

[0257]

[0003] Further embodiments relate to methods for monitoring a disease, condition, or any treatment thereof (e.g., myopathy or myopathy treatment) in a subject having or at risk of having the disease or condition, the method comprising: (a) obtaining a biological sample from the subject; (b) determining the level of pro / latent myostatin in the biological sample using an antibody that detects pro / latent myostatin; and (c) repeating steps (a) and (b) on one or more occasions. Although myostatin has been used as a biomarker for muscle atrophy, currently available commercial methods and reagents (e.g., antibodies used in ELISA and Western blot) are either not specific for myostatin, detect only mature myostatin, or do not detect myostatin at all. Thus, provided herein are methods and reagents (e.g., antibodies) for detecting pro / latent myostatin for diagnostic purposes in the context of a disease and / or condition (e.g., muscle atrophy). In one example, the level of pro / latent myostatin can be used to detect or monitor the progression of a disease or condition in a subject or a biological sample derived therefrom. As another example, the level of pro / latent myostatin may be measured in a subject or a biological sample derived therefrom to monitor response to treatment for a disease or condition. It should be understood that the level of pro / latent myostatin may be monitored for any suitable period of time, which may vary depending on the disease or condition the subject has or any treatment regimen the subject may be subjected to.

[0258] Another embodiment relates to a diagnostic composition comprising any one of the above-described antibodies, antigen-binding fragments, polynucleotides, vectors, or cells, and optionally suitable means for detection. Antibodies are suitable for use in immunoassays, for example, which can be used in liquid phase or bound to a solid-phase carrier. Examples of immunoassays that may utilize antibodies are competitive and non-competitive immunoassays in direct or indirect formats. Examples of such immunoassays are enzyme-linked immunoassays (ELISAs), radioimmunoassays (RIAs), sandwich (immunometric assays), flow cytometry, Western blot assays, immunoprecipitation assays, immunohistochemistry, immunomicroscopy, lateral flow immunochromatographic assays, and proteomics arrays. Antigens and antibodies may be bound to a number of 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 either fixed or suspended in solution (e.g., beads).

[0259] In further embodiments, the antibodies (including antigen-binding fragments) provided herein can also be used in methods for assessing pro / latent myostatin expression in a subject by obtaining a biological sample from the subject, which can be a tissue sample, a blood sample, or any other suitable bodily fluid sample. The procedure can include contacting the blood sample (whole blood, serum, plasma), tissue sample, or a protein sample isolated therefrom, with the antibody under conditions that allow for 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 that allow for binding of the antibody to pro / latent myostatin protein, if the antigen is present in the sample, and for the formation of 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, plate well, membrane bath, cell culture dish, microscope slide, etc. 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 polymeric membrane (e.g., nitrocellulose strips, polyvinylidene fluoride (PVDF) membranes, etc.) Other suitable solid supports may be used.

[0260] In some embodiments, the antibody is immobilized to the solid support prior to contacting with the antigen. In other embodiments, antibody immobilization is performed after formation of the binding complex. In still other embodiments, the antigen is immobilized to the solid support prior to formation of the binding complex. 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 secondary antibody directed against the antigen. In some embodiments, the primary antibody is itself detectably labeled and is therefore a detection reagent.

[0261] In one aspect, the detection method includes the steps of immobilizing the antibody on a solid support; subjecting the sample (e.g., a biological sample or an isolated protein sample) to the antibody of the antigen, if the antigen is present in the sample; the solid support under conditions that allow binding of the detectably labeled antibody to the antigen-binding immobilized antibody; removing excess sample from the solid support; applying a detectably labeled antibody under conditions that allow binding of the detectably labeled antibody to the antigen-binding immobilized antibody; washing the solid support; and assaying for the presence of label on the solid support.

[0262] In some embodiments, the antigen is immobilized on a solid support, such as a PVDF membrane, before contacting it with the antibody in the reaction chamber (e.g., a membrane bath). 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 secondary antibody directed against the antigen. In some embodiments, the detection reagent comprises a detectably labeled secondary antibody directed against the primary antibody. As disclosed herein, a detectable label 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 primary antibody is itself detectably labeled and is therefore the detection reagent. Suitable detectable labels are described herein and will be readily apparent to those skilled in the art.

[0263] Thus, diagnostic kits suitable for home or clinical use (point-of-care service) are provided that include (a) a detectably labeled antibody and / or an unlabeled antibody as an antigen-binding reagent (e.g., a pro / 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 an antibody and / or pro / latent myostatin immobilized on a solid support. Any of the solid supports described herein are suitable for incorporation into the diagnostic kit. In a preferred embodiment, the solid support is the surface of a reaction chamber in a plate well. Typically, the plate wells are in a multiwell plate having a number of wells selected from 6, 12, 24, 96, 384, and 1536, but are not limited to such. 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 kit composition will be readily apparent to those skilled in the art.

[0264] The following specific embodiments are, therefore, to be construed as merely illustrative, and not limiting of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein. [Example]

[0265] Example 1: Antibody generation and selection Antibody Overview Ab2 is a fully human anti-pro / latent myostatin monoclonal antibody of the IgG4 / lambda isotype that binds to human promyostatin and latent myostatin with high affinity (Kd = 3420 pM by ForteBio BLI). The antibody can inhibit proteolytic activation of pro / latent myostatin with IC50 values ​​in the 0.5 micromolar range (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 using antibody display was performed to identify higher affinity variants Ab4 and Ab6. Affinity-optimized variants are similarly constructed in a human IgG4 / lambda isotype framework. [Table 2]

[0266] Parent antibody platform and identification The parental Ab1 antibody was identified through the selection of a naive phage display library using pro- and latent myostatin as the primary antigen 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 selection round consisted of pre-clearing (to remove nonspecific phage antibodies), incubation with antigen, washing, elution, and amplification. Selection was performed through multiple rounds using both solid-phase (biotinylated antigen coated on immunotubes) and solution-phase (biotinylated antigen captured using streptavidin-coated beads) panning strategies.

[0267] A total of 10,000 individual scFv clones were screened for binding to pro- or latent myostatin in two separate runs. The first program utilized pro / latent myostatin as the antigen, while the second run used latent myostatin as the antigen. DNA sequencing of the scFv clones of interest identified 216 unique clones. Positive binding scFv clones were counterscreened for binding to pro-GDF11 as well as a panel of unrelated proteins to confirm specificity for pro / latent myostatin. From the panel of unique scFv clones, 101 (out of 134 GDF8-specific clones) were converted to full-length IgG (IgG1 isotype) for further characterization.

[0268] The full-length IgG antibodies were further characterized by ELISA for binding to human and murine pro- and latent forms of myostatin and GDF11. Antibodies were also screened for binding to the myostatin prodomain, pro-TGFβ (human and murine), myostatin mature growth factor, GDF11 mature growth factor, activin A growth factor, and pro-activin A. Lead antibodies were selected based on cross-reactivity with human and murine pro- and latent forms of myostatin but not interacting with GDF11, activin, or TGFβ proteins.

[0269] Two forms of epitope binning were used. First, chimeric constructs were designed and generated in which part of the prodomain of myostatin was swapped with part of GDF11. These chimeric proteins were assayed for interaction with the screening antibodies by ELISA. Epitope binning was performed using a ForteBio BLI instrument in which biotinylated pro / latent myostatin antibodies were immobilized on a streptavidin-coated biosensor chip, and antibody cross-blocking was assessed by sensor response. These epitope binning experiments, along with data from ELISA binding experiments, revealed significant differences in the interaction of the antibodies with the pro / latent myostatin antibodies. This allowed us to separate our functionally active lead antibodies (see below) into three distinct epitope groups (see Table 3). [Table 3] *Statistically significant by one-way ANOVA and Dunnett. Ab8 does not bind to latent myostatin, only to promyostatin. Murine pro / latent myostatin preparations have approximately 40% latent material, which is why it is highly reactive in functional assays. This reduces the apparent efficacy. ND: Not yet decided

[0270] Several biochemical and cellular assays were established to assess the ability of antibodies to bind to pro- and latent myostatin and inhibit its activation. Binding kinetics to pro- and latent myostatin were measured using a ForteBio Octet assay, in which biotinylated substrate proteins were immobilized on a streptavidin-coated sensor chip. The equilibrium dissociation constants of candidates from the screening are shown in Table 3.

[0271] To measure the ability of IgG to inhibit myostatin signaling, a myostatin activation assay was developed. Conditioned medium was generated from cells overexpressing either mTll2 (a tolloid protease required for myostatin activation) or furin (a proprotein convertase that cleaves mature growth factors from the prodomain). After preincubation with the test antibody, pro / latent myostatin or latent myostatin was incubated with either a mixture of mTll2 and furin-conditioned medium (pro-myostatin) or mTll2-conditioned medium (latent myostatin). After an overnight proteolytic reaction, the release of mature growth factors was measured using a CAGA-based reporter assay in 293T cells. The antibodies were further confirmed by dose-response in the same assay, and the results are shown in Table 3.

[0272] Five parent antibodies (Table 3) consistently demonstrated strong selectivity and activity in all of the above assays and were further selected for further characterization in vivo (discussed in Example 2). Although Ab8 does not recognize latent myostatin, for consistency, we summarize the binding and activity of these antibodies against pro / latent myostatin.

[0273] 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 us to track the myostatin prodomain fragment (boxed area) generated after mTll2 cleavage. As the concentration of Ab1 increased, a dose-dependent decrease in the production of this fragment was observed. This experiment demonstrates that the antibody to epitope Bin1 acts by blocking the cleavage of pro- and latent myostatin by the Tolloid family of proteases.

[0274] Based on the in vitro and in vivo activity of the active anti-pro / latent myostatin antibody, Ab1 was selected as a lead for further optimization, including affinity maturation, germlining, and manufacturability analysis.

[0275] Ab1 optimization The Ab1 antibody was selected for further optimization. The affinity for pro / latent myostatin was optimized using yeast display. Furthermore, the sequence of Ab1 was germlined to reduce the potential immunogenic potential of non-germlined amino acid positions within the human variable region framework.

[0276] Affinity optimization of Ab1 by yeast display The Ab1 parent antibody was optimized for binding to pro / latent myostatin using a yeast-based scFv display approach. Briefly, three different scFv libraries were generated in which point mutations were introduced into selected CDR positions based on the amino acid frequencies observed in the natural human antibody repertoire using antibody deep sequencing corresponding to the human framework utilized by Ab1. Each library contained scFvs based on the Ab1 sequence with one point mutation introduced into each CDR, such that each resulting heavy or light chain variant had one substitution in each CDR, for a total of three substitutions. The three libraries were used for FACS-based sorting and selection to identify pools of clones with higher binding affinity to pro / latent myostatin (Figure 23). Direct binding of yeast-expressed scFv clones was used to select antibodies for conversion to full-length IgG expressed in mammalian cell culture.

[0277] Many of the high-affinity scFv clones identified in the yeast run contained substitutions at position 28 of the heavy chain. Some clones incorporated a non-canonical N-glycosylation motif within CDRH1 by substituting threonine for asparagine. Because N-glycosylation within the variable region of an antibody can be undesirable, any clones containing a glycosylation motif were further substituted to contain an alanine at this position.

[0278] The binding kinetics for pro- and latent myostatin were then assessed by octet for each of the affinity-optimized constructs and compared to that of the parent Ab1 (discussed in Example 2). All clones showed significantly increased binding affinity for myostatin, and two, Ab3 and Ab5, were selected based on their selective binding profiles compared to GDF11.

[0279] Primary sequence and framework of anti-pro / latent myostatin antibodies A sequence alignment of the variable regions of the parent Ab1 and its affinity-optimized variants is shown below. Complementarity-determining regions (CDRs) are defined using Kabat (underlined) and IMGT nomenclature (bold). Substitutions from the parent Ab1 are shown in lowercase (below and Figures 24A-24B). [ka]

[0280] Rationale for antibody engineering and isotype selection In some embodiments, antibodies useful for blocking myostatin lack effector function. For this reason, an IgG4-Fc region was selected for the humanized construct. 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 inefficient or nonexistent antibody-dependent cell-mediated cytotoxicity (ADCC).

[0281] To avoid potential difficulties with Fab arm exchange known to occur with native IgG4 mAbs, Ab1 and its variants were engineered to bind to the Fab arm at serine 228 ( The Fc sequence was engineered with a stabilizing "Adair" mutation (Angal, 1993) in which residue 241 (EU numbering: Kabat numbering) was converted to proline to create an IgG1-like (CPPCP (SEQ ID NO: 58)) hinge sequence. This engineered Fc sequence is used in the generation of the approved antibodies Keytruda, Mylotarg, and Tysabri, as well as several current late-stage clinical candidate mAbs.

[0282] Germlining and immunogenicity risk assessment 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 one stabilizing mutation (described above) to prevent Fab arm exchange. IgG4 Fc is not expected to have measurable binding to Fc gamma receptors (see Example 2).

[0283] The variable framework region of Ab1, isolated from a fully human naive phage library, 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 lowercase. [ka] [ka]

[0284] To mitigate potential immunogenicity, additional variants of the Ab1 molecule were generated in which non-germline framework residues were replaced with their corresponding germline amino acids. In some embodiments, the substitutions for Ab1 can be similarly applied to Ab3 and Ab4, or any antibody disclosed herein for which germlining is appropriate.

[0285] Sequence alignments of the variable regions of Ab1 and its affinity-optimized variants are shown below: A.) Heavy chain, B.) Light chain. Complementarity-determining regions (CDRs) are defined using Kabat (underlined) and IMGT nomenclature (bold). Substitutions in the framework regions present in the parent Ab1 are shown in lowercase. [ka] [ka]

[0286] Three of the five substitutions were found to be away from the CDR regions, thus resulting in Because the proline at position 2 of the light chain packs against CDRL3, substituting this proline with the germline serine actually improves binding to pro / latent myostatin by stabilizing the conformation of the CDR.

[0287] The entire antibody is >99% human (calculated by subtracting the % of non-germline AA excluding CDRH3 from 100%). There is no chemical conjugation. The heavy chain CDRH2 sequence contains a possible isomerization tendency (Asp-Gly) that is also present in the germline IgHV3-30 sequence.

[0288] Example 2: Pharmacological characterization In vitro pharmacological assays A total of 24 optimized Ab1 variants were expressed, purified as IgG4, and assayed for improved binding and functional activity. Changes to these molecules included germline mutations to the parent variable regions, along with CDR mutations that confer increased binding to pro / latent myostatin in affinity maturation screens (see Example 1).

[0289] The Ab1 variants were screened in several different ELISA-based assays to reassess binding to promyostatin and latent myostatin proteins (human, murine, and cynomolgus monkey) along with a large-scale screen of negative control proteins to ensure that nonspecific binding was not introduced as a result of affinity maturation. Negative controls included GDF11 proteins (pro-GDF11, latent GDF11, and mature GDF11), TGFβ proteins, and activin proteins (pro-activin). Additionally, the antibodies were evaluated for multispecificity (potential for rapid clearance) in a screen similar to a previously published screen (Hotzel et al., 2012). Any antibodies that significantly interacted with the negative control proteins or baculovirus particles in the multispecificity screen were not further considered candidates for the development program.

[0290] Twenty-four optimized variants of Ab1 were also evaluated in a promyostatin activation assay to determine their functional potency and compare EC50 values ​​from dose-response curves with the parent Ab1 antibody. Most antibodies had comparable or improved EC50 values, with a few showing reduced potency in this assay. Antibodies with reduced potency in the activity assay were excluded from further analysis.

[0291] Three variants of Ab1 were identified that showed improved binding to promyostatin and latent myostatin while retaining specificity for promyostatin and latent myostatin. Binding and activity data for these three variants and the parent Ab1 molecule are summarized in Tables 4-7, and the sequences are shown in Example 1. [Table 4] [Table 5] [Table 6] [Table 7]

[0292] Cell-based ex vivo and in vivo bioactivity assays The Ab1-optimized variants were evaluated in a dose-response study in a GDF8 activation assay. In these experiments, 0.5 μM promyostatin was preincubated with increasing amounts of the test substance. After this preincubation step, conditioned medium from HEK293 cells overexpressing mTll2 and furin protease was added to release mature growth factors from promyostatin. After overnight incubation at 30°C, the material was added to 293T cells harboring a SMAD-based luciferase reporter plasmid, and the activity of the released material was recorded. The data from the screening are shown in Figure 4.

[0293] Selectivity for myostatin over other TGFβ family members The selectivity of the candidate antibodies was also assessed by both binding and functional assays to confirm the absence of cross-reactivity with 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. ELISA assays demonstrated that this antibody binds to a construct consisting of the myostatin prodomain, and epitope mapping studies determined that the parent Ab1 molecule recognizes an epitope on the prodomain of promyostatin and latent myostatin. Although the myostatin prodomain and GDF11 share less than 50% identity, and significant cross-reactivity is not expected, the specificity of the lead antibody was carefully evaluated.

[0294] A sensitive assay was developed to detect interactions between the antibody of interest and a negative control reagent. In this assay, biotinylated pro-GDF11 or biotinylated pro-myostatin was immobilized on a ForteBio BLI streptavidin-coated sensor chip, which was applied to wells containing 30 μg / mL of antibody. Interaction of the analyte with the protein immobilized on the chip was measured by the magnitude of the biosensor chip response. Biosensor responses (saturating signals for pro-GDF8) after 5 minutes of association were compared between the two antigens and expressed as a percent response for GDF8 binding. All antibodies had minimal interactions with pro-GDF11 compared to the robust binding events measured for pro-myostatin. [Table 8]

[0295] Antibody candidates were also evaluated in a GDF11 activation assay. In this assay, 50 nM pro-GDF11 was pre-incubated with increasing concentrations of antibody. After pre-incubation, conditioned medium from HEK293 cells overexpressing BMP-1 (a tolloid family protease) and PCSK5 (a furin family member specific for GDF11) was added to activate pro-GDF11 via proteolysis. 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 pro-GDF11 activation, while a positive control antibody conferred dose-dependent inhibition of GDF11 activation.

[0296] The binding affinity of antibody candidates was determined using a ForteBio Octet Qke dip and read label-free assay system utilizing bio-layer interferometry. In each experiment, the antigen was immobilized on a biosensor (streptavidin-coated biosensor for pro-GDF8, pro-GDF11, and pro-activin; direct amine coupling for all others), and the antibody / construct was presented at a high concentration (50 μg / mL) in solution to measure the binding interaction.

[0297] Antibody binding affinities were determined using a ForteBio Octet QKe dip and readout system utilizing biolayer interferometry, a label-free assay system. Human pro-GDF8, latent GDF8, pro-GDF11, and pro-activin were biotinylated and immobilized on streptavidin-coated biosensors (ForteBio). Mature growth factors were immobilized by direct amine coupling to an amine-reactive chip according to the manufacturer's instructions (ForteBio). In each experiment, the antibody / construct was presented 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 generated as described. [Table 9A]

[0298] The results from the antigen binding studies are summarized in Table 9a. Experiments with no detectable binding are indicated with a minus sign (-). There were several calculated Kd values ​​fitted to the data with poor binding responses, which are listed in the table as weak nonspecific binding ( * )

[0299] Because the pro-GDF8 sample used in Table 9a contained approximately 10-15% latent GDF8, separate experiments were performed to specifically confirm the binding of pro-GDF8 to human and murine GDF8 antigens. Furthermore, primed GDF8, in which pro-GDF8 was proteolytically cleaved by both proprotein convertase and thrombin protease, was also evaluated for its binding affinity to Ab2 and ABMyo. For these experiments, a homogeneous preparation of human pro-GDF8 was purified from stably integrated 293 cells cultured in the presence of 30 μM decanoyl-RVKR-CMV. Primed human GDF8 was produced by in vitro cleavage of pro-GDF8 using conditioned medium from mTll2-overexpressing cells and purified furin protease. In binding experiments with these proteins, 150 nM of Ab2 or AbMyo was used to saturate the immobilization sites of a human Fc capture chip (ForteBio), and association and dissociation of 150 nM of analyte was assessed.

[0300] Analysis of binding affinity to murine proteins was also evaluated and is reported in Table 9b. Murine pro-GDF8 was generated by negative selection using an antibody (AbMyo2) that specifically recognizes latent and mature GDF8 to remove all mature and latent murine GDF8 from the sample. 50 nM of antibody was used to saturate an anti-human Fc capture chip (ForteBio). All antibodies were initially tested against a single concentration of 200 nM of murine pro-GDF8, murine latent GDF8, and mature GDF8. If binding was observed, the antibody was immobilized as described previously, and Kd values ​​were determined by using the analyte in a 3-fold dilution titration from 200 to 0.82 nM. Kd was determined using a global fit with ForteBio's data analysis software 8.2. For binding to mature myostatin, 5 μg / mL growth factor β was used in acetate buffer at pH 5. The antibody (R&D Systems) was coupled to an amine-reactive sensor chip (ForteBio). All antibodies were initially tested at 333 nM for binding to this myostatin-coupled sensor. Antibodies that showed binding were then tested at 3-fold diluted concentrations ranging from 333 to 1.37 nM. A global fit was used to determine the Kd of the interaction using ForteBio's data analysis software 8.2. [Table 9B]

[0301] Results from the antigen binding studies are summarized in Table 9b. Experiments with no detectable binding are indicated with a minus sign (-). Some values ​​labeled <1E-12 have very slow dissociation rates, making it impossible to quantify high affinity. Surprisingly, AbMyo was unable to recognize recombinant pro-GDF8, which differs from results reported by Latres et al. (2015), who reported the association of AbMyo with pro-GDF8 in immunoprecipitation experiments from serum of antibody-treated mice, which may have resulted in artifacts. Another surprising result was the interaction of Ab2 with primed GDF8, i.e., a complex of GDF8 and the tolloid-cleaved prodomain. This result was unexpected because Ab2 blocks tolloid cleavage of the prodomain, suggesting that the interaction of Ab2 with pro-GDF8 and latent GDF8 does not require an intact tolloid cleavage site.

[0302] Evaluation of Fc region functionality In some embodiments, anti-pro / latent myostatin therapy involves binding to a soluble target (pro / latent myostatin) and preventing activation via proteolysis. In some embodiments, antibody-dependent cell-mediated cytotoxicity and complement fixation are not involved in this process. Ab1 and related variants were engineered to contain an IgG4-Fc region. IgG4 antibodies are generally understood to lack effector function due to their weak binding to complement component C1q and Fcγ receptors.

[0303] To demonstrate reduced effector function capacity, Ab1 and related antibodies were tested for binding to CD64 (FcgRI) and C1q by ELISA. For comparison, IgG1 variants of Ab1 were also prepared. In this assay, all IgG4 antibodies showed significantly weaker binding (10-20 times weaker) to CD64 and C1q compared to IgG1. Relative binding values ​​at EC50 are listed in Table 10. [Table 10]

[0304] The apparent binding affinities of Ab1 and its related variants to CD64 and C1q are similar to other IgG4 clinical candidate antibodies and are significantly reduced compared to antibodies of the IgG1 isotype. Therefore, based on the biology of IgG4 antibodies, it is concluded that anti-pro / latent myostatin antibodies do not induce appreciable effector functions in vivo.

[0305] Efficacy in animal models Based on in vitro characterization, four antibodies (Ab7, Ab1, Ab8, and Ab9) were selected for in vivo testing. The purpose of the study was to evaluate the ability of these four candidate antibodies to regulate muscle mass in mice. Five groups of 10 female SCID mice were administered the test substance via intraperitoneal (IP) injection once a week on days 0, 7, 14, 21, 28, and 35. Prior to administration of the test substance (day 0), all animals underwent grip strength assessment. Grip strength assessment was also performed on the final day of the study (day 42). On day 0, blood was collected via retro-orbital bleeding for complete blood count (CBC) assessment. After administration, animals were evaluated daily for weight and general health observations. On day 42, after grip strength assessment, animals were sacrificed by CO2 overdose, and blood was collected by cardiac puncture for CBC assessment. Additional blood was collected for plasma preparation. Various tissues were isolated and weighed. The muscles harvested were gastrocnemius, pectoralis, soleus, triceps, tibialis anterior, quadriceps (rectus femoris), and diaphragm. The organs harvested were heart, kidney, spleen, liver, and inguinal white adipose tissue. All tissues were weighed and flash-frozen, except for the gastrocnemius, which was fixed in formalin (leg 1) and OCT (leg 2) for histological analysis.

[0306] summary The mean daily weight change data for animals in Study SCH-02 are shown in Figure 6. Animals in all five groups gained weight every week. Animals treated with antibody Ab1 showed the greatest weight gain (14.6%), as depicted in Figure 6. Only animals treated with Ab1 showed a statistically significant increase in mean weight change compared to animals in the vehicle (PBS) control group (Figure 6).

[0307] The weights of the excised muscles are plotted in Figures 7 and 8. Animals treated with Ab1 showed a statistically significant increase in gastrocnemius (Figure 7A) and diaphragm (Figure 8B) weights, 27.6% and 49.8%, respectively, compared with vehicle (PBS) control-treated animals. Additional muscles from Ab1-treated animals showed increased weights compared with PBS controls, but these differences were not statistically significant. There were no statistically significant differences between treatment groups with regard to mean tissue weights of the heart, spleen, kidney, liver, and adipose tissue.

[0308] SCID dose-response study In the in vivo study (described above), animals treated with Ab1 at 25 mg / kg once weekly for 6 weeks showed statistically significant increases in body and muscle weights (gastrocnemius and diaphragm) compared with animals treated with vehicle (PBS). This muscle-building activity of Ab1 was then investigated in more detail in a dose-response study in SCID mice. This study examined whether the magnitude of the effect on muscle mass could be increased by increasing the dose of Ab1 to as high as 60 mg / kg / week, and whether the magnitude of the effect on muscle mass could be decreased by decreasing the dose to as low as 2 mg / kg / week. In this study, the activity of Ab1 was compared with two additional antibodies (Ab8, originally tested in the study above, and Ab10).

[0309] Ten groups of ten female SCID mice were administered the test article by intraperitoneal (IP) injection (10 ml / kg) twice weekly on days 0, 3, 7, 10, 14, 17, 21, and 24. The doses of test article were as follows: Ab1 (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). Control groups received PBS and IgG control (30 mg / kg). Treatment groups are listed in Table 11. Animals were 10 weeks old at the start of the study. Body weights were measured on day -4 and twice weekly throughout the study corresponding to the days of administration. Body composition parameters (fat mass, lean mass, and water content) were measured by EchoMRI (QNMR) on days -4, 7, 14, 21, and 28. Thirty days after the first dose of antibody, animals were sacrificed by CO2 overdose, and blood was collected by cardiac puncture for CBC evaluation and plasma preparation. Furthermore, at the end of the study, various tissues were isolated and weighed. The muscles collected were the gastrocnemius, soleus, tibialis anterior, quadriceps (rectus femoris), and diaphragm. Muscles were excised from both the left and right legs of the test mice. For analysis, the weights of individual muscles from both legs were summed to calculate the average muscle weight in grams. Other tissues collected were the heart, kidney, spleen, liver, and adipose tissue. All tissues were weighed and then flash-frozen, except for the gastrocnemius, which was fixed in formalin (left leg) and OCT (right leg) for histological analysis. [Table 11]

[0310] The mean percent weight change and mean percent lean mass change data for animals treated with vehicle (PBS), IgG control, and different doses of Ab1 are shown in Figure 9. Animals treated with Ab1 at doses of 20 and 60 mg / kg / week showed a significant increase in body weight, 15.3% and 14.4%, respectively, on study day 28 compared with IgG control-treated animals (Figure 9A). All four groups of animals treated with Ab1 (doses of 60, 20, 6, and 2 mg / kg / week) showed a statistically significant increase in lean mass, 14.1%, 12.4%, 17.1%, and 15.5%, respectively, on study day 28 compared with IgG control-treated animals (Figure 9B).

[0311] The weights of four muscles (quadriceps, gastrocnemius, tibialis anterior, and diaphragm) are plotted in Figure 10. The soleus muscle was also excised, but due to the small size of the muscle, the data set was highly variable. Animals treated with all doses of Ab1 showed a statistically significant increase in muscle weight compared to IgG control animals (Figure 10). The mean percent change in muscle mass compared to the IgG control is shown above the corresponding bar on each muscle graph. The mean percent change in quadriceps weight ranged from 20.5% at the highest dose to 10.7% at the lowest dose (Figure 10A). The mean percent weight change in gastrocnemius weight ranged from 17.7% at the highest dose to 15.9% at the lowest dose (Figure 10B). The mean percent weight change in tibialis anterior weight ranged from 24.0% at the highest dose to 18.0% at the lowest dose (Figure 10C). The mean percent weight change in diaphragm weight 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.

[0312] Ab1 treatment in a dexamethasone-induced muscle atrophy model Considering the ability of anti-myostatin antibody Ab1 to build muscle mass in healthy SCID mice, we determined whether Ab1 treatment could also protect animals from treatments that induce muscle atrophy. A corticosteroid-induced muscle atrophy model 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 individual hindlimb muscle mass. In the following experiment, animals were treated with different doses of Ab1 to determine whether they could protect against this dexamethasone-induced muscle atrophy.

[0313] In this study, eight groups of 10 male mice (C57BL / 6) were enrolled at 13.5 weeks of age. Starting on study day 0, mice received either regular drinking water (Groups 1-4) or water containing dexamethasone (Groups 5-8). Test substances were administered via intraperitoneal (IP) injection (10 ml / kg) twice weekly on days 0, 3, 7, and 10. Test substances and doses were as follows: PBS (Groups 1 and 5), 10 mg / kg IgG control (Groups 2 and 6), 10 mg / kg Ab1 (Groups 3 and 7), and 1 mg / kg Ab1 (Groups 4 and 8). Treatment groups are listed in Table 12. Body weights were measured at least twice weekly throughout the study. Body composition parameters (fat mass, lean mass, and water content) were measured by EchoMRI (QNMR) on days -1, 6, and 13. Fourteen days after the first dose of antibody, the animals were sacrificed by CO2 overdose, and blood was collected by cardiac puncture for plasma preparation. Furthermore, at the end of the study, various tissues were isolated and weighed. The collected muscles were the gastrocnemius, soleus, tibialis anterior, quadriceps (rectus femoris), and diaphragm. Muscles were excised from both the left and right legs of the test mice. For analysis, the weights of individual muscles from both legs were summed to calculate the average muscle weight in grams. Other tissues collected were the heart, kidney, spleen, liver, and adipose tissue. All tissues were weighed and then flash-frozen, except for the gastrocnemius, which was fixed in formalin (left leg) and OCT (right leg) for histological analysis. [Table 12]

[0314] In this experiment, we determined whether treatment of mice with anti-myostatin antibody Ab1 could protect them from corticosteroid-induced muscle atrophy. Body weight was measured twice weekly throughout the study, and lean mass was measured by QNMR on days -1, 6, and 13. Mean percent weight change and lean 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. On day 14, no significant differences in mean percent weight change were observed between any of these treatment groups (Figure 11A). Treating mice with dexamethasone for 2 weeks significantly reduced lean mass (Groups 5 and 6) compared with the control group (Group 1) given regular drinking water (Figure 11B). However, mice treated with both dexamethasone and 20 mg / kg / week of antibody Ab1 (Group 7) did not show significant differences in lean mass change on day 14 compared with the control group (Group 1). Animals treated with 20 mg / kg / week of Ab1 showed a significant difference in percent lean mass change on day 14 compared to either of the dexamethasone-treated control groups (Groups 5 and 6), whereas 2 mg / kg / week did not.

[0315] At the end of the two-week treatment with dexamethasone and test substances, individual muscles were excised and weighed. Weight data for two muscles (gastrocnemius and quadriceps) are plotted in Figures 12A-12B. Animals receiving dexamethasone through their drinking water and similarly receiving either PBS or an IgG control antibody showed significant atrophy of the gastrocnemius and quadriceps muscles (Groups 5 and 6) compared with the non-diseased control group (Group 1). Animals treated with both dexamethasone and Ab1 at 20 mg / kg / week (Group 7) showed significant differences in muscle weight compared with either of the dexamethasone-treated control groups (Groups 5 and 6), whereas the 2 mg / kg / week group showed no significant differences. Furthermore, mice treated with both dexamethasone and Ab1 at 20 mg / kg / week (Group 7) showed no significant differences in gastrocnemius and quadriceps muscle weight compared with the non-diseased control group (Group 1). The percentage difference in mean muscle weight for each group compared to the mean muscle weight of the control groups (Group 1, PBS, and water) is shown in Figures 12C-12D. The percentage loss of gastrocnemius muscle mass induced by dexamethasone treatment in the PBS and IgG control groups was 16.5% and 18.9%, respectively. In contrast, animals treated with both dexamethasone and 20 mg / kg / week Ab1 experienced only a 4.0% loss in gastrocnemius muscle mass, which was not statistically different from the non-diseased control group (Group 1). Animals treated with both dexamethasone and 2 mg / kg / week Ab1 (Group 8) experienced only a 10% loss in gastrocnemius muscle mass, and the muscle mass loss in this group was P < 0.05. This was not statistically different from the reductions in the BS and IgG control groups (Groups 5 and 6). Similar results were seen for the quadriceps muscle (Figure 12D).

[0316] Ab1 treatment in a cast-induced muscle atrophy model Given the ability of the anti-myostatin antibody Ab1 to build muscle mass in healthy SCID mice, we investigated whether Ab1 treatment could also protect animals from treatments that induce muscle atrophy. A disuse atrophy model was established by immobilizing the right leg of mice in a plaster cast for two weeks. Immobilizing the right leg in a plaster cast for this period, with the foot in a plantar flexed position, induced a significant loss of individual hindlimb muscle mass. In the following experiment, animals were treated with different doses of Ab1 to determine the extent to which they were protected from this cast-induced muscle atrophy. [Table 13]

[0317] In this study, eight groups of 10 male mice (C57BL / 6) were enrolled at 14.5 weeks of age. Starting on study day 0, mice were anesthetized and had a cast placed on the right hind limb with the foot in a plantar flexed position (Groups 5-8). Control groups (Groups 1-4) were also anesthetized but did not have the hind limb casted. Test substances were administered via intraperitoneal (IP) injection (10 ml / kg) twice weekly on days 0, 3, 7, and 10. Test substances and doses were as follows: PBS (Groups 1 and 5), 10 mg / kg IgG control (Groups 2 and 6), 10 mg / kg Ab1 (Groups 3 and 7), and 1 mg / kg Ab1 (Groups 4 and 8). Treatment groups are listed in Table 13. Body weights were measured at least twice weekly throughout the study. Body composition parameters (fat mass, lean mass, and water content) were measured by EchoMRI (QNMR) on days -1, 7, and 14. 14 days after the first dose of antibody, animals were sacrificed by CO2 overdose and blood was collected by cardiac puncture for plasma preparation.

[0318] Additionally, various tissues were isolated and weighed. The muscles harvested were the gastrocnemius, soleus, plantaris, tibialis anterior, and quadriceps (rectus femoris). Individual muscle weights were collected from the right hind limb of the animals for analysis. Other tissues harvested were the heart, adipose tissue, and spleen. All tissues were weighed and then flash-frozen, except for the gastrocnemius, which was fixed in formalin for histological analysis.

[0319] summary In this experiment, we tested whether treatment of mice with the anti-myostatin antibody Ab1 could protect them from disuse muscle atrophy induced by cast immobilization of the right hindlimb. Body weight was measured twice weekly during the study, and lean mass was measured by QNMR on days -1, 7, and 14. Mean percent weight change and mean percent lean mass change data for animals in the non-diseased control group (Group 1) and the groups immobilized for 2 weeks (Groups 5-8) are shown in Figure 13. Cast immobilization of the right hindlimb did not have any negative effect on weight gain (Figure 13A), and any differences in lean mass between groups were not significant (Figure 13B).

[0320] At the end of the two-week study, individual muscles were excised and weighed. Weight data for two muscles (gastrocnemius and quadriceps) are plotted in Figures 14A-14B. Animals with legs immobilized and similarly administered either PBS or an IgG control antibody showed significant atrophy of the gastrocnemius and quadriceps muscles (Groups 5 and 6) compared with the non-immobilized control group (Group 1). Animals immobilized and administered 20 mg / kg / week of Ab1 (Group 7) showed significant differences in muscle weight compared with either of the immobilized control groups (Groups 5 and 6), whereas 2 mg / kg / week did not. Furthermore, casted mice treated with 20 mg / kg / week of antibody Ab1 (Group 7) showed no significant differences in gastrocnemius and quadriceps muscle weight compared with the non-immobilized control group (Group 1). The percentage difference in mean muscle weight for each group compared to the mean muscle weight of the non-immobilized control group (Group 1) is shown in Figures 14C-14D. The percent loss of gastrocnemius muscle mass induced by cast immobilization in the PBS and IgG control groups was 22.8% and 23.5%, respectively. In contrast, the loss of gastrocnemius muscle mass in cast-immobilized mice treated with 20 mg / kg / week of Ab1 was only 10.0%. This difference was found to be statistically different from the loss in the PBS and IgG control antibody-administered control groups (Groups 5 and 6). The loss in muscle mass in cast-immobilized mice treated with 2 mg / kg / week of Ab1 was not statistically different from that in the PBS and IgG control groups (Groups 5 and 6). Similar results were seen for the quadriceps muscle (Figure 14D).

[0321] The domain structure of promyostatin and latent myostatin is shown in Figure 16A, with protease cleavage sites indicated. An example of partial proprotein convertase-cleaved promyostatin on an SDS PAGE gel is shown in Figure 16B. Under reducing conditions, the protein bands consisted of the promyostatin monomer (approximately 50 kD), the prodomain (approximately 37 kD), and the growth factor (12.5 kD).

[0322] Ab1 specifically bound to promyostatin and latent myostatin, with no binding observed to other members of the TGF-β superfamily, particularly the corresponding forms of GDF11 (Figure 17A). Ab1 was administered at high concentration (50 μg / mL) to ForteBio BLI chips coated with the indicated antigens, and on- and off-rates were measured to obtain approximate Kd values. The magnitude of the biosensor response, indicating binding events, is graphically represented by the black bars, with the calculated Kd shown in orange. Furthermore, Ab1 blocks the activation of promyostatin but not pro-GDF11 (Figure 17B).

[0323] SCID dose-response study with Ab1, Ab2, Ab4, and Ab6 Previous in vivo studies with Ab1 demonstrated that Ab1 can increase muscle mass in healthy animals and prevent muscle loss in mouse muscle atrophy models (dexamethasone- and cast-induced atrophy). Antibody engineering efforts identified three antibodies with better in vitro characteristics than Ab1. In this study, the in vivo activity of these antibodies was compared with that of the previously established Ab1 at three different doses in SCID mice.

[0324] Fourteen groups of eight female SCID mice were treated twice weekly at 0, 3, 7, 10, 14, and 17 days. Test substances were administered by intraperitoneal (IP) injection (10 ml / kg) on ​​days 0, 7, 14, 21, and 24. The doses of test substances were as follows: Ab1, Ab2, Ab4, and Ab6 were administered at three different doses (10 mg / kg, 1 mg / kg, and 0.25 mg / kg), and the IgG control antibody was administered at 10 mg / kg. Treatment groups are listed in Table 14. Animals were 10 weeks old at the start of the study. Body weights were measured twice weekly throughout the study, corresponding to the dosing days. Body composition parameters (fat mass, lean mass, and water content) were measured by EchoMRI (QNMR) on days 0, 7, 14, 21, and 28. 28 days after the first dose of antibody, animals were sacrificed by CO2 overdose, and blood was collected by cardiac puncture for plasma preparation.

[0325] Additionally, various tissues were isolated and weighed. The muscles harvested were the gastrocnemius, soleus, tibialis anterior, quadriceps (rectus femoris), extensor digitorum longus, and diaphragm. Muscles were excised from both the left and right legs of the test mice. For analysis, the weights of individual muscles from both legs were summed to calculate the average muscle weight in grams. Other tissues harvested were the heart, kidney, spleen, liver, and adipose tissue. All tissues were weighed and then flash-frozen, except for the left gastrocnemius, which was fixed in formalin for histological analysis. [Table 14]

[0326] The data for the mean percent lean mass change (from day 0) for animals treated with vehicle (PBS), IgG control, and different doses of Ab1, Ab2, Ab4, and Ab6 are shown in Figure 15. At the 20 mg / kg / week dose level of Ab1, Ab2, Ab4, and Ab6, Treated animals had a significant increase in lean mass compared to IgG control and vehicle (PBS) treated animals on days 21 and 28 of the study. Animals treated with Ab1 and Ab2 at the 2 mg / kg / week dose level also had significant changes in lean mass on days 21 and 28 of the study. Animals treated with Ab1, Ab2, Ab4, and Ab6 at the 0.5 mg / kg / week dose level were not significantly different from the control group in lean mass.

[0327] Muscles were harvested and weighed at the end of the study (day 28). Quadriceps (rectus femoris) and gastrocnemius (gastrocnemius) muscle weights are plotted in Figures 18A and 18B. Animals treated with Ab1, Ab2, Ab4, and Ab6 at a dose level of 20 mg / kg / week had significant increases in gastrocnemius and quadriceps (rectus femoris) muscle weights compared with animals treated with vehicle (PBS). Animals treated with Ab2 and Ab4 at a dose level of 2 mg / kg / week also had significant changes in gastrocnemius muscle weight. Animals treated with Ab2 at a dose level of 2 mg / kg / week also had significant changes in quadriceps muscle weight. For animals treated with Ab1, Ab2, Ab4, and Ab6 at a dose level of 0.5 mg / kg / week, muscle mass did not change significantly from the control group. The percent changes in gastrocnemius and quadriceps (rectus femoris) weights (compared to the vehicle group) for animals treated with different doses of Ab1, Ab2, Ab4, and Ab6 are summarized in FIG. 18C.

[0328] Duration of effect studies with Ab1 in SCID mice The ability of Ab1 to increase lean mass in SCID mice was tested after a single dose and three weekly doses. Seven groups of eight female SCID mice were administered the test substance via intraperitoneal (IP) injection (10 ml / kg) once on day 0 (Groups 1-4) or once weekly on days 0, 7, and 14 (Groups 5-7). See Table 15. Antibodies (IgG control, Ab1, and AbMyo) were administered at a dose of 10 mg / kg. Animals were 10-11 weeks old at the start of the study. Body weights were measured twice weekly throughout the study, corresponding to the days of administration. Body composition parameters (fat mass, lean mass, and water content) were measured by Echo MRI (QNMR) on days 0, 7, 14, and 21. [Table 15]

[0329] The mean percent lean mass change data for animals treated with vehicle (PBS), IgG control, Ab1, and AbMyo are shown in Figure 19. Data are expressed as change in lean mass from day 0 of the study. After a single dose of test article, animals treated with Ab1 (Group 3) on day 21 lost 2.5% of their body mass (1.2%) compared to IgG. Control animals (compared to Group 1) had a significant increase in lean mass that was indistinguishable from the changes in lean mass after three doses of Ab1 (Group 6). These changes in lean mass were also similar to those seen in animals treated with a single dose of AbMyo (Group 4) or three weekly doses of AbMyo (Group 7).

[0330] Example 3 Chemistry / Pharmaceutical Sciences Ab2 is a humanized monoclonal antibody of the IgG4 subtype with a serine-to-proline substitution at position 228. This creates an IgG1-like hinge sequence and minimizes the incomplete formation of interchain disulfide bridges characteristic of IgG4. The complete amino acid sequences of the heavy and light chains of Ab2 are shown below. The complementarity-determining regions (CDRs) are underlined. The bolded NST sequence represents the N-linked glycosylation consensus sequence; the bolded DP sequence represents a potential cleavage site. The bolded NX sequence represents a potential deamidation site; the bolded DX sequence represents a potential isomerization site; the bolded methionine represents a potential methionine oxidation site. The bolded Q represents the predicted N-terminal pyroglutamic acid (Figures 21A-21B).

[0331] Molecular modeling of Ab1 identified several potential post-translational modification sites. Two asparagines in the light chain and seven asparagines in the heavy chain are susceptible to deamidation. Two of these residues are located within the CDR regions of the heavy chain.

[0332] Native IgG4 mAbs may have incomplete formation of inter-heavy chain disulfide bridges, where two half molecules (each containing one heavy chain and one light chain) are maintained in the intact antibody structure by non-covalent interactions. IgG4 molecules may be prone to half-molecule exchange in vitro and in vivo, and the level of half molecules must be consistent between production batches. A Ser to Pro substitution in the backbone of the IgG4 structure results in an IgG1-like hinge sequence, which allows the formation of inter-chain disulfide bonds and significantly stabilizes the antibody structure. The integrity and stability of the hinge region are monitored during development with extensive characterization using assays such as non-reducing capillary electrophoresis and quantification of free sulfhydryls. The potential for chain exchange in Monitor in vivo.

[0333] summary Provided herein is a pro / latent myostatin-specific antibody that blocks the activation of promyostatin and / or latent myostatin. Administration of this activation-blocking antibody to healthy mice increases lean body mass and muscle size, with only a single dose required to maintain muscle-building effects for one month. Furthermore, administration of the antibody protects healthy mice from muscle atrophy in two separate muscle wasting models. These data demonstrate that blocking myostatin activation promotes robust muscle growth and prevents muscle atrophy in vivo, representing an alternative mechanism for therapeutic intervention in muscle wasting.

[0334] Example 4 Analysis of pro- and latent myostatin in muscular atrophy Western blots were performed to determine the presence of pro- and latent myostatin in muscle tissue and the circulation under muscle atrophy and normal conditions. The standard model of muscle atrophy involves treating mice with 2.5 mg / kg / week of dexamethasone (administered in drinking water), with muscle and plasma harvested after 2 weeks of treatment. This model typically results in a 15-25% loss of muscle mass over the course of treatment. Control muscle and plasma were simultaneously harvested from mice not treated with dexamethasone. The rectus femoris, tibialis anterior, and soleus muscles were analyzed. Muscles were excised, flash-frozen in liquid nitrogen, and stored at -80°C until ready for use. Muscle lysates were prepared by grinding followed by lysis in T-PER buffer supplemented with protease and phosphatase inhibitors. Plasma was collected by standard methods and stored at -80°C.

[0335] Multiple samples containing the same protein concentration were separated on a PAGE gel and Western blotted onto a PVDF membrane. For muscle lysates, 10–50 ng of total protein was loaded onto the gel. Plasma was diluted 1:10 with PBS, and 10 μl of each sample was loaded onto the gel. 0.1–1 ng of recombinant pro- and latent myostatin was also loaded onto the gel as size standards. Identification of myostatin protein was achieved using an antibody (AF1539, R&D Systems) that recognizes the myostatin prodomain. This analysis shows that promyostatin is the predominant form in muscle, while latent myostatin is the predominant form in plasma (Figure 25). Furthermore, it was shown that promyostatin is increased in muscle tissue and latent myostatin is decreased in plasma in mice with dexamethasone-induced muscle atrophy.

[0336] To confirm these results, Western blots were repeated using fluorescent labeling and detection (Azure Biosystems). The relative levels of each myostatin form were quantified in the plasma and rectus femoris and tibialis anterior muscles of normal and dexamethasone-treated mice. These data confirm 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).

[0337] These data provide a model for myostatin "flux" in normal and diseased muscle. As shown, in normal muscle (Figure 28A), promyostatin is produced in the muscle and converted to latent myostatin via cleavage by the furin protease. This can occur either inside or outside the cell (Anderson et al., 2008). A percentage of the latent myostatin in the muscle is then released into the circulation, forming a circulating pool of latent myostatin. In muscle atrophy, active myostatin growth factor increases, driving muscle atrophy. This increase is thought to be caused by upregulation of promyostatin levels in muscle and increased conversion of latent myostatin to active growth factor (Figure 28B). The data outlined here directly support the first step of this model, demonstrating an increase in promyostatin in muscle. The data also support the second step, as a decrease in muscle mass was observed in dexamethasone-treated mice, indicating increased production of mature myostatin without a concomitant increase in latent myostatin in muscle. Thus, plasma myostatin levels decreased, suggesting increased conversion to mature myostatin.

[0338] Example 5 Immunoprecipitation of murine serum and muscle tissue Immunoprecipitations were performed to determine the presence of promyostatin in the circulation and to examine the binding of Ab2 and AbMyo to endogenous myostatin precursors in serum and muscle. Ab2 recognizes the predominant form of myostatin in muscle. The results, shown in Figure 27, demonstrate that a pool of serum promyostatin co-precipitates with Ab2, suggesting the presence of extracellular promyostatin in vivo. In addition to binding to promyostatin, latent myostatin, and other partially processed forms of myostatin in serum, Ab2 immunoprecipitated promyostatin from muscle extracts. In contrast, AbMyo efficiently binds to latent myostatin and partially processed precursors in serum, but does not detectably interact with promyostatin in muscle. Given that muscle is the site of myostatin signaling, this provides important insight into the mechanism of action of Ab2. Advantages may arise.

[0339] Homogenized muscle lysates were prepared as follows: Frozen mouse quadriceps muscles were pulverized using a CryoPrep pulverizer (Covaris, Woburn, MA). The pulverized muscle was then resuspended at a concentration of 50 mg / mL in M-Per buffer (ThermoFisher Scientific) with EDTA-free 1xHalt™ protease and phosphatase inhibitor cocktail. The tissue was then pulverized using a plastic pestle (Bio-Plas Cat#4030-PB) and further homogenized by repeated pipetting using a cut-off pipette tip. The muscle samples were then incubated at 4°C for 30 minutes with end-over-end rotation. Finally, the samples were centrifuged at 16,100 g for 10 minutes to pellet the insoluble fraction. The soluble fraction was aspirated and used in the following experiments.

[0340] For immunoprecipitation, Ab2, IgG Ctl, or AbMyo antibodies were covalently conjugated to agarose beads using the Thermo Scientific Pierce™ Co-immunoprecipitation Kit according to the manufacturer's instructions. 75 μg of each antibody was conjugated to 50 μL of bead slurry, with 30 μg of antibody utilized for each immunoprecipitation. Immunoprecipitations were performed on 3 mL of pooled normal mouse serum (Bioreclamation) or 1.05 mL of homogenized soluble mouse quadriceps muscle prepared as described above. The antibody-conjugated beads and sample were incubated overnight at 4°C with end-over-end rocking. After incubation, beads were recovered by passing the entire sample volume through the spin filter included in the co-immunoprecipitation kit using a QIAvac24Plus vacuum manifold (Qiagen). The beads were then washed three times with 200 μL of IP lysis / wash buffer and once with 100 μL of 1× conditioning buffer according to the kit instructions, eluted with 50 μL of elution buffer for 5 minutes, and then mixed with 5 μL of 1 M Tris, pH 9.5 in a collection tube.

[0341] Myostatin species precipitated by the test antibodies were visualized by Western blot using AF1539 (R&D systems) ab124721 (Abcam), Alexa Fluor® 680 AffiniPure donkey anti-sheep IgG (H+L), (Jackson ImmunoResearch), and IRDye® 800CW donkey anti-rabbit IgG (H+L) (LI-COR Biosciences) Thermo Scientific. SEA BLOCK blocking buffer was used for blocking and primary antibody incubation.

[0342] Example 6 Increased muscle mass and changes in myostatin protein expression in rats treated with Ab2 Study design Female Sprague-Dawley rats aged 7–8 weeks were administered a single intravenous dose of Ab2 (10 mg / kg), a nonfunctional human IgG control antibody (10 mg / kg), or an equivalent volume of phosphate-buffered saline (PBS). Serum samples were collected from three rats per group over the course of the experiment at 4 h, 48 h, 7 days, 14 days, 21 days, and 28 days post-dose. Collections were performed using standard methods, and samples were stored at −80°C. Lean mass was measured by quantitative nuclear magnetic resonance (qNMR) at baseline (pre-dose on day 0) and at days 7, 14, 21, and 28 (eight rats per group). At the end of the experiment (day 28), skeletal muscle (rectus femoris, tibialis anterior, and soleus) was harvested, weighed, and snap-frozen in liquid nitrogen for storage at −80°C.

[0343] result Drug exposure in serum samples was assessed using an ELISA specific for human IgG as a reference standard. The levels of Ab2 and IgG control antibodies were measured with known amounts of each drug used. As shown in Figure 29, both Ab2 and IgG control antibodies were detected in rat serum 4 hours after injection. As the experiment progressed, circulating drug levels of Ab2 increased compared to the IgG control, averaging 17.1 μg / ml of drug in serum at the end of the experiment.

[0344] The pharmacodynamic effects of Ab2 treatment were assessed by measuring lean mass (by qNMR) over the course of the experiment and by determining the weight of excised muscle at the end of the experiment. Figure 30A shows lean mass measurements over the course of the experiment. Rats treated with Ab2 show a clear increase in lean mass compared to rats treated with PBS or a human IgG control antibody. Muscle mass was measured by harvesting and weighing all skeletal muscles at the end of the experiment (day 28). As shown in Figure 30B, rats treated with Ab2 show a 14% and 11% increase in rectus femoris and tibialis anterior muscle mass, respectively. Together, these data demonstrate that treatment of rats with a single dose of Ab2 results in a long-lasting increase in muscle mass.

[0345] Relative levels of pro- and latent myostatin were determined by quantitative Western blot of muscle lysates or serum samples. Muscle lysates were prepared from flash-frozen muscle samples by grinding followed by lysis in T-PER buffer supplemented with protease and phosphatase inhibitors. After lysis, samples containing the same concentration of protein were separated on a PAGE gel and Western blotted onto a low-fluorescence PVDF membrane. For muscle lysates, 10–50 ng of total protein was loaded onto the gel. Plasma was diluted 1:10 with PBS, and 10 μl of each sample was loaded onto the gel. 0.1–1 ng of recombinant pro- and latent myostatin was also loaded onto the gel as a size standard. Identification of myostatin protein was achieved using an antibody (AF1539, R&D Systems) that recognizes the prodomain of latent myostatin, followed by detection with a fluorescently labeled secondary antibody. For all Western blot analyses, a minimum of three samples per group were analyzed.

[0346] Treatment with Ab2 increased serum levels of latent myostatin by approximately 20-fold compared to rats treated with the IgG control (Figure 31A). These data are consistent with the effects seen with other antibody drugs and reflect binding of the drug target in the circulation. In rat muscle (rectus femoris), Ab2 treatment resulted in a 1.9-fold increase in the latent form of myostatin (compared to IgG control-treated rats). No statistically significant changes were observed in promyostatin. These data indicate that Ab2 binds to its target, pro / latent myostatin, and alters the processing of myostatin in muscle and in the circulation. We also observed that Ab2 treatment increased latent myostatin, but not promyostatin, in rat muscle (Figure 31B).

[0347] Example 7 Increased muscle mass and changes in myostatin protein expression in mice treated with Ab2 and comparison with a comparator anti-myostatin antibody Test Design Ten-week-old male SCID mice received a single intraperitoneal dose (5 mg / kg) of either Ab2, a nonfunctional human IgG control antibody, or a comparator antibody (AbMyo) that acts by blocking myostatin / receptor interaction. Serum and skeletal muscle samples were collected at 1, 4, 48, 7, 14, 21, 28, and 56 days post-dose throughout the course of the experiment. Serum collection was performed using standard methods, and samples were stored at -80°C. Skeletal muscle (rectus femoris, tibialis anterior, and soleus) was harvested, weighed, and flash-frozen in liquid nitrogen for storage at -80°C. Lean mass was measured by quantitative nuclear magnetic resonance (qNMR) at baseline (pre-dose on day 0) and weekly throughout the course of the experiment.

[0348] result The pharmacodynamic effects of Ab2 treatment were assessed by measuring lean mass (by qNMR) over the course of the experiment. Figure 32 shows lean mass measurements over the course of the experiment. Mice treated with Ab2 show a clear increase in lean mass compared to mice treated with a human IgG control antibody. During the first 3 weeks of the experiment, mice treated with the comparator antibody (AbMyo) show lean mass comparable to the Ab2 group. However, by 28 days after administration, mice treated with AbMyo do not maintain their increased lean mass. In contrast, mice in the Ab2-treated group maintain their increased lean mass throughout the course of the experiment (56 days). These data suggest that Ab2 has a longer duration of action than AbMyo.

[0349] Drug exposure in serum samples was measured using a human IgG-specific ELISA, with known amounts of each drug used as a reference standard. As shown in Figure 33, both Ab2 and the comparator antibody (AbMyo) were detected in the serum as early as 1 hour after injection, and levels of both antibodies exceeding 1 μg / ml were detectable throughout the experiment. However, Ab2 exhibited a significantly longer half-life and estimated area under the curve (AUC1NF) than AbMyo, suggesting that Ab2 exhibits significantly greater exposure than AbMyo at similar doses.

[0350] Relative levels of pro- and latent myostatin were determined by quantitative Western blot of muscle lysates or serum samples. Muscle lysates were prepared from flash-frozen muscle samples by grinding followed by lysis in T-PER buffer supplemented with protease and phosphatase inhibitors. After lysis, samples containing the same concentration of protein were separated on a PAGE gel and Western blotted onto a low-fluorescence PVDF membrane. For muscle lysates, 10–50 ng of total protein was loaded onto the gel. Plasma was diluted 1:10 with PBS, and 10 μl of each sample was loaded onto the gel. 0.1–1 ng of recombinant pro- and latent myostatin was also loaded onto the gel as a size standard. Identification of myostatin protein was achieved using an antibody (AF1539, R&D Systems) that recognizes the prodomain of latent myostatin, followed by detection with a fluorescently labeled secondary antibody. For all Western blot analyses, a minimum of three samples per group were analyzed.

[0351] Serum myostatin was measured by fluorescent Western blot in drug-treated mice and controls. Despite increased serum exposure of Ab2, 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 greater than target levels that increased serum exposure of Ab2 does not result in a greater increase in circulating latent myostatin than observed in the AbMyo group.

[0352] Myostatin levels in muscle (rectus femoris) were also assessed by fluorescent Western blot. Relative levels of latent and promyostatin were measured in mouse muscle lysates by fluorescent Western blot. Latent myostatin is elevated in both Ab2- and AbMyo-treated muscles (Figure 35A). However, while the increase in latent myostatin in AbMyo-treated muscles returns to baseline by day 28, latent myostatin in Ab2-treated muscles remains elevated at least until this time (P < 0.003 vs. AbMyo treatment). A similar trend is observed for promyostatin (Figure 35B), although the difference is not statistically significant (P = 0.068). These data suggest a longer duration of action for Ab2 at the site of drug action, skeletal muscle.

[0353] Example 8 Ab2 increases muscle force production In this example, the myogenic effects of Ab2 were evaluated. Briefly, male C57BL / 6J mice were intraperitoneally administered IgG (20 mg / kg), Ab2 (20 mg / kg) containing the constant region of the mouse IgG1 isotype, or PBS once a week for 4 weeks ( n = 10 per group).

[0354] At the end of the experiment, the muscles were excised and weighed, and in vitro muscle performance of the extensor digitorum longus (EDL) was measured using a 305C muscle lever system (Aurora Scientific Inc., Aurora, CAN) adapted to a horizontal perfusion bath. The muscles were placed in ice-cold physiological buffer solution and sutured to the proximal tendon with silk suture. The muscles were placed in the horizontal bath of the 305C muscle lever system and perfused with physiological buffer oxygenated with 95% O2 / 5% CO2 and maintained at 37°C.

[0355] The suture was tied to a fixed post on one side and to a lever arm on the other. To ensure that the suture was tight and the maximum force generated was stable, a series of 1 Hz and 100 Hz electrical field stimuli (0.2 ms pulses, 100 ms elapsed time) were applied at a frequency of 0.01 Hz through platinum electrodes placed on the lateral side of the muscle. Once stabilized, direct muscle stimulation—force versus frequency—was measured. Platinum wire electrodes were attached proximal and distal to the muscle ampulla.

[0356] Twitch tension was monitored with 1 ms pulses and the voltage was increased until maximal force was achieved, followed by a series of 1 ms pulses of 250 ms duration at increasing frequencies (1, 10, 20, 40, 60, 80, 100, 150 Hz, followed by a final stimulus at 1 Hz).

[0357] As shown in Figure 36A, after 4 weeks of treatment with Ab2, muscle mass and function increased: mean EDL weight increased by 33%, and mean gastrocnemius and quadriceps weights increased by 19%.

[0358] As shown in Figure 36B, after four weekly doses of Ab2, maximal force production increased by 30%.

[0359] While several embodiments of the present disclosure have been described and illustrated herein, various other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein will readily occur to those skilled in the art, and each such variation and / or modification is considered to be within the scope of the present disclosure. More generally, all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and those skilled in the art will readily understand that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications for which the teachings of the present disclosure are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Accordingly, the foregoing embodiments are presented by way of example only, and it should be understood that, within the scope of the appended claims and their equivalents, the present disclosure may be practiced other than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, and / or methods is within the scope of the present disclosure, unless such features, systems, articles, materials, and / or methods are mutually inconsistent.

[0360] The indefinite articles "a" and "an," as used in this specification and in the claims, should be understood to mean "at least one," unless clearly indicated to the contrary.

[0361] The term "and / or," as used in the specification and claims, should be understood to mean "either or both" of the elements so conjoined, i.e., the elements conjointly present in some instances and separately present in other instances. Unless otherwise indicated, other elements may optionally be present other than the elements specifically identified by the term "and / or," whether related or unrelated to such elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended terms such as "comprising," can refer, in one embodiment, 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), etc.

[0362] As used in this specification and the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating listed items, "or" or "and / or" should be interpreted inclusively, i.e., including at least one, but also more than one, of several elements or a list of elements, and optionally including additional unlisted items. Only terms such as "only" or "exactly one," or, when used in the claims, "consisting of," that clearly indicate the contrary, refer to the inclusion of exactly one element of several elements or a list of elements. Generally, the term "or" as used herein will only be interpreted to indicate exclusive alternatives (i.e., "one or the other, but not both") when preceded by an exclusive term such as "either," "one of," "only one of," or "exactly one of." When used in the claims, "consisting essentially of" has its ordinary meaning as used in the field of patent law.

[0363] As used in this specification and claims, the phrase "at least one," when referring to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but does not necessarily include at least one of every element specifically set forth in the list of elements, nor does it exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to those specifically identified 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") can refer, in one embodiment, to at least one A, optionally including more than one A, and no B (optionally including elements other than B); in another embodiment, to at least one B, optionally including more than one B, and no A (optionally including elements other than A); in yet another embodiment, to at least one A, optionally including more than one A, and at least one B, optionally including more than one B (optionally including other elements); and so forth.

[0364] In the claims, as well as in the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," etc., are to be understood as open-ended, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" are closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0365] The use of ordinal numbers such as "first," "second," "third," etc. in the claims to modify claim elements does not, by itself, imply any priority, preference, or ordering of one claim element over another claim element or the chronological order in which method actions are performed, but rather serves to distinguish one claim element having a particular name from another element having the same name (other than the use of ordinal numbers) to distinguish claim elements. It is used simply as a display for

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, the antibody or antigen-binding fragment thereof 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; the antibody comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3; a) CDRH1 comprises the amino acid sequence of SEQ ID NO: 1; CDRH2 comprises the amino acid sequence of SEQ ID NO:4; CDRH3 comprises the amino acid sequence of SEQ ID NO: 10; CDRL1 comprises the amino acid sequence of SEQ ID NO: 12; CDRL2 comprises the amino acid sequence of SEQ ID NO: 18; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 22; wherein said CDR sequences are numbered according to the Kabat numbering system; or b) CDRH1 comprises the amino acid sequence of SEQ ID NO:2; CDRH2 comprises the amino acid sequence of SEQ ID NO:5; CDRH3 comprises the amino acid sequence of SEQ ID NO: 10; CDRL1 comprises the amino acid sequence of SEQ ID NO: 13; CDRL2 comprises the amino acid sequence of SEQ ID NO: 19; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 22; wherein said CDR sequences are numbered according to the IMGT numbering system; An antibody or an antigen-binding fragment thereof.

2. The antibody or antigen-binding fragment thereof according to claim 1, The antibody or antigen-binding fragment is an IgG ４ containing a constant domain, An antibody or an antigen-binding fragment thereof.

3. The antibody or antigen-binding fragment thereof according to claim 2, The IgG ４ The constant domains have a Ser to Pro backbone substitution that creates an IgG1-like hinge and allows interchain disulfide bond formation. An antibody or an antigen-binding fragment thereof.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the light chain variable region is attached at its C-terminus to a Cλ light chain constant domain; An antibody or an antigen-binding fragment thereof.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, The antibody or antigen-binding fragment a heavy chain comprising the amino acid sequence of SEQ ID NO: 50, wherein the N-terminal glutamine (Q) is a pyroglutamic acid residue; and a light chain comprising the amino acid sequence of SEQ ID NO: 51, wherein the N-terminal glutamine (Q) is a pyroglutamic acid residue; Including, An antibody or an antigen-binding fragment thereof.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody or antigen-binding fragment binds to pro / latent myostatin with reduced affinity at acidic pH compared to physiological pH; An antibody or an antigen-binding fragment thereof.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, The antibody or antigen-binding fragment prevents the formation of mature myostatin via proteolysis by thrombin protease. An antibody or an antigen-binding fragment thereof.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, The antibody or antigen-binding fragment prevents proteolytic activation of pro / latent myostatin to mature myostatin. An antibody or an antigen-binding fragment thereof.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the antibody or antigen-binding fragment cross-reacts with human and murine pro / latent myostatin; An antibody or an antigen-binding fragment thereof.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the antibody or antigen-binding fragment does not bind to GDF11 or activin; An antibody or an antigen-binding fragment thereof.

11. 1. A pharmaceutical composition for use in treating a subject having a myopathy or metabolic disorder, comprising: The composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and a pharmaceutically acceptable carrier. Pharmaceutical compositions.

12. A pharmaceutical composition for use according to claim 11, comprising: The myopathy includes spinal muscular atrophy. Pharmaceutical compositions.

13. A pharmaceutical composition for use according to claim 11, comprising: The myopathy includes muscular dystrophy. Pharmaceutical compositions.

14. 14. A pharmaceutical composition for use according to claim 13, comprising: The muscular dystrophy includes Duchenne muscular dystrophy, Becker muscular dystrophy, or facioscapulohumeral muscular dystrophy. Pharmaceutical compositions.

15. A pharmaceutical composition for use according to claim 11, comprising: The metabolic disorder comprises obesity, type II diabetes, or Prader-Willi syndrome. Pharmaceutical compositions.

16. 1. A pharmaceutical composition for use in treating a subject with osteogenesis imperfecta, comprising: The composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and a pharmaceutically acceptable carrier. Pharmaceutical compositions.

17. 1. A pharmaceutical composition for use in treating a subject with amyotrophic lateral sclerosis (ALS), comprising: The composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and a pharmaceutically acceptable carrier. Pharmaceutical compositions.

18. A pharmaceutical composition for use according to any one of claims 11 to 17, comprising: The composition is formulated for subcutaneous or intravenous administration. Pharmaceutical compositions.

19. A pharmaceutical composition for use according to any one of claims 11 to 18, comprising: The composition is (i) a lyophilized composition, (ii) a liquid composition, or (iii) a frozen composition. Pharmaceutical compositions.

20. 20. A pharmaceutical composition for use according to claim 19, comprising: The composition is frozen at a temperature below or equal to -65°C. Pharmaceutical compositions.

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