Anti-myosin / latent myostatin antibodies and their uses

Abstract

This disclosure relates to antibodies that specifically bind to tropomyostatin and / or latent myostatin, and their uses.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680065184.7, filed on September 15, 2016, entitled "Anti-myosotherm / latent myosotherm antibody and its use".

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 219,094, filed September 15, 2015, entitled “Anti-myosotherm / latent myosotherm antibody and its use herein,” the contents of which are incorporated herein by reference for all purposes. Technical Field

[0004] Embodiments of this disclosure may include regulators of growth factor activity. In some embodiments, such regulators may include antibodies and may modulate the activity and / or biology of TGF-β family members. Background Technology

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

[0006] In some embodiments, aspects of this disclosure relate to antibodies that specifically bind to forms of myostatin (e.g., proMyostatin and / or latent Myostatin). For example, antibodies provided herein specifically bind to one or more of the pro- and / or latent forms of myostatin, such as proMyostatin and / or latent Myostatin. In some aspects, this disclosure is based on the unexpected discovery that antibodies provided herein specifically bind to pure or substantially pure proGDF8 (also known as proMyostatin). In some embodiments, antibodies provided herein inhibit myostatin signaling. In some embodiments, inhibition of myostatin signaling can be used to increase muscle mass or prevent muscle atrophy. In some embodiments, antibodies provided herein bind to myostatin and prevent myostatin from being cleaved by proprotein convertases and / or tolloid proteases. In some embodiments, prevention of cleavage of proMyostatin or latent Myostatin prevents myostatin activation. A further aspect of this disclosure relates to antibodies having a pH-sensitive affinity for antigens. In some embodiments, such pH-sensitive antibodies are effective for clearing antigens from serum. Furthermore, in some embodiments, the antibodies provided herein are sweeping antibodies that can effectively clear antigens (e.g., tropomyostatin and / or latent myostatin) from serum.

[0007] This disclosure includes 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) containing a sequence as shown in any of SEQ ID NO:10-11. In some embodiments, the antibody specifically binds to tropomyostatin / latent myostatin. In some embodiments, the light chain variable domain comprises a complementarity-determining region 3 (CDRL3) containing a sequence as shown in any of SEQ ID NO:22-23. In another embodiment, the antibody comprises six complementarity-determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises any of the sequences shown in SEQ ID NO:1-3, CDRH2 comprises any of the sequences shown in SEQ ID NO:4-9, CDRH3 comprises any of the sequences shown in SEQ ID NO:10-11, CDRL1 comprises any of the sequences shown in SEQ ID NO:12-17, CDRL2 comprises any of the sequences shown in SEQ ID NO:18-21, and CDRL3 comprises any of the sequences shown in SEQ ID NO:22-23.

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

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

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

[0011] In another embodiment, the antibody comprises a heavy chain variable domain sequence as shown in any of SEQ ID NO:25-29. In some embodiments, the antibody comprises a light chain variable domain sequence as shown in any of SEQ ID NO:30-35.

[0012] Other aspects of this disclosure include antibodies that specifically bind to tropomyostatin / 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) containing a sequence as shown in any of SEQ ID NO:22-23. In some embodiments, the antibody comprises the light chain variable domain sequence of SEQ ID NO:30.

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

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

[0015] Another aspect of this disclosure includes antibodies that competitively bind to tropomyostatin / latent myostatin against the antibodies described above. In some embodiments, the antibody binds to tropomyostatin / latent myostatin at the same epitope as the antibodies described above. In another embodiment, the antibody binds to tropomyostatin / latent myostatin at a ratio of less than 10. -6 The equilibrium dissociation constant Kd of M competitively binds to tropomyostatin / latent myostatin. In other embodiments, the Kd of the antibody is in the range of 10. -11 M to 10 -6 Within the range of M.

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

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

[0018] Further aspects of this disclosure include antibodies that specifically bind to tropomyostatin / latent myostatin and inhibit the formation of mature myostatin via tolloid protease hydrolysis. In some embodiments, the antibody inhibits the formation of mature myostatin via tolloid protease hydrolysis with an IC50 of less than 1 μM. In some embodiments, the antibody is cross-reactive with human and mouse tropomyostatin / latent myostatin. In other embodiments, the antibody specifically binds to tropomyostatin / latent myostatin compared to GDF11 or activin. In another embodiment, the antibody specifically binds to tropomyostatin / latent myostatin compared to mature myostatin.

[0019] Another aspect of this disclosure includes a method for reducing myostatin receptor activation in cells present in a culture medium containing tropomyostatin / latent myostatin, the method comprising delivering the antibody to the culture medium in an amount that effectively inhibits the proteolytic activation of tropomyostatin / latent myostatin. In some embodiments, the culture medium further contains a precursor protein convertase. In other embodiments, the culture medium further contains a tolloid protease. In another embodiment, the antibody is delivered to the culture medium in an amount that effectively inhibits the proteolytic activation of tropomyostatin / latent myostatin by the tolloid protease. In some embodiments, the cells are in vitro. In other embodiments, the cells are in vivo.

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

[0021] Another aspect of this disclosure includes methods for treating subjects suffering from age-related diseases or conditions. Exemplary age-related diseases or conditions include, but are not limited to, sarcopenia (age-related muscle loss), frailty, and androgen deficiency.

[0022] Another aspect of this disclosure includes methods for treating subjects suffering from diseases or conditions associated with disuse atrophy / trauma. Exemplary diseases or conditions associated with disuse atrophy / trauma include, but are not limited to, myasthenia gravis, hip / joint replacement, hip fracture, stroke, bedriddenness, SCI, rotator cuff injury, knee replacement, fracture, and burns related to time spent in the intensive care unit (ICU).

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

[0024] Another aspect of this disclosure includes methods for treating subjects suffering from diseases or conditions associated with cachexia. Exemplary diseases or conditions associated with cachexia include, but are not limited to, cancer, chronic heart failure, acquired immunodeficiency syndrome (AIDS), chronic obstructive pulmonary disease (COPD), and chronic kidney disease (CKD).

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

[0026] Another aspect of this disclosure includes methods for treating a subject suffering from a disease or condition related to 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, other diseases or conditions related to metabolic disorders and / or body composition may also be within the scope of this disclosure.

[0027] Another aspect of this disclosure includes methods for treating a subject suffering from a disease or condition associated with congenital myopathy. Exemplary congenital myopathy includes, but is not limited to, X-linked myotubular myopathy, autosomal dominant central nucleus myopathy, autosomal recessive central nucleus myopathy, linear myopathy, and congenital fibrous asymmetric myopathy.

[0028] Another aspect of this disclosure includes methods for treating subjects suffering from diseases or conditions associated with muscular dystrophy. Exemplary muscular dystrophys include, but are not limited to, Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy (FSH), and limb-girdle muscular dystrophy.

[0029] Another aspect of this disclosure includes methods for treating subjects suffering from gynecological urinary-related diseases or conditions, speech disorders (stenosis), extraocular myopathy, carpal tunnel syndrome, Guillain-Barré syndrome, or osteosarcoma.

[0030] 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.

[0031] In some embodiments, 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.

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

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

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

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

[0036] Further aspects of this disclosure include isolated nucleic acids comprising sequences as shown in any of SEQ ID NO:38-49.

[0037] Another aspect of this disclosure includes isolated cells containing the isolated nucleic acids described above.

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

[0039] In one aspect, this document discloses isolated antibodies comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO:25 and a light chain variable region containing the amino acid sequence of SEQ ID NO:31. In one embodiment, the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO:50. In another embodiment, the antibody comprises a light chain containing the amino acid sequence of SEQ ID NO:51.

[0040] In another aspect, this article discloses isolated antibodies comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDRH1 sequence containing SEQ ID NO:1, a CDRH2 sequence containing SEQ ID NO:6, and a CDRH3 sequence containing SEQ ID NO:11; and the light chain variable region comprises a CDRL1 sequence containing SEQ ID NO:14, a CDRL2 sequence containing SEQ ID NO:20, and a CDRL3 sequence containing SEQ ID NO:23.

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

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

[0043] In another aspect, this article discloses isolated antibodies comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDRH1 sequence containing SEQ ID NO:1, a CDRH2 sequence containing SEQ ID NO:8, and a CDRH3 sequence containing SEQ ID NO:11; and the light chain variable region comprises a CDRL1 sequence containing SEQ ID NO:16, a CDRL2 sequence containing SEQ ID NO:20, and a CDRL3 sequence containing SEQ ID NO:23.

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

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

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

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

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

[0049] In one embodiment, the antibody is cross-reactive with human and mouse tropomyostatin / latent myostatin. In another embodiment, the antibody binds to tropomyostatin / latent myostatin but not to GDF11 or activin.

[0050] In one aspect, this document discloses a method for reducing myostatin receptor activation in cells present in a culture medium containing tropomyostatin / latent myostatin, the method comprising delivering an antibody described herein to the culture medium in an amount that effectively inhibits the proteolytic activation of tropomyostatin / latent myostatin. In one embodiment, the culture medium contains a precursor protein convertase. In another embodiment, the culture medium contains a tolloid protease. In one embodiment, the cells are in vitro. In another embodiment, the cells are in vivo.

[0051] In another aspect, this article discloses a method for treating subjects with myopathy, which involves administering an effective amount of the antibody disclosed herein to the subject.

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

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

[0054] In one implementation, administration results in improved muscle strength in the subject. In another implementation, administration results in improved metabolic status in the subject.

[0055] 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.

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

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

[0058] In another aspect, this document discloses pharmaceutical compositions comprising the antibodies disclosed herein and pharmaceutically acceptable carriers. 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 below or equal to -65°C.

[0059] In another aspect, this document discloses syringes comprising the pharmaceutical compositions described herein.

[0060] In another aspect, this article discloses isolated nucleic acids encoding antibodies comprising a heavy chain variable region containing the nucleic acid sequence of SEQ ID NO:39 and a light chain variable region containing the nucleic acid sequence of SEQ ID NO:45.

[0061] In another aspect, this article discloses isolated nucleic acids encoding antibodies comprising heavy chain variable regions and light chain variable regions, wherein the heavy chain variable regions comprise CDRH1 sequence containing SEQ ID NO:1, CDRH2 sequence containing SEQ ID NO:6, and CDRH3 sequence containing SEQ ID NO:11; and the light chain variable regions comprise CDRL1 sequence containing SEQ ID NO:14, CDRL2 sequence containing SEQ ID NO:20, and CDRL3 sequence containing SEQ ID NO:23.

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

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

[0064] In another embodiment, this document discloses isolated nucleic acids encoding antibodies comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDRH1 sequence containing SEQ ID NO:1, a CDRH2 sequence containing SEQ ID NO:8, and a CDRH3 sequence containing SEQ ID NO:11; and the light chain variable region comprises a CDRL1 sequence containing SEQ ID NO:16, a CDRL2 sequence containing SEQ ID NO:20, and a CDRL3 sequence containing SEQ ID NO:23.

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

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

[0067] In another aspect, this article discloses isolated cells containing the isolated nucleic acids described herein.

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

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

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

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

[0072] 4. An isolated antibody comprising:

[0073] The heavy chain variable region contains the CDRH1 sequence containing SEQ ID NO:1, the CDRH2 sequence containing SEQ ID NO:6, and the CDRH3 sequence containing SEQ ID NO:11; and

[0074] The light chain variable region contains the CDRL1 sequence containing SEQ ID NO:14, the CDRL2 sequence containing SEQ ID NO:20, and the CDRL3 sequence containing SEQ ID NO:23.

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

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

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

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

[0079] 9. An isolated antibody comprising

[0080] The heavy chain variable region contains the CDRH1 sequence containing SEQ ID NO:1, the CDRH2 sequence containing SEQ ID NO:8, and the CDRH3 sequence containing SEQ ID NO:11; and

[0081] The light chain variable region contains the CDRL1 sequence containing SEQ ID NO:16, the CDRL2 sequence containing SEQ ID NO:20, and the CDRL3 sequence containing SEQ ID NO:23.

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

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

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

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

[0086] 14. An isolated antibody as described in any one of items 1-13, wherein the antibody is a human antibody.

[0087] 15. An isolated antibody as described in any one of items 1-14, wherein said antibody contains an IgG4 constant domain.

[0088] 16. An isolated antibody as described in any one of items 1-14, wherein the antibody comprises an IgG4 constant domain having a Ser to Pro backbone replacement, which generates an IgG1-like hinge and allows the formation of interchain disulfide bonds.

[0089] 17. The isolated antibody as described in any one of items 1-16, wherein the antibody specifically binds to tropomyostatin / latent myostatin.

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

[0091] 19. The isolated antibody as described in any one of items 1-18, wherein the antibody inhibits the formation of mature myostatin via proteolysis by a tolloid protease.

[0092] 20. The isolated antibody as described in item 19, wherein the antibody inhibits the formation of mature myostatin via proteolysis by tolloid protease at an IC50 of less than 1 μM.

[0093] 21. An isolated antibody as described in item 19 or 20, wherein the antibody is cross-reactive with human and murine myostatin / latent myostatin.

[0094] 22. An isolated antibody as described in any one of items 19-21, wherein the antibody binds to tropomyostatin / latent myostatin but not to GDF11 or activin.

[0095] 23. A method for reducing myostatin receptor activation in cells present in a culture medium containing tropomyostatin / latent myostatin, the method comprising delivering an antibody according to any one of claims 1-22 to the culture medium in an amount that effectively inhibits the proteolytic activation of said tropomyostatin / latent myostatin.

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

[0097] 25. A pharmaceutical composition comprising an antibody as described in any one of claims 1-22 and a pharmaceutically acceptable carrier.

[0098] 26. The pharmaceutical composition as described in claim 25, wherein the composition is a lyophilized composition.

[0099] 27. The pharmaceutical composition as described in claim 25, wherein the composition is a liquid composition.

[0100] 28. The pharmaceutical composition as described in claim 25, wherein the composition is frozen.

[0101] 29. The pharmaceutical composition of claim 28, wherein the composition is frozen at a temperature of -65°C or below.

[0102] 30. A syringe comprising the pharmaceutical composition of any one of claims 25-29.

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

[0104] 32. An isolated nucleic acid encoding an antibody containing the following:

[0105] The heavy chain variable region contains the CDRH1 sequence containing SEQ ID NO:1, the CDRH2 sequence containing SEQ ID NO:6, and the CDRH3 sequence containing SEQ ID NO:11; and

[0106] The light chain variable region contains the CDRL1 sequence containing SEQ ID NO:14, the CDRL2 sequence containing SEQ ID NO:20, and the CDRL3 sequence containing SEQ ID NO:23.

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

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

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

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

[0111] 37. An isolated nucleic acid encoding an antibody containing the following:

[0112] The heavy chain variable region contains the CDRH1 sequence containing SEQ ID NO:1, the CDRH2 sequence containing SEQ ID NO:8, and the CDRH3 sequence containing SEQ ID NO:11; and

[0113] The light chain variable region contains the CDRL1 sequence containing SEQ ID NO:16, the CDRL2 sequence containing SEQ ID NO:20, and the CDRL3 sequence containing SEQ ID NO:23.

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

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

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

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

[0118] 42. An isolated cell comprising the isolated nucleic acid as described in any one of claims 31-41. Attached Figure Description

[0119] Figure 1A-1B The structure of the myostatin domain and the assembly of tropomyostatin are displayed. Figure 1A This protein is shown to be myostatin secreted by the genoprotein, which has an inhibitory prodomain and a subsequent C-terminal growth factor domain. The genoprotein exists as a disulfide-linked dimer. Figure 1B This diagram shows a precursor protein assembled in an inactive conformation, where the pre-domain (dark gray) is assembled around the growth factor (light gray) in a “sleeve.” This graphic is an adaptation from the structure of latent TGFβ1 (Shi et al., Nature 2011).

[0120] Figure 2 The activation of myostatin involves two distinct protease events, resulting in three major myostatin species. The biosynthetic precursor protein, tropomyostatin, is processed by two separate proteases. Cleavage of tropomyostatin (and proGDF11) is achieved via precursor protein convertases such as Furin / PACE3 (…). Coupling of basic amino acid cleavage enzyme (Paired Basic Amino Acid Cleaving Enzyme)3) or PCSK5 (precursor protein convertase, subtilisin / Kexin type 5) cleaves the conserved RXXR site between the pre-domain and the mature growth factor. This cleavage produces a latent complex in which the mature growth factor is shielded by the pre-domain and does not bind to its receptor. Activation and release of the active growth factor are accomplished post-cleavage by other proteases from the BMP / tolloid family, such as TLL-2 (Tolloid-like protein 2) or BMP1 (bone morphogenetic protein 1). These cleavage events produce the mature form of myostatin, which can be called active myostatin or mature myostatin.

[0121] Figures 3A-3C Ab1 demonstrates that it blocks the cleavage of tropomyostatin by members of the tolloid family of proteases. Latent myostatin samples pre-incubated with increased amounts of Ab1 were analyzed in the myostatin activation assay. Results were obtained via a reporter assay (…). Figure 3A After analyzing myostatin release, the sample was then run under reducing conditions and detected by Western blotting using an antibody generated against the pre-domain of myostatin. Figure 3B The ~18kDa band (boxed) corresponds to the ARM portion of the pre-domain generated after tolloid cleavage, decreasing proportionally with increasing Ab1 dose. Latency and tropomyostatin standards (loaded 45ng) showed migration of tropomyostatin at ~50kDa and the pre-domain at ~37kDa. Figure 3C The activation of myostatin involves two distinct protease events, resulting in three major myostatin species. The biosynthetic precursor protein, tropomyostatin, is processed by two separate proteases. Cleavage of tropomyostatin (and proGDF11) occurs via precursor protein convertases such as Furin / PACE3 (a basic amino acid lyase 3) or PCSK5 (a precursor protein convertase, subtilisin / Kexin type 5), which cleave the conserved RXXR site between the predomain and the mature growth factor. This cleavage produces a latent complex in which the mature growth factor is shielded from binding to its receptor by the predomain. See also Figure 3B This demonstrates the potential inhibition of proteases, thereby blocking further cleavage of tropomyostatin. Activation and release of active growth factors are accomplished post-cleavage by other proteases from the BMP / tolloid family, such as TLL-2 (Tolloid-like protein 2) or BMP1 (bone morphogenetic protein 1).

[0122] Figure 4 This study demonstrates the performance of the parental Ab1 antibody and other candidates in a cell-based reporter assay. Mature growth factor release was measured in 293T cells using a CAGA-based reporter assay following overnight proteolytic reactions with enzymes from both the precursor protein convertase and tolloid protease families. Results were compared to control reactions to calculate the fraction of tropomyostatin or proGDF11 released in this assay. The standard deviation of the means for three replicates is shown, but is not visible on the plot for most data points due to their low amplitude.

[0123] Figure 5 The diagram illustrates that antibodies Ab1, Ab2, Ab4, and Ab6 do not inhibit proGDF11 activation.

[0124] Figure 6 This presentation shows the results of an experiment assessing the change in mean percentage body weight. Animals were weighed daily and the change in percentage weight from day 0 was calculated. Data are presented as group mean ± SEM. Mean percentage changes in each group relative to the PBS control group at day 42 of the study were analyzed using one-way ANOVA followed by Holm-Sidak's posterior test, **p < 0.01.

[0125] Figures 7A-7D This shows the results of a test that assesses tissue weight. Figure 7A This shows the average weight of the gastrocnemius muscle. Figure 7B This shows the average weight of the pectoral muscles. Figure 7C This displays the average weight of the soleus muscle. Figure 7D The average triceps weight is displayed. Statistical assessment relative to the solvent control group (group 1) was performed using one-way ANOVA followed by Holm-Sidak's posterior test. Data are presented as group means ± SEM. **p<0.01. Bars indicate groups 1-5 from left to right.

[0126] Figures 8A-8C This shows the results of a test that assesses tissue weight. Figure 8A This shows the average weight of the tibialis anterior muscle. Figure 8B This shows the average weight of the diaphragm. Figure 8C The average quadriceps weight is displayed. Statistical assessment relative to the solvent control group (group 1) was performed using one-way ANOVA followed by Holm-Sidak's posterior test. Data are presented as group means ± SEM. *p<0.05. Bars indicate groups 1-5 from left to right.

[0127] Figures 9A-9B This shows the results of a trial that assessed changes in average percentage body weight and lean body mass. Figure 9A It is a graph showing the percentage weight change from day 0 in animals weighed twice a week throughout the study. Figure 9B In this study, animals underwent EchoMRI (QNMR) to measure body composition on days -4, 7, 14, 21, and 28, and the change in percentage lean body mass from day 0 was calculated. Data are presented as group means ± SEM. For both body weight and lean body mass, the mean percentage change in each group relative to the IgG control group (group 2) on day 28 of the study was analyzed using one-way ANOVA followed by Holm-Sidak's posterior test. ***p<0.0005, **p<0.005, *p<0.05, ns (not significant).

[0128] Figure 10A-10D It is a graph showing the test results for assessing muscle weight. Figure 10A Showing average quadriceps weight. Figure 10B This displays the average weight of the gastrocnemius muscle. Figure 10C Showing average tibialis anterior muscle weight, and Figure 10D Mean diaphragm weight is shown. The percentage difference in mean muscle weight between the Ab1 treatment group and the IgG control group is indicated above each bar. Statistical assessment relative to the IgG control group (group 2) was performed using one-way ANOVA followed by Holm-Sidak's posterior test. Data are presented as group means ± SEM. ****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05, ns (not significant).

[0129] Figure 11A-11B This shows the results of a trial that assessed changes in average percentage body weight and lean body mass. Figure 11A This shows the percentage change in weight from day 0, calculated from animals weighed twice a week throughout the study. Figure 11B Animals underwent EchoMRI (QNMR) to measure body composition on days -1, 6, and 13, and to calculate the change in percentage lean body mass from day -1. PBS = phosphate-buffered saline, Dex = dexamethasone, IgG(20) = IgG control antibody administered at 20 mg / kg / wk, Ab1(20) = Ab1 antibody administered at 20 mg / kg / wk, and Ab1(2) = Ab1 antibody administered at 2 mg / kg / wk. Data are presented as group mean ± SEM. The mean percentage changes in each group on day 14 (for body weight) and day 13 (for lean body mass) were analyzed using one-way ANOVA followed by Dunnett's multiple comparison test relative to group 1 (****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05) and relative to group 5 using one-way ANOVA followed by Dunnett's multiple comparison test (++++p<0.0001, +++p<0.0005, ++p<0.005, +p<0.05). The results were not significant (ns).

[0130] Figure 12A-12D It is a graph showing the test results for assessing the weight of different muscles. Figure 12A Displays the average weight of the gastrocnemius muscle (grams). Figure 12B Displays average quadriceps weight (grams). Figure 12C Showing the mean percentage change in gastrocnemius muscle weight relative to control animals (group 1) treated with PBS (IP) and normal drinking water, and Figure 12D Showing the mean percentage change in quadriceps weight relative to control animals (group 1) treated with PBS (IP) and normal drinking water. PBS = phosphate-buffered saline, Dex = dexamethasone, IgG (20) = IgG control antibody administered at 20 mg / kg / wk, Ab1 (20) = Ab1 antibody administered at 20 mg / kg / wk, and Ab1 (2) = Ab1 antibody administered at 2 mg / kg / wk. For Figure 12A-12B The error bars represent the standard deviation (SD). For Figure 12C-12DError bars represent the standard error (SEM) of the mean. Statistical assessments relative to group 1 (****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05) and relative to group 5 (++++p<0.0001, +++p<0.0005, ++p<0.005, +p<0.05) were performed using one-way ANOVA followed by Dunnett's multiple comparison test. ns (not significant). Bars from left to right indicate PBS, water; PBS, dex; IgG control; Ab1(20); and Ab1(2).

[0131] Figures 13A-13B This shows the results of a trial that assessed changes in average percentage body weight and lean body mass. Figure 13A This shows the percentage change in weight from day 0 for animals weighed twice a week throughout the study. Figure 13B The change in percentage lean body mass from day -1 is shown from animals that underwent EchoMRI (QNMR) to measure body composition on days -1, 7, and 14. PBS = phosphate-buffered saline, IgG (20) = IgG control antibody administered at 20 mg / kg / wk, Ab1 (20) = Ab1 antibody administered at 20 mg / kg / wk, and Ab1 (2) = Ab1 antibody administered at 2 mg / kg / wk. Data are presented as group mean ± SEM.

[0132] Figures 14A-14D This shows the results of an experiment that assesses muscle weight. Figure 14A This shows the average gastrocnemius muscle weight (in grams) of the leg in a cast. Figure 14B This shows the average quadriceps weight (in grams) of the leg in a cast. Figure 14C Showing the mean percentage change in gastrocnemius muscle weight relative to control animals treated with PBS (IP) and not treated with plaster cast (group 1), and Figure 14D Shows the mean percentage change in quadriceps weight relative to control animals (group 1) treated with PBS (IP) and not treated with plaster (group 1). PBS = phosphate-buffered saline, IgG (20) = IgG control antibody administered at 20 mg / kg / wk, Ab1 (20) = Ab1 antibody administered at 20 mg / kg / wk, and Ab1 (2) = Ab1 antibody administered at 2 mg / kg / wk. For Figures 14A-14B The error bars represent the standard deviation (SD). For Figure 14C-14DError bars represent the standard error (SEM) of the mean. Statistical assessments relative to group 1 (****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05) and relative to group 5 (++++p<0.0001, +++p<0.0005, ++p<0.005, +p<0.05) were performed using one-way ANOVA followed by Dunnett's multiple comparison test. ns (not significant). Bars from left to right indicate PBS, no plaster; PBS, plaster; IgG control (2), plaster; Ab1 (20), plaster; and Ab1 (2), plaster.

[0133] Figure 15 The results of the trial assessing changes in lean body mass on day 21 (top right) and day 28 (top left) are shown. It also describes the percentage change in lean body mass at three different doses of the tested antibody (20 mg / kg / wk (bottom left), 2 mg / kg / wk (bottom center), and 0.5 mg / kg / wk (bottom right)), a PBS control, and an IgG control. Statistical assessments relative to group 1 (****p<0.0001, ***p<0.005, **p<0.01, *p<0.05) and relative to the IgG control were performed using one-way ANOVA followed by Dunnett's multiple comparison test. For the two graphs above, the bars from left to right represent: PBS; IgG Ctrl 20 mg / kg / wk; Ab1 20 mg / kg / wk; Ab1 2 mg / kg / wk; Ab1 0.5 mg / kg / wk; Ab2 20 mg / kg / wk; Ab2 2 mg / kg / wk; Ab2 0.5 mg / kg / wk; Ab4 20 mg / kg / wk; Ab4 2 mg / kg / wk; Ab4 0.5 mg / kg / wk; Ab6 20 mg / kg / wk; Ab6 2 mg / kg / wk; and Ab6 0.5 mg / kg / wk. For the bottom left graph (20 mg / kg / wk), the data points corresponding to day 28 after administration, from top to bottom, correspond to Ab1, Ab4, Ab2, Ab6, IgG control, and PBS. For the lower middle graph (2 mg / kg / wk), the data points corresponding to day 28 after administration, from top to bottom, correspond to Ab2, Ab1, Ab6, Ab4, IgG control, and PBS. For the lower right graph (0.5 mg / kg / wk), the data points corresponding to day 28 after administration, from top to bottom, correspond to IgG control, Ab1, Ab2, PBS, Ab4, and Ab6.

[0134] Figures 16A-16B Domain structure and evaluation of myostatin precursor form. Figure 16A The domain structures of tropomyostatin and latent myostatin were shown, indicating the protease cleavage sites. Figure 16B This image shows partial precursor protein convertase cleavage of tropomyostatin on an SDS-PAGE gel. Under reducing conditions, the protein band consists of tropomyostatin monomer (~50 kD), predomain (~37 kD), and growth factor (12.5 kD).

[0135] Figures 17A-17B This indicates that Ab1 is specific for myostatin. Figure 17A Ab1 was shown to specifically bind to tropomyosin and latent myostatin, with no binding observed to other members of the TGFB superfamily, most notably the corresponding form of GDF11. Ab1 was applied at a high concentration (50 μg / mL) to a Forte-Bio BLI tip coated with the indicated antigen, and binding and dissociation rates were measured to obtain approximate Kd values. The magnitude of the biosensor response (indicating binding events) is represented by a black bar graph, and the calculated Kd is indicated in orange. Figure 17B Ab1 showed that it blocked the activation of tropomyostatin but not proGDF11. The release of mature growth factors in 293T cells was measured using a CAGA-based reporter assay following overnight proteolytic reactions with enzymes from both the precursor protein convertase and tolloid protease families. Results were compared to control responses to calculate the fraction of tropomyostatin or proGDF11 released in this assay.

[0136] Figures 18A-18C This shows the SCID dose response of the candidate antibody. Figure 18A Showing the muscle weight of the gastrocnemius muscle and Figure 18B This shows the muscle weight of the quadriceps. Figure 18C Shows the percentage change in average muscle mass compared to the PBS control. Figures 18A-18B The bars in the image, from left to right, are: PBS; IgG Ctrl 20 mg / kg / wk; Ab1 20 mg / kg / wk; Ab1 2 mg / kg / wk; Ab1 0.5 mg / kg / wk; Ab2 20 mg / kg / wk; Ab2 2 mg / kg / wk; Ab2 0.5 mg / kg / wk; Ab4 20 mg / kg / wk; Ab4 2 mg / kg / wk; Ab4 0.5 mg / kg / wk; Ab6 20 mg / kg / wk; Ab6 2 mg / kg / wk; and Ab6 0.5 mg / kg / wk.

[0137] Figure 19 This study shows the results of a duration of action study comparing Ab1 to an existing myostatin antibody (AbMyo). PBS was used as a negative control; IgG was used as a positive control. Changes in lean body mass were examined after 21 days under different dosing regimens.

[0138] Figure 20 This is a schematic diagram illustrating an experiment to reconstruct myostatin activation in vitro.

[0139] Figures 21A-21B The heavy chain of the humanized monoclonal antibody (Ab2) showing the proline subtype of IgG4 with replaced serine ( Figure 21A ;SEQ ID NO:50) and light chains ( Figure 21B (SEQ ID NO: 51). This produces an IgG1-like hinge sequence and minimizes the incomplete formation of interchain disulfide bridges (a characteristic of IgG4). Complementarity-determining region (CDR) is underlined. The bold NST sequence: N-linked glycosylation concordance sequence site; the bold DP sequence is a potential cleavage site; the bold NX sequence, where X can be S, T, or G, is a potential deamidation site; the bold DX sequence, where X can be G, S, T, or SDG, is a potential isomerization site; the bold methionine is a potential methionine oxidation site; the bold Q is the expected N-terminal pyroglutamic acid.

[0140] Figure 22 This is a schematic diagram illustrating the reduced immunogenicity risk through germlining. 24H4 (WT) contains five non-germline amino acids within the frame region, as shown in the schematic diagram.

[0141] Figures 23A-23C Optimization of Ab1 was demonstrated. Optimized candidates that specifically bind to tropomyostatin were selected, resulting in dozens of clones with enhanced affinity. FACS was performed to demonstrate the affinity for Ab1 (…). Figure 23A Compared to yeast cloning ( Figure 23B The combination of ) increases. Figure 23C The variants showing mature affinity also exhibit a slower dissociation rate via octet.

[0142] Figures 24A-24B Showing the heavy chain variable region of parent Ab1 with affinity-optimized variants Ab3 and Ab5 ( Figure 24A ) and light chain variable region ( Figure 24B Sequence alignment. The sequence identifiers from top to bottom correspond to SEQ ID NO: 24, 26, 28 ( Figure 24A The sequence identifiers from top to bottom correspond to SEQ ID NO: 30, 32, 34 (). Figure 24B The complementarity-determining region (CDR) is defined using Kabat (underline) and IMGT nomenclature (bold). Substitutions relative to parent Ab1 are shown in light gray.

[0143] Figure 25The expression of tropomyostatin and latent myostatin in the muscle and plasma of normal and muscularly atrophied mice is shown.

[0144] Figure 26 This displays the quantification of changes in tropomyostatin and latent myostatin in muscle and plasma. The bars, from left to right, show tropomyostatin, latent myostatin, tropomyostatin, latent myostatin, and latent myostatin.

[0145] Figure 27 This shows that Ab2 uniquely recognizes tropomyostatin and latent myostatin, thus binding the major form of myostatin in both serum and muscle. Non-reduced protein blots of the pre-domain (dark gray) and mature growth factor (light gray) are shown. Recombinant tropomyostatin (rProMyostatin) migration on the gel is shown for both tropomyostatin and the pre-domain (latent myostatin), highlighted by arrows. In serum, both Ab2 and AbMyo bind latent myostatin (pre-domain band) and various partially processed precursors, but only Ab2 recognizes tropomyostatin (upper band). In muscle, Ab2 precipitates tropomyostatin, while no interaction between AbMyo and tropomyostatin is observed in muscle tissue.

[0146] Figures 28A-28B This provides a model of myostatin flow in normal and atrophied muscles. In normal muscle ( Figure 28A Tropostatin is produced in muscle and converted into latent myostatin by cleavage by furin proteases (which can be present intracellularly or extracellularly). Latent myostatin in certain parts of the muscle is then released into the circulation, thus forming a circulating pool of latent myostatin. In muscle atrophy ( Figure 28B The increase in active myostatin growth factor is caused by the upregulation of tropomyostatin levels in muscle and the increased conversion of latent myostatin to active growth factor. As a result, circulating latent myostatin decreases as the muscle pool of latent myostatin is redirected to mature myostatin via mTLL2 cleavage.

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

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

[0149] Figures 31A-31B This shows the levels of tropomyosin / latent myostatin in Ab2-treated rats. Figure 31A The Ab2 treatment (top line) showed that it increased the level of latent myostatin in rat serum by ~20-fold. Figure 31B The results show that Ab2 treatment in rat muscle (rectus femoris) resulted in a 1.9x increase in the latent form of myostatin. The bars from left to right correspond to tropomyostatin, latent myostatin, tropomyostatin, and latent myostatin. No statistically significant changes in tropomyostatin were observed in rat muscle. These data were derived from quantitative Western blot analysis of n=3 samples / groups.

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

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

[0152] Figure 34Serum myostatin was measured using Western blotting in drug-treated mice and in controls. Despite increased serum Ab2 exposure, serum latent myostatin levels were similar in both Ab2- and AbMyo-treated mice. These data suggest that circulating levels of the free drug were sufficiently excessive relative to the target level so that increased serum Ab2 exposure did not result in a greater increase in circulating latent myostatin compared to that observed in the AbMyo group. Data sets from left to right correspond to IgG, Ab2, AbMyo, IgG, Ab2, and AbMyo.

[0153] Figures 35A-35B The relative levels of latent myostatin and tropomyostatin were measured in mouse myolysis products using Western blotting. Figure 35A The study showed that latent myostatin levels were elevated in both Ab2- and AbMyo-treated muscles. However, the elevation of latent myostatin in AbMyo-treated muscles returned to baseline by day 28, while those in Ab2-treated muscles remained elevated until at least this time (P<0.003, relative to AbMyo-treatment). Figure 35B Similar trends were observed for tropomyostatin, although the difference between the Ab2 and AbMyo treatment groups was not statistically significant at day 28 (P = 0.068).

[0154] Figure 36A This demonstrates the effect of Ab2 treatment on muscle mass and function in mice.

[0155] Figure 36B This demonstrates the effect of Ab2 treatment on maximal force generation in mice. Detailed Implementation

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

[0157] The activation and release of mature growth factors are accomplished through several discrete protease cleavage events, which are outlined in... Figure 2The first cleavage step of tropomyostatin and proGDF11 occurs via a precursor protein convertase, which cleaves at a conserved RXXR site between the predomain and the mature growth factor. This cleavage produces a latent complex in which the mature growth factor is shielded from its receptor by the predomain. Activation and release of mature, active myostatin growth factor are then completed following cleavage by further proteases from the BMP / tolloid family, such as mTLL-2. Figure 2 ).

[0158] Exemplary proGDF8 sequences from humans, rats, mice, and cynomolgus monkeys are provided below. In these proGDF8 sequences, the precursor protein convertase cleavage site is indicated in bold and the tolloid protease site is indicated by underlining. In some embodiments, the precursor protein convertase cleavage site comprises amino acid residues 240-243 of SEQ ID NO:52-55. In some embodiments, the tolloid protease site comprises amino acid residues 74-75 of SEQ ID NO:52-55. It should be understood that the exemplary proGDF8 sequences provided herein are not intended to be limiting, and additional proGDF8 sequences from other species (including any of their variants) are within the scope of this disclosure.

[0159] proGDF8 (person):

[0160]

[0161] proGDF8 (rat):

[0162]

[0163] proGDF8 (mouse):

[0164]

[0165] proGDF8 (Crab-eating Monkey):

[0166]

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

[0168] In some embodiments, this document provides a tropomyostatin / latent myostatin antibody that specifically binds to a chimeric construct comprising a growth factor domain and the N-terminal propeptide portion of GDF11 and the C-terminal propeptide portion of GDF8. This chimeric construct (as shown below) is referred to as GDF11Arm8.

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

[0170] MDMRVPAQLLGLLLLWFSGVLGDYKDDDDKHHHHHHLEVLFQGPAEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETP TTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRNLGL DCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS

[0171] The role of myostatin in myopathy

[0172] Skeletal muscle accounts for approximately 40% of body weight and is a dynamic organ, renewing itself at a rate of 1–2% per day. Muscle atrophy is a highly regulated catabolic process that occurs during disuse (e.g., disuse atrophy) and / or in response to increased systemic inflammation (cachexia). In disuse atrophy (which can occur during prolonged periods of immobility, such as bed rest), muscle loss occurs rapidly. For example, during a one-week hospital stay, patients lose an average of ~1.3 kg of muscle mass.

[0173] Muscle atrophy causes significant pathology in a wide range of clinical conditions. In denervated diseases such as amyotrophic lateral sclerosis (ALS) or spinal muscular atrophy (SMA) and hereditary diseases including muscular dystrophy, loss of muscle strength and function is a highly disabling clinical manifestation for which there is insufficient treatment. In cachexia syndromes caused by kidney failure, AIDS, heart disease, or cancer, muscle atrophy often impairs the successful treatment of the primary condition. Muscle loss also results from the natural aging process and, in its most severe form, is classified as sarcopenia, a common condition in older adults that is increasingly recognized as a pathology requiring intervention. Finally, the primary driver of muscle atrophy is disuse. Immobilization causes rapid and significant muscle loss in a large class of conditions such as hip fractures, selective joint replacements, spinal cord injuries, critical myopathy, and stroke. Although their etiologies vary, these indications share the characteristic of muscle weakness, which leads to severe disability, prolonged physical rehabilitation and recovery times, and impaired quality of life.

[0174] There is an unmet medical need in muscular atrophy disorders. Therefore, in some embodiments, methods for treating muscular atrophy are provided herein. In some embodiments, the methods provided herein relate to the treatment of primary myopathy. In some embodiments, the methods provided herein relate to the treatment of secondary myopathy (where muscle loss is secondary to the pathology of the disease), such as, for example, denervation disorders, hereditary myasthenia gravis, and cachexia. In some embodiments, the methods provided herein for treating primary myopathy such as disuse atrophy (e.g., associated with hip fractures or spinal cord injuries (SCI)) result in improvements in muscle mass, strength, and function in the subject.

[0175] Inhibition of the myostatin pathway

[0176] Several myostatin pathway antagonists exist at various stages of clinical development for the treatment of muscle-related conditions. These pathway antagonists target mature growth factors or their type II receptors, and most antagonize the signaling of multiple TGFβ family members. For example, several 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. This disclosure relates to the understanding that blocking these factors other than myostatin could potentially limit the patient population that can be safely treated due to unacceptable side effects.

[0177] Therefore, this article provides antibodies capable of binding to tropomyostatin and / or latent myostatin (thereby inhibiting myostatin activity) and their use in treating myopathy-related diseases and disorders. In some embodiments, given the pervasiveness of the latent complex in circulation, this article provides treatments that specifically target the more abundant and longer-lived myostatin precursors (e.g., tropomyostatin and latent myostatin) rather than mature growth factors. Not wishing to be limited to any particular theory, the antibodies provided herein prevent the proteolytic activation of tropomyostatin and / or latent myostatin into mature myostatin (considered the “active” form of myostatin) capable of activating the myostatin pathway (e.g., by binding to type I (ALK4 / 5) and type II (ACTRIIA / B) receptors).

[0178] As used herein, the term "tropomyostatin / latent myostatin" refers to tropomyostatin, latent myostatin, or both. In some embodiments, an anti-tropomyostatin / latent myostatin antibody specifically binds to tropomyostatin. In some embodiments, an anti-tropomyostatin / latent myostatin antibody specifically binds to latent myostatin. In some embodiments, an anti-tropomyostatin / latent myostatin antibody specifically binds to both latent myostatin and tropomyostatin. It should be understood that "latent myostatin" and "tropomyostatin" may also be referred to herein as "latent GDF8" and "proGDF8," respectively.

[0179] As used herein, the term "mature myostatin" refers to the mature, biologically active form of myostatin. In some embodiments, mature myostatin is capable of binding to and / or activating the myostatin receptor. Activation and release of mature myostatin from its proto-myostatin form in vivo is accomplished through several separate protease cleavage events. First, proto-myostatin is cleaved by a precursor protein convertase to produce latent myostatin, in which mature myostatin is shielded from binding to its receptor by a portion of its prodomain. Activation and release of mature myostatin are then completed following cleavage of latent myostatin by additional proteases from the BMP / tolloid family, such as mTLL-2. See, for example, Figure 1A , 1B And 2. As used herein, the term "mature myostatin" can refer to both full-length mature myostatin and fragments of full-length mature myostatin that retain biological activity. Exemplary mature myostatin sequences, variants thereof, and methods for generating mature myostatin are well known in the art and are described in more detail herein.

[0180] The term "tropomyostatin," also known as "proGDF8," refers to the inactive form of mature myostatin, comprising disulfide-linked homodimers, each molecule of which contains an amino-terminal prodomain covalently bound to a carboxyl-terminal mature myostatin domain. In one embodiment, "tropomyostatin" is not cleaved by a precursor protein convertase or a protease from the BMP / tolloid family. Exemplary tropomyostatin sequences, variants thereof, and methods for generating tropomyostatin are well known in the art and are described in more detail herein.

[0181] 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 non-covalently bound to a carboxyl-terminal mature myostatin domain. In one embodiment, "latent myostatin" is produced from tropomyostatin that has been cleaved by a precursor protein convertase but not by a protease from the BMP / tolloid family. In another embodiment, "latent myostatin" can be produced by combining the prodomain and the carboxyl-terminal mature myostatin domain in vitro and allowing them to fold correctly. See, for example, Sengle et al., J. Biol. Chem., 286(7):5087-5099, 2011. Exemplary latent myostatin sequences, variants thereof, and methods for generating latent myostatin are well known in the art and are described in more detail herein.

[0182] As used herein, the term "tropomyostatin / latent myostatin" refers to tropomyostatin, latent myostatin, or both. In one embodiment, the antibody disclosed herein binds to tropomyostatin. In another embodiment, the antibody disclosed herein binds to latent myostatin. In yet another embodiment, the antibody disclosed herein binds to both tropomyostatin and latent myostatin.

[0183] As used herein, the terms "pure tropomyostatin" or "pure pro-GDF8" refer to a composition containing tropomyostatin that contains little or no other forms of myostatin, such as latent myostatin and mature myostatin. In one embodiment, the antibody disclosed herein specifically binds to pure tropomyostatin. In other words, such an antibody binds to tropomyostatin in a composition lacking other forms of myostatin, latent myostatin, and mature myostatin.

[0184] As used herein, the term "precursor protein convertase cleavage site" refers to the site where tropomyostatin is cleaved by a precursor protein convertase. In one embodiment, the precursor protein convertase cleavage site is a conserved RXXR site between the predomain and the bioactive domain or mature myostatin. See, for example, Figure 1A , 1B And 2.

[0185] As used herein, the term "BMP / tolloid protease family cleavage site" refers to the site where latent myostatin is cleaved by a member of the BMP / tolloid protease family. In one embodiment, the BMP / tolloid protease family member is mTLL-2. See, for example, Figure 1A , 1B And 2.

[0186] Antibodies that bind to tropomyostatin / latent myostatin

[0187] This disclosure is at least in part based on the unexpected discovery that certain tropomyostatin / latent myostatin-specific antibodies (e.g., antibodies referred to herein as Ab1) prevent the proteolytic activation of tropomyostatin / latent myostatin into mature myostatin. Furthermore, using such antibodies to inhibit myostatin activation has effectively increased muscle mass in a dexamethasone and plaster-induced muscle atrophy mouse model. Aspects of this disclosure provide antibodies (e.g., antibody-antigen binding fragments) that bind to tropomyostatin / latent myostatin and inhibit its proteolytic activation into mature myostatin.

[0188] An antibody (which may be used interchangeably in the plural form) is an immunoglobulin molecule capable of specifically binding to a target (such as carbohydrates, polynucleotides, lipids, peptides, etc.) through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. As used herein, the term “antibody” encompasses not only complete (e.g., full-length) polyclonal or monoclonal antibodies, but also its antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv), single chains (scFv), mutants thereof, fusion proteins containing antibody moieties, humanized antibodies, chimeric antibodies, biantibodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified conformation of immunoglobulin molecules containing an antigen recognition site with the desired specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies include any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and antibodies need not be of any particular type. Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the antibody heavy chain constant domain. There are five main types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Several of these types can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different types of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different types of immunoglobulins are well known.

[0189] As used herein, "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to tropomyostatin / latent myostatin is substantially free of antibodies that specifically bind to antigens other than tropomyostatin / latent myostatin). However, isolated antibodies that specifically bind to tropomyostatin / latent myostatin may have cross-reactivity with other antigens, such as tropomyostatin / latent myostatin molecules from other species. Furthermore, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0190] As used herein, the term "human antibody" is intended to include antibodies and fragments thereof having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of this disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo), such as in CDRs and, particularly, CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species, such as a mouse, is grafted onto a human frame sequence.

[0191] The term "epitope" includes any polypeptide determinant capable of specifically binding to immunoglobulins or T-cell receptors. In some embodiments, epitope determinants comprise chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments, may have specific three-dimensional structural features and / or specific charge features. An epitope is an antigenic region that is bound by an antibody. In some embodiments, it is referred to as a specifically binding antigen when the antibody preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0192] The antibodies described herein bind to tropomyostatin / latent myostatin, thereby inhibiting the proteolytic activation of tropomyostatin / latent myostatin into mature myostatin. In some cases, the antibodies described herein can inhibit the proteolytic activation of tropomyostatin / latent myostatin by at least 20%, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some cases, the antibodies described herein can inhibit the proteolytic cleavage of tropomyostatin by a precursor protein converting enzyme (e.g., furin protease) by at least 20%, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some cases, the antibodies described herein can inhibit the proteolytic cleavage of tropomyostatin or latent myostatin by tolloid proteases (e.g., mTLL2) by at least 20%, such as 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. The inhibitory activity of anti-tropomyostatin / latent myostatin antibodies can be measured by conventional methods, such as Western blot analysis as described in Example 1 and Figure 3. However, it should be understood that alternative methods can be used to measure the inhibitory activity of anti-tropomyostatin / latent myostatin antibodies against the proteolytic cleavage of tropomyostatin / latent myostatin. In some embodiments, inhibition of tropomyostatin / latent myostatin cleavage (e.g., by precursor protein convertase and / or tolloid protease) can be reflected as an inhibition constant (Ki), which provides a measure of antagonist efficacy and is the concentration of the antagonist (e.g., anti-tropomyostatin / latent myostatin antibody) required to reduce protease activity (e.g., precursor protein convertase or tolloid protease activity) by half, independent of enzyme or substrate concentration.

[0193] In some embodiments, the precursor protein convertase comprises (i) a catalytic domain that hydrolyzes the peptide bonds of a protein containing a precursor protein convertase cleavage site, and (ii) a binding pocket that binds to rTGF having a precursor protein convertase cleavage site. Examples of precursor protein convertases used according to this disclosure include, but are not limited to, PCSK5 / 6, PACE4, PACE7, and PACE3 (e.g., furin protease). In some embodiments, the precursor protein convertase is derived from any mammal, including but not limited to humans, monkeys, or rodents (e.g., mice, rats, hamsters).

[0194] In some embodiments, the precursor protein convertase is homologous to a precursor protein convertase selected from PCSK5 / 6, PACE4, PACE7, and PACE3 (e.g., furin protease). For example, the precursor protein convertase may be at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least about 99.9% identical to PCSK5 / 6, PACE4, PACE7, or PACE3 (e.g., furin protease).

[0195] In some embodiments, the precursor protein convertase cleavage site is an amino acid sequence that can be cleaved by precursor protein convertases (e.g., PCSK5 / 6, PACE4, PACE7, and PACE3). In some embodiments, the precursor protein convertase cleavage site comprises the amino acid sequence RXXR, where R is arginine and X is any amino acid. In some embodiments, the precursor protein 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, the precursor protein convertase cleavage site comprises the amino acid sequence RVRR (SEQ ID NO: 57), where R is arginine and V is valine. Exemplary precursor protein convertase cleavage sites for human, rat, mouse, and cynomolgus monkey myostatin are shown in bold in SEQ ID NO: 52-55. In some embodiments, the precursor protein convertase cleavage site comprises the amino acid sequence RSRR (SEQ ID NO: 56).

[0196] In some embodiments, the tolloid proteases used according to this disclosure include, but are not limited to, BMP-1, mTLL-1, and mTLL-2. The tolloid proteases can be obtained from any mammal, including but not limited to humans, monkeys, or rodents (e.g., mice, rats, hamsters). In some embodiments, the tolloid proteases are homologous to tolloid proteases selected from BMP-1, mTLL-1, and mTLL-2. For example, the tolloid proteases may be at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least about 99.9% identical to BMP-1, mTLL-1, and mTLL-2.

[0197] In some embodiments, the tolloid protease cleavage site is an amino acid sequence that can be cleaved by a tolloid (e.g., BMP-1, mTLL-1, and mTLL-2). Exemplary tolloid protease cleavage sites for human, rat, mouse, and cynomolgus monkey myostatin are underlined in SEQ ID NO:52-55. In some embodiments, the tolloid cleavage site comprises the amino acid sequence QR, where Q is glutamine and R is arginine.

[0198] In some embodiments, the antibodies described herein are capable of binding to tropomyostatin / latent myostatin, thereby inhibiting myostatin activity. In some cases, the antibodies described herein can inhibit myostatin signaling by at least 20%, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some embodiments, the inhibition of myostatin signaling can be measured by conventional methods, for example, using the myostatin activation assay as described in Example 1. However, it should be understood that other methods can be used to measure myostatin signaling activity.

[0199] It should be understood that the extent of myostatin proteolytic cleavage by, for example, precursor protein convertases and / or tolloid proteases can be measured and / or quantified using any suitable method. In some embodiments, the extent of myostatin proteolytic cleavage is measured and / or quantified using enzyme-linked immunosorbent assay (ELISA). For example, ELISA can be used to measure the level of released growth factors (e.g., mature myostatin). As another example, antibodies that specifically bind to tropomyostatin, latent myostatin, and / or mature myostatin can be used in ELISA to measure the level of a specific form of myostatin (e.g., tropomyostatin / latent / mature-myostatin) to quantify the extent of myostatin proteolytic cleavage. In some embodiments, the extent of myostatin proteolytic cleavage is measured and / or quantified using immunoprecipitation followed by SDS-PAGE or mass spectrometry of trypsin peptides, anisotropic fluorescence-based techniques, FRET analysis, hydrogen-deuterium exchange mass spectrometry, and / or NMR spectroscopy.

[0200] In some embodiments, antibodies, also known as immunoglobulins, are tetraglycosylated proteins composed of two light chains (L) each approximately 25 kDa and two heavy chains (H) each approximately 50 kDa. Two types of light chains, called λ and κ, can be found in antibodies. Depending on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be classified into five major classes: A, D, E, G, and M, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Each light chain typically includes an N-terminal variable (V) domain (V0). L ) and constant (C) domain (C L Each heavy chain typically includes an N-terminal V-domain (V... H ), three or four C structural domains (C H 1-3) and the hinge area. Closest to V H C H The structure domain is named C H 1. V H and V LThe domain consists of four relatively conserved sequences called the framework region (FR1, FR2, FR3, and FR4), which form the backbone of three regions (complementarity-determining regions, CDRs) for the hypervariable sequence. The CDRs contain most of the residues responsible for the specific interaction between the antibody and the antigen. The CDRs are referred to as CDR1, CDR2, and CDR3. Therefore, the CDR components on the heavy chain are called CDRH1, CDRH2, and CDRH3, while the CDR components on the light chain are called CDRL1, CDRL2, and CDRL3. CDRs often refer to the Kabat CDR, as described in *Sequences of Proteins of Immunological Interest*, US Department of Health and Human Services (1991), eds. Kabat et al. Another criterion used to characterize antigen-binding sites is the hypervariable loop 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. Another standard is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). Implementations described for the Kabat CDR can alternatively be implemented using similar relationships relative to the Chothia hypervariable ring or the AbM-defined ring, or any combination of these methods.

[0201] In some embodiments, the anti-myosin / latent myosin antibody and the nucleic acid molecule encoding the antibody of this disclosure include the CDR amino acid sequence shown in Table 1.

[0202] Table 1.

[0203]

[0204] In Table 1, the single sequences of CDRH3 and CDRL3 reflect Kabat and IMGT.

[0205] In some embodiments, the anti-myositis / latent myostatin conjugate (e.g., antibody) of this disclosure comprises any antibody (including an antigen-binding fragment) comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or combinations thereof, as provided for any of the antibodies shown in Table 1. In some embodiments, the anti-myositis / latent myostatin conjugate comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of any of the antibodies shown in Table 1. This disclosure also includes any nucleic acid sequence encoding a molecule comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3 as provided for any of the antibodies shown in Table 1. The CDR3 domains of the antibody heavy and light chains can play a particularly important role in the antibody's binding specificity / affinity to the antigen. Therefore, the anti-myosin / latent myosin binding agent or its nucleic acid molecule of the present disclosure may include at least the heavy chain and / or light chain CDR3 of the antibody as shown in Table 1.

[0206] This disclosure relates to monoclonal antibodies or antigen-binding fragments that bind to tropomyostatin / latent myostatin protein and include six complementarity-determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3.

[0207] In some embodiments, CDRH1 comprises any of the sequences shown in SEQ ID NO:1-3. In some embodiments, CDRH2 comprises any of the sequences shown in SEQ ID NO:4-9. In some embodiments, CDRH3 comprises any of the sequences shown in SEQ ID NO:10-11. CDRL1 comprises any of the sequences shown in SEQ ID NO:12-17. In some embodiments, CDRL2 comprises any of the sequences shown in SEQ ID NO:18-21. In some embodiments, CDRL3 comprises any of the sequences shown in SEQ ID NO:22-23.

[0208] In some embodiments (e.g., for the anti-tropomyostatin / latent myostatin antibody Ab1 shown in Table 1), CDRH1 contains the sequence shown as shown in SEQ ID NO:1 or 2, CDRH2 contains the sequence shown as shown in SEQ ID NO:4 or 5, CDRH3 contains the sequence shown in SEQ ID NO:10, CDRL1 contains the sequence shown in SEQ ID NO:12 or 13, CDRL2 contains the sequence shown in SEQ ID NO:18 or 19, and CDRL3 contains the sequence shown in SEQ ID NO:22, and the antibody binds to tropomyostatin / latent myostatin.

[0209] In some embodiments (e.g., for the anti-tropomyostatin / latent myostatin antibody Ab3 shown in Table 1), CDRH1 contains the sequence shown as shown in SEQ ID NO:1 or 3, CDRH2 contains the sequence shown as shown in SEQ ID NO:6 or 7, CDRH3 contains the sequence shown in SEQ ID NO:11, CDRL1 contains the sequence shown in SEQ ID NO:14 or 15, CDRL2 contains the sequence shown in SEQ ID NO:20 or 21, and CDRL3 contains the sequence shown in SEQ ID NO:23, and the antibody binds to tropomyostatin / latent myostatin.

[0210] In some embodiments (e.g., for anti-tropomyostatin / latent myostatin antibody Ab5 as shown in Table 1), CDRH1 comprises the sequence shown in SEQ ID NO:1 or 3, CDRH2 comprises the sequence shown in SEQ ID NO:8 or 9, CDRH3 comprises the sequence shown in SEQ ID NO:11, CDRL1 comprises the sequence shown in SEQ ID NO:16 or 17, CDRL2 comprises the sequence shown in SEQ ID NO:20 or 21, and CDRL3 comprises the sequence shown in SEQ ID NO:23, and the antibody binds to tropomyostatin / latent myostatin. In some instances, any anti-tropomyostatin / latent myostatin binder (e.g., antibody) of this disclosure comprises any antibody (including antigen-binding fragment) having one or more CDR (e.g., CDRH or CDRL) sequences substantially similar to those of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3. For example, an antibody may include one or more CDR sequences (SEQ ID NO: 1-23) as shown in Table 1, which contain a variation of up to 5, 4, 3, 2, or 1 amino acid residues compared to the corresponding CDR region in any of SEQ ID NO: 1-23. The complete amino acid and nucleic acid sequences of the heavy chain variable regions and light chain variable regions of the antibodies listed in Table 1 are provided below.

[0211] Heavy chain variable region - Ab1 parent

[0212] QIQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHYYGMDVWGQGTTVTVSS(SEQ ID NO:24)

[0213] CAGATCCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGATTTTTGGAGTGGTCGCACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO:38)

[0214] Heavy chain variable region - Ab2 germline

[0215] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHYYGMDVWGQGTTVTVSS(SEQ ID NO:25)

[0216] CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGATTTTTGGAGTGGTCGCACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO:39)

[0217] Heavy chain variable region - Ab3 parent

[0218] QIQLVQSGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVISYDGSIKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHKYGMDVWGQGTTVTVSS(SEQ ID NO:26)

[0219] CAGATCCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCGCCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTATCAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGATTTTTGGAGTGGTCGCACAAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO:40)

[0220] Heavy chain variable region - Ab4 germline

[0221] QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVISYDGSIKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHKYGMDVWGQGTTVTVSS(SEQ ID NO:27)

[0222] CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCGCCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTATCAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGATTTTTGGAGTGGTCGCACAAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO:41)

[0223] Heavy chain variable region - Ab5 parent

[0224] QIQLVQSGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHKYGMDVWGQGTTVTVSS(SEQ ID NO:28)

[0225] CAGATCCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCGCCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAATAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGATTTTTGGAGTGGTCGCACAAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO:42)

[0226] Heavy chain variable region - Ab6 germline

[0227] QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHKYGMDVWGQGTTVTVSS(SEQ ID NO:29)

[0228] CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCGCCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAATAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGATTTTTGGAGTGGTCGCACAAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO:43)

[0229] Light chain variable region - Ab1 parent

[0230] QPVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVHWYQQLPGTAPKLLIYSDNQRPSGVPDRFSGSKSGTSASLVISGLQSDDEADYYCAAWDDSLNGVFGGGTKLTVL(SEQ ID NO:30)

[0231] CAGCCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATACTGTCCACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATAATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGTCATCAGTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA(SEQ ID NO:44)

[0232] Light chain variable region - Ab2 germline

[0233] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVHWYQQLPGTAPKLLIYSDNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGVFGGGTKLTVL(SEQ ID NO:31)

[0234] CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATACTGTCCACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATAATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA(SEQ ID NO:45)

[0235] Light chain variable region - Ab3 parent

[0236] QPVLTQPPSASGTPGQRVTISCSGSTSNIGSNTVHWYQQLPGTAPKLLIYSDDQRPSGVPDRFSGSKSGTSASLVISGLQSDDEADYYCAAWDESLNGVFGGGTKLTVL(SEQ ID NO:32)

[0237] CAGCCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTAATACTGTCCACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATGATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGTCATCAGTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGATGAGAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA(SEQ ID NO:46)

[0238] Light chain variable region - Ab4 germline

[0239] QSVLTQPPSASGTPGQRVTISCSGSTSNIGSNTVHWYQQLPGTAPKLLIYSDDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDESLNGVFGGGTKLTVL(SEQ ID NO:33)

[0240] CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTAATACTGTCCACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATGATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGAGAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA(SEQ ID NO:47)

[0241] Light chain variable region - Ab5 parent

[0242] QPVLTQPPSASGTPGQRVTISCSGSSSNIGGNTVHWYQQLPGTAPKLLIYSDDQRPSGVPDRFSGSKSGTSASLVISGLQSDDEADYYCAAWDESLNGVFGGGTKLTVL(SEQ ID NO:34)

[0243] CAGCCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAGGAAATACTGTCCACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATGATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGTCATCAGTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGATGAGAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA(SEQ ID NO:48)

[0244] Light chain variable region - Ab6 germline

[0245] QSVLTQPPSASGTPGQRVTISCSGSSSNIGGNTVHWYQQLPGTAPKLLIYSDDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDESLNGVFGGGTKLTVL(SEQ ID NO:35)

[0246] CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAGGAAATACTGTCCACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTGATGATCAGCGCCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGAGAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA(SEQ ID NO:49)

[0247] Ab2 - heavy chain

[0248] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVRFLEWSHYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:50)

[0249] Ab2 - light chain

[0250] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGSNTVHWYQQLPGTAPKLLIYSDNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGVFGGGTKLTVLG QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQID NO:51)

[0251] In some embodiments, the anti-myosin / latent myosin antibody of this disclosure comprises any antibody containing a heavy chain variable domain of any one of SEQ ID NO: 24-29 or a light chain variable domain of any one of SEQ ID NO: 30-35. In some embodiments, the anti-myosin / latent myosin antibody of this disclosure comprises any antibody containing a heavy chain variable domain and a light chain variable domain pair of SEQ ID NO: 24 and 30; 25 and 31; 26 and 32; 27 and 33; 28 and 34; or 29 and 35.

[0252] This disclosure provides anti-myosin / latent myostatin antibodies having heavy chain variable and / or light chain variable amino acid sequences homologous to any heavy chain variable and / or light chain variable amino acid sequences described herein. In some embodiments, the anti-myosin / latent myostatin antibody comprises at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) of the heavy chain variable sequence or light chain variable sequence of any of the heavy chain variable sequences in SEQ ID NO:24-29 or any of the light chain variable sequences in SEQ ID NO:30-35. In some embodiments, the homologous heavy chain variable and / or light chain variable amino acid sequences are not altered within any CDR sequences provided herein. For example, in some embodiments, the degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) may be present in heavy chain variable and / or light chain variable sequences other than any CDR sequences provided herein.

[0253] The "percentage identity" of two amino acid sequences was determined using an algorithm as described in Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990 (an improvement on the algorithm described in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993). This algorithm is integrated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the target protein molecule. In cases where there are gaps between the two sequences, BLAST with gaps can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using BLAST and BLAST with gaps, the default parameters of the corresponding procedures (e.g., XBLAST and NBLAST) can be used.

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

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

[0256] The anti-myosin / latent myosin binder disclosed herein may optionally comprise an antibody constant region or a portion thereof. For example, V L The structural domain can be attached to a light chain constant domain such as Cκ or Cλ at its C-terminus. Similarly, V H The domain or a portion thereof may be attached to all or part of the heavy chain, such as IgA, IgD, IgE, IgG, and IgM, and any isotype subclass. Antibodies may include suitable constant regions (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Therefore, antibodies within the scope of this disclosure may include V in combination with any suitable constant region. H and V L The domain or its antigen-binding portion.

[0257] In some implementations, V H and / or V L Domains can be reconstructed as germline sequences; for example, the FRs of these domains are mutated using conventional molecular biology techniques to match those produced through germline cells. For example, V... H and / or V L The domains can be recovered as phylogenetic sequences of IgHV3-30 (SEQ ID NO:36) and / or IgLV1-44 (SEQ ID NO:37), respectively. It should be understood that any V H and / or V LThe domain can be reconstructed into any suitable phylogenetic sequence. In other implementations, the FR sequence remains distinct from the common phylogenetic sequence.

[0258] IgHV3-30

[0259] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR(SEQ ID NO:36)

[0260] IgLV1-44

[0261] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNG(SEQ ID NO:37)

[0262] In some embodiments, the anti-myosin / latent myosin antibody or antigen-binding fragment may or may not include the frame region of the antibody shown in SEQ ID NO:24-35. In some embodiments, the anti-myosin antibody is a mouse antibody and includes a mouse frame region sequence.

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

[0264] Antibodies that "specifically bind" to a target antigen bind to the target antigen with a higher affinity than they bind to non-target antigens, are more likely to bind to the target antigen, and / or bind to the target antigen for a longer duration. In some embodiments, antibodies that specifically bind to tropomyostatin / latent myostatin are disclosed herein. In some embodiments, any antibody provided herein binds to or near the tolloid cleavage site or tolloid docking site of tropomyostatin / latent myostatin. In some embodiments, if the antibody binds to 15 or fewer amino acid residues within the tolloid cleavage site or tolloid docking site, the antibody binds to or near the tolloid cleavage site or tolloid docking site. In some embodiments, any antibody provided herein binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues within the tolloid cleavage site or tolloid docking site. In some embodiments, the antibody binds to or near the tolloid cleavage site of GDF8. For example, the antibody may bind to the amino acid sequence PKAPPLRELIDQYDVQRDDSSDGSLEDDDYHAT (SEQ ID NO:62) shown in SEQ ID NO:62. In other embodiments, any antibody provided herein binds to or near the precursor protein convertase cleavage site or precursor protein convertase docking site of tropomyostatin / latent myostatin. In some embodiments, if the antibody binds to 15 or fewer amino acid residues of the precursor protein convertase cleavage site or precursor protein convertase docking site, the antibody binds to or near the precursor protein convertase cleavage site or precursor protein convertase docking site. In some embodiments, any antibody provided herein binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the precursor protein convertase cleavage site or precursor protein convertase docking site. In some embodiments, the antibody binds to or near the precursor protein convertase cleavage site of GDF8. For example, an antibody can bind to the amino acid sequence GLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRC (SEQ ID NO:63) shown in SEQ ID NO:63.

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

[0266] In some embodiments, antibodies bind to antigens but cannot effectively eliminate them from plasma. Therefore, 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., sweep antibodies) have a pH-sensitive affinity for the antigen. Such pH-sensitive antibodies bind to antigens in plasma at neutral pH and dissociate from the antigen in acidic endosomes, thus reducing antibody-mediated antigen accumulation and / or promoting antigen clearance from plasma.

[0267] This disclosure relates to sweep antibodies. As used herein, a “sweep antibody” refers to an antibody having both pH-sensitive antigen binding and binding at least a threshold level to nascent Fc receptors (FcRn) on the cell surface at neutral or physiological pH. In some embodiments, the sweep antibody binds to nascent Fc receptors (FcRn) at neutral pH. For example, the sweep antibody binds to FcRn at a pH in the range of 7.0-7.6. In some embodiments, the sweep antibody may bind to the antigen at the antigen-binding site and bind to the cellular FcRn via the Fc portion of the antibody. In some embodiments, the sweep antibody may then be neutralized, thereby releasing the antigen in an acidic endosome, where it may be degraded. In some embodiments, the sweep antibody, no longer binding to the antigen, may then be returned to the serum via cellular release (e.g., via exocytosis).

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

[0269] In some embodiments, the Fc moiety of the sweep antibody binds to FcRn. In some embodiments, the Fc moiety of the sweep antibody is subjected to a 10-1 concentration at pH 7.4. -3 M-10 -8 Kd-bound FcRn in the M range. In some embodiments, the sweep antibody is subjected to a 10-1 pH at 7.4.-3 M-10 -7 M, 10 -3 M-10 -6 M, 10 -3 M-10 -5 M, 10 -3 M-10 -4 M, 10 -4 M-10 -8 M, 10 -4 M-10 -7 M, 10 -4 M-10 -6 M, 10 -4 M-10 -5 M, 10 -5 M-10 -8 M, 10 -5 M-10 -7 M, 10 -5 M-10 -6 M, 10 -6 M-10 -8 M, 10 -6 M-10 -7 M or 10 -7 M-10 -8 The Kd region of the M-scan antibody binds to FcRn. In some embodiments, FcRn binds to the CH2-CH3 hinge region of the sweep antibody. In some embodiments, FcRn binds to the same region as protein A or protein G. In some embodiments, FcRn binds to a different binding site than FcγR. In some embodiments, amino acid residue AA in the Fc region of the sweep antibody is required for binding to FcRn. In some embodiments, amino acid residue AA in the Fc region of the sweep antibody affects binding to FcRn.

[0270] In some embodiments, any antibody provided herein is engineered to bind FcRn with higher affinity. In some embodiments, any antibody provided herein is engineered to bind FcRn with higher affinity at pH 7.4. In some embodiments, the sweep antibody has increased affinity for FcRn to extend its pharmacokinetic (PK) properties compared to its conventional counterpart. For example, in some embodiments, the sweep antibody causes fewer adverse reactions due to its efficacy at lower doses. In some embodiments, the sweep antibody is administered at a lower frequency. In some embodiments, the sweep antibody transcytosis into certain tissue types is increased. In some embodiments, the sweep antibody enhances the efficiency of transplacental delivery. In some embodiments, the sweep antibody is produced at a lower cost.

[0271] In some embodiments, any antibody provided herein is engineered to bind FcRn with a lower affinity. In some embodiments, any antibody provided herein is engineered to bind FcRn with a lower affinity at pH 7.4. In some embodiments, the sweep antibody has a reduced affinity for FcRn to shorten its pharmacokinetic (PK) properties compared to its conventional counterpart. For example, in some embodiments, the sweep antibody clears FcRn more rapidly for imaging and / or radioimmunotherapy. In some embodiments, the sweep antibody promotes the clearance of endogenous pathogenic antibodies as a treatment for autoimmune diseases. In some embodiments, the sweep antibody reduces the risk of adverse pregnancy outcomes that can be caused by transplacental transport of maternally derived fetal-specific antibodies.

[0272] In some embodiments, the sweep antibody has reduced affinity for the antigen at a low pH compared to neutral or physiological pH (e.g., pH 7.4). In some embodiments, the sweep antibody has reduced affinity for the antigen at an acidic pH (e.g., pH in the range of 5.5-6.5) compared to physiological pH (e.g., pH 7.4). It should be understood that any antibody provided herein can be engineered to dissociate from the antigen according to pH changes (e.g., pH-sensitive antibody). In some embodiments, the sweep antibody provided herein is engineered to bind to the antigen according to pH. In some embodiments, the sweep antibody provided herein is engineered to bind to FcRn according to pH. In some embodiments, the sweep antibody provided herein is internalized via endocytosis. In some embodiments, the sweep antibody provided herein is internalized via FcRn binding. In some embodiments, the endocytotic sweep antibody releases the antigen in the endosome. In some embodiments, the sweep antibody is recycled back to the cell surface. In some embodiments, the sweep antibody remains attached to the cell. In some embodiments, the endocytotic sweep antibody is recycled back into the plasma. It should be understood that the Fc portion of any antibody provided herein can be engineered to have different FcRn binding activities. In some embodiments, the FcRn binding activity affects the time it takes for the antigen to be cleared by the sweep antibody. In some embodiments, the sweep antibody may be a long-acting or rapid-acting sweep antibody.

[0273] In some embodiments, converting a conventional therapeutic antibody to a cleansing antibody reduces the effective dose. In some embodiments, converting a conventional therapeutic antibody to a cleansing 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 cleansing antibody reduces the effective dose by at least 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, 15 times, 20 times, 50 times, or 100 times.

[0274] In some embodiments, the appropriate dose of the sweep antibody for treatment can be selected empirically. In some embodiments, high doses of sweep antibody can saturate the FcRn, resulting in antibodies that stabilize the antigen in the serum without internalization. In some embodiments, low doses of sweep antibody may not be therapeutically effective. In some embodiments, the sweep antibody is administered once daily, once weekly, every two weeks, every three weeks, every four weeks, every six weeks, every eight weeks, every ten weeks, every twelve weeks, every sixteen weeks, every twenty weeks, or every twenty-four weeks.

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

[0276] Some aspects of this disclosure are based on the understanding that the affinity (e.g., expressed as Kd) of any anti-troponin / latent myostatin antibody provided herein is sensitive to changes in pH. In some embodiments, the antibodies provided herein have an increased Kd for binding to troponin / latent myostatin compared to relatively high pH (e.g., in the range of 4.0–6.5) at relatively low pH levels (e.g., in the range of 7.0–7.4). In some embodiments, the antibodies provided herein have a Kd of 10 at pH 4.0–6.5. -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8The M-range Kd binds to tropomyostatin / latent myostatin. In some embodiments, the antibodies provided herein have 10 Kd at pH 7.0-7.4. -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 The Kd for binding to tropomyostatin / latent myostatin is in the range M. In some embodiments, the antibodies provided herein have a Kd for binding to tropomyostatin / 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 higher at a pH of 4.0-6.5 compared to a pH of 7.0-7.4.

[0277] In some embodiments, this document provides tropomyostatin / latent myostatin antibodies that nonspecifically bind to epitopes within the amino acid sequence shown in SEQ ID NO:64. In some embodiments, the tropomyostatin / latent myostatin antibodies provided herein nonspecifically bind to the same epitopes as those described in Tables 2a, 11a, 11b, or 13 of International Patent Application Publication No. WO2016 / 098357, published on June 23, 2016, and based on International Patent Application No. PCT / JP2015 / 006323 filed on December 18, 2015. In some embodiments, the tropomyostatin / latent myostatin antibodies provided herein do not compete or cross-compete for binding to the same epitopes as those described in Tables 2a, 11a, 11b, or 13 of International Patent Application Publication No. WO 2016 / 098357, published on June 23, 2016, and based on International Patent Application Publication No. PCT / JP2015 / 006323 filed on December 18, 2015. In some embodiments, the tropomyostatin / latent myostatin antibodies provided herein nonspecifically bind to the same epitopes as those of antibodies comprising the VH and VL pairs described in Tables 2a, 11a, 11b, or 13 of International Patent Application Publication No. WO 2016 / 098357, published on June 23, 2016, and based on International Patent Application Publication No. PCT / JP2015 / 006323 filed on December 18, 2015. In some embodiments, the tropomyostatin / latent myostatin antibodies provided herein do not compete or cross-competitively bind to the same epitopes as antibodies containing the VH and VL pairs described in Tables 2a, 11a, 11b or 13 of International Patent Application Publication No. WO 2016 / 098357, published on June 23, 2016, and based on International Patent Application No. PCT / JP2015 / 006323 filed on December 18, 2015.

[0278] polypeptide

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

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

[0281] Antibodies competing with anti-myosin / latent myosin antibodies

[0282] This disclosure relates to antibodies that compete with or cross-compete with any antibodies provided herein. As used herein, the term "competitive" with respect to antibodies means that a first antibody binds to an epitope of a protein (e.g., latent myostatin) in a manner sufficiently similar to the binding of a second antibody, such that the binding of the first antibody to its epitope is detectably reduced in the presence of the second antibody compared to the binding of the first antibody in its absence. An alternative, but not necessarily one, is that the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody. That is, the first antibody may inhibit the binding of the second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its corresponding epitope. However, antibodies are said to "cross-compete" with each other for binding to their respective epitopes when each antibody detectably inhibits the binding of other antibodies to their epitopes or ligands (whether to the same, greater, or lesser extent). Both competitive and cross-compete antibodies are within the scope of this 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 a portion thereof), those skilled in the art will understand that such competing and / or cross-competing antibodies are included and can be used in the methods and / or compositions provided herein.

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

[0284] In another embodiment, the antibody is at a concentration of less than 10. -6 The equilibrium dissociation constant Kd between the antibody and the protein M competes or cross-competitively binds to any antigen provided herein (e.g., tropomyosperidin / latent myostatin). In other embodiments, the antibody is at 10 -11 M-10 -6 M-range Kd competition or cross-competition combination with any antigen provided in this article.

[0285] This disclosure relates to antibodies that compete with any antibodies provided herein for binding to tropomyostatin / latent myostatin. In some embodiments, the antibody binds to tropomyostatin / latent myostatin at the same epitope as any antibody provided herein. For example, in some embodiments, any antibody provided herein binds to or near the tolloid cleavage site or tolloid docking site of tropomyostatin / latent myostatin. In other embodiments, any antibody provided herein binds to or near the precursor protein convertase cleavage site or precursor protein convertase docking site of tropomyostatin / latent myostatin. In another embodiment, the antibody binds at a concentration of less than 10 -6 The antibody M competes with the equilibrium dissociation constant Kd between tropomyostatin / latent myostatin for binding to tropomyostatin / latent myostatin. In other embodiments, the antibody competing with any antibody provided herein uses a 10 -11 M-10 -6 Kd-binding tropomyostatin / latent myostatin in the M range.

[0286] Any antibody presented herein can be characterized using any suitable method. For example, one method is to identify the epitope to which the antigen binds, or “epitope mapping.” Many suitable methods exist for locating and characterizing epitopes on proteins, including resolving the crystal structure of antibody-antigen complexes, competition analysis, gene fragment expression analysis, and synthetic peptide-based analysis, 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 another instance, epitope mapping can be used to determine the sequence to which the antibody binds. Epitopes can be linear epitopes, i.e., contained in a single amino acid extension, or conformational epitopes formed by the three-dimensional interactions of amino acids, which may not necessarily be contained in a single extension (a linear sequence of the primary structure). Peptides of varying lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used for antibody binding analysis. In yet another instance, the epitope to which the antibody binds can be determined in systematic screening by using overlapping peptides derived from the target antigen sequence and measuring antibody binding. Based on gene fragment expression analysis, the open reading frame encoding the target antigen is randomly or fragmented through specific genetic reconstruction, and the reactivity of the expressed antigen fragment with the antibody to be tested is determined. For example, the gene fragment can be generated by PCR and then transcribed and translated into a protein in vitro in the presence of radioactive amino acids. The binding of the antibody to the radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified using a large library (phage library) of random peptide sequences displayed on the surface of phage particles. Alternatively, a defined library of overlapping peptide fragments can be used to determine binding with the test antibody in a simple binding assay. In other instances, mutagenesis of the antigen-binding domain, domain exchange assays, and alanine scan mutagenesis can be performed to identify sufficient and / or necessary residues for epitope binding. For example, domain exchange assays can be performed using mutants of the target antigen in which various fragments of the tropomyostatin / latent myostatin polypeptide have been replaced (exchanged) with sequences from closely related but antigenically different proteins (such as another member of the TGFβ protein family, e.g., GDF11). By evaluating the binding of antibodies to mutant tropomyostatin / latent tropomyostatin, the importance of specific antigen fragments to antibody binding can be assessed.

[0287] Alternatively, competitive analysis can be performed using other antibodies known to bind to the same antigen to determine whether an antibody binds to the same epitope as other antibodies. Competitive analysis is well known to those skilled in the art.

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

[0289] Production of antibodies binding to tropomyostatin / latent myostatin

[0290] Various methods can be used to obtain the antibodies or antigen-binding fragments thereof disclosed herein. For example, antibodies can be produced using recombinant DNA methods. Monoclonal antibodies can also be produced by generating hybridomas according to known methods (see, for example, Kohler and Milstein (1975) Nature, 256:495-499). 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) assays, to identify one or more hybridomas that produce antibodies that specifically bind to the specified antigen. The specified antigen in any form can be used as an immunogen, for example, a recombinant antigen, a naturally occurring form, any variant or fragment thereof, and its antigenic peptide (e.g., any epitope described herein as a linear epitope or as a conformational epitope within the backbone). An exemplary method for preparing antibodies includes screening protein expression libraries, such as phage or ribosome display libraries, that express the antibody or fragments thereof (e.g., scFv). Phage display is described in, for example, Ladner et al., US Pat. No. 5, 223, 409; Smith (1985) Science 228: 1315-1317; Clackson et al. (1991) Nature, 352: 624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597 WO92 / 18619; WO 91 / 17271; WO 92 / 20791; WO 92 / 15679; WO 93 / 01288; WO 92 / 01047; WO 92 / 09690 and WO 90 / 02809.

[0291] In addition to using a display library, a specified antigen (e.g., tropomyostatin) can be used to immunize non-human animals, such as rodents, for example, mice, hamsters, or rats. In one embodiment, the non-human animal is a mouse.

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

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

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

[0295] In some implementations, once the vector has been incorporated into a suitable host, the host can be maintained under conditions suitable for high-level expression of the nucleotide sequence, and, as needed, the immunoglobulin light chain, heavy chain, light / heavy chain dimer, or intact antibody, antigen-binding fragment, or other immunoglobulin forms can be collected and purified; see Beychok, Cells of Immunoglobins Synthesis, Academic Press, NY, (1979). Thus, the introduction of the polynucleotide or vector into the cell subsequently produces antibodies or antigen-binding fragments. Furthermore, transgenic animals, preferably mammals, containing the aforementioned host cells can be used for large-scale production of antibodies or antibody fragments.

[0296] The transformed host cells can be grown in a fermenter and cultured using any suitable technique to achieve optimal cell growth. Once expressed, the whole antibody, its dimer, single light and heavy chains, other immunoglobulin forms, or antigen-binding fragments can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column chromatography, column chromatography, gel electrophoresis, etc.; see Scopes, "Protein Purification", Springer Verlag, NY (1982). The antibody or antigen-binding fragment can then be isolated from the growth medium, cell lysis products, or cell membrane fractions. For example, the isolation and purification of bacterially expressed antibodies or antigen-binding fragments can be carried out in any conventional manner, such as, for example, preparative chromatographic separation and immunoassay, as those involving the use of monoclonal or polyclonal antibodies targeting, for example, a constant region of the antibody.

[0297] This disclosure relates to hybridomas, which provide an indefinitely extended source of monoclonal antibodies. As an alternative to obtaining immunoglobulins directly from hybridoma cultures, immortalized hybridoma cells can be used as a source of rearranged heavy and light chain loci for subsequent expression and / or genetic manipulation. The rearranged antibody genes can be reverse transcribed from suitable mRNA to produce cDNA. In some embodiments, heavy chain constant regions can be exchanged with or eliminated together with heavy chain constant regions of different isotypes. Variable regions can be linked to encode single-stranded Fv regions. Multiple Fv regions can be linked to confer binding capacity to more than one target or can employ chimeric combinations of heavy and light chains. Any suitable method can be used for cloning antibody variable regions and generating recombinant antibodies.

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

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

[0300] The polynucleotide encoding the antibody or antigen-binding fragment can be, for example, DNA, cDNA, RNA, or synthetically produced DNA or RNA, or a chimeric nucleic acid molecule resulting from recombination of any of these polynucleotides, individually or in combination. In some embodiments, the polynucleotide is part of the vector. Such a vector may contain further genes, such as marker genes, that allow selection of the vector in a suitable host cell and under suitable conditions.

[0301] In some embodiments, the polynucleotide is operatively linked to an expression control sequence to allow expression in prokaryotic or eukaryotic cells. Expression of the polynucleotide involves transcription into translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. These may include regulatory sequences that promote transcription initiation and poly-A signals that promote transcription termination and transcript stability. Additional regulatory elements may include transcription and translation enhancers and / or naturally associated or heterologous promoter regions. Possible regulatory elements allowing expression in prokaryotic host cells include, for example, the PL, Lac, Trp, or Tac promoters in *E. coli*, and examples of regulatory elements allowing expression in eukaryotic host cells are the AOX1 or GAL1 promoters in yeast or the CMV-promoter, SV40-promoter, RSV-promoter (Laurel's sarcoma virus), CMV-enhancer, SV40-enhancer, or globulin introns in mammalian and other animal cells.

[0302] In addition to elements responsible for transcription initiation, these regulatory elements may also include transcription termination signals downstream of the polynucleotide, such as SV40-poly-A sites or tk-poly-A sites. Furthermore, depending on the expression system employed, a lead sequence capable of directing the polypeptide into a cellular compartment or secreting it into a culture medium can be added to the coding sequence of the polynucleotide and has been previously described. The lead sequence is assembled in a suitable phase with translation, initiation, and termination sequences, and preferably a lead sequence capable of directing the secretion of the translated protein or a portion thereof into, for example, an extracellular culture medium. Optionally, a heteropolynucleotide sequence encoding a fusion protein comprising a C- or N-terminal identifying peptide conferring desired properties (e.g., stability or simplified purification of the expressed recombinant product) can be used.

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

[0304] In some embodiments, the expression control sequence is provided as a eukaryotic promoter system in a vector capable of transforming or transfecting eukaryotic host cells, but control sequences intended for prokaryotic hosts may also be used. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpesviruses, or bovine papillomaviruses can be used to deliver polynucleotides or vectors to a target cell population (e.g., to express antibodies or antigen-binding fragments for cell engineering). A variety of suitable methods can be used to construct recombinant viral vectors. In some embodiments, the polynucleotide and vector can be reconstructed into liposomes for delivery to target cells. Vectors containing polynucleotides (e.g., coding sequences for variable domains of the heavy and / or light chains of immunoglobulin chains and expression control sequences) can be transferred into host cells using suitable methods that vary depending on the type of cell host.

[0305] Modification

[0306] The antibody or antigen-binding fragments of this disclosure can be modified with detectable labels, including but not limited to enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radioactive substances, positron-emitting metals, non-radioactive paramagnetic metal ions, and affinity labels for detecting and separating tropomyosin / latent myostatin. Detectable substances can be directly conjugated or conjugated to the peptides of this disclosure using suitable techniques, or indirectly conjugated or conjugated via intermediates (e.g., linkers). Non-restricted examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, or acetylcholinesterase; non-restricted examples of suitable cofactor complexes include streptavidin / biotin and avidin / biotin; non-restricted examples of suitable fluorescent substances include biotin, umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazineamine luciferin, dansyl chloride, or phycoerythrin; examples of luminescent substances include luminol; non-restricted examples of bioluminescent substances include luciferase, luciferin, and jellyfish luminescent protein; and examples of suitable radioactive substances include radioactive metal ions, such as alpha emitters or other radioactive isotopes, such as 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 The detectable substance can be directly conjugated or conjugated with the anti-myosin / latent myosin antibody of this disclosure using suitable techniques, or indirectly conjugated or conjugated via an intermediate (e.g., a linker). The anti-myosin / latent myosin antibody conjugated with the detectable substance can be used for diagnostic analyses as described herein.

[0307] Pharmaceutical Composition

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

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

[0310] In some instances, the pharmaceutical compositions described herein comprise liposomes containing an anti-myosin / latent myosin antibody, 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 US Pat. Nos. 4,485,045 and 4,544,545. Liposomes with increased cycle times are disclosed in US Pat. No. 5,013,556. Particularly useful liposomes can be prepared by a reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter having a defined pore size to produce liposomes with the desired diameter.

[0311] Anti-myosin / latent myosin antibodies can also be encapsulated in microcapsules, for example, prepared by coagulation techniques or by interfacial polymerization, such as hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or crude emulsions. Exemplary techniques have been previously described; see, for example, Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

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

[0313] Pharmaceutical compositions intended for internal administration must be sterile. This is readily achieved, for example, by filtration through a sterile filter membrane. Therapeutic antibody compositions are generally housed in containers with sterile access ports, such as intravenous solvent bags or vials with stoppers that can be punctured by a hypodermic needle.

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

[0315] To prepare solid compositions such as tablets, the active ingredient can be mixed with a pharmaceutical carrier, such as conventional tableting ingredients like corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or resin, and other pharmaceutical diluents, such as water, to form a solid preformation composition comprising a homogeneous mixture of the compounds of this disclosure or their non-toxic, pharmaceutically acceptable salts. When these preformation compositions are referred to as homogeneous, it means that the active ingredient is uniformly dispersed throughout the composition, allowing the composition to be readily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules. This solid preformation composition is then subdivided into unit dosage forms of the aforementioned type containing from 0.1 mg to approximately 500 mg of the active ingredient of this disclosure. Tablets or pills of the novel composition can be coated or additionally compounded to provide a dosage form that offers the advantage of prolonged action. For example, tablets or pills can comprise an internal dose component and an external dose component, the latter being a coating on the former. The two components can be separated by an enteric coating, which counteracts disintegration in the stomach and allows the internal components to enter the duodenum intact or delay release. A variety of materials can be used for such enteric coatings or coatings, including various polymeric acids and mixtures of these materials such as shellac, cetyl alcohol, and cellulose acetate.

[0316] Suitable surfactants include, in particular, nonionic agents such as polyoxyethylene sorbitan (e.g., Tween). TM 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span TM 20, 40, 60, 80, or 85). Compositions containing surfactants conveniently contain 0.05-5% surfactant, and may be 0.1-2.5%. It should be understood that other ingredients, such as mannitol or other pharmaceutically acceptable media, may be added if desired.

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

[0318] The emulsion composition can be made by mixing anti-myosin antibody with Intralipid TM Those prepared from its components (soybean oil, lecithin, glycerin, and water).

[0319] Pharmaceutical compositions for inhalation or inhalation include solutions and suspensions and powders in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as given above. In some embodiments, the composition is administered via oral or nasal inhalation to achieve a local or systemic effect.

[0320] Compositions in preferably sterile, pharmaceutically acceptable solvents can be nebulized using a gas. The nebulized solution can be inhaled directly from the nebulizer or the nebulizer can be connected to a mask, tent, or intermittent positive pressure ventilator. Solution, suspension, or powder compositions can be administered from a device that delivers the formulation in a suitable manner (preferably orally or nasally).

[0321] Uses of anti-myosin / latent myostatin antibodies for the treatment of diseases / disorders

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

[0323] This disclosure includes methods for treating a subject with myopathy, the method comprising administering an effective amount of the aforementioned antibody to the subject. In some embodiments, the myopathy is primary myopathy. In another embodiment, primary myopathy includes disuse atrophy. In other embodiments, disuse atrophy is associated with hip fracture, selective joint replacement, critical myopathy, spinal cord injury, or stroke. In some embodiments, the myopathy is secondary myopathy, wherein muscle loss is secondary to the pathology of the disease. In other embodiments, secondary myopathy includes denervation, hereditary myasthenia gravis, or cachexia. In another embodiment, secondary myopathy is denervation associated with amyotrophic lateral sclerosis or spinal muscular atrophy. In some embodiments, secondary myopathy is hereditary myasthenia gravis associated with muscular dystrophy. In other embodiments, secondary myopathy is cachexia associated with kidney failure, AIDS, heart disease, cancer, or aging.

[0324] Another aspect of this disclosure includes methods for treating subjects suffering from age-related diseases or conditions. Exemplary age-related diseases or conditions include, but are not limited to, sarcopenia (age-related muscle loss), frailty, and androgen deficiency.

[0325] Another aspect of this disclosure includes methods for treating subjects suffering from diseases or conditions associated with disuse atrophy / trauma. Exemplary diseases or conditions associated with disuse atrophy / trauma include, but are not limited to, myasthenia gravis, hip / joint replacement, hip fracture, stroke, bedriddenness, SCI, rotator cuff injury, knee replacement, fracture, and burns related to time spent in the intensive care unit (ICU).

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

[0327] Another aspect of this disclosure includes methods for treating subjects suffering from diseases or conditions associated with cachexia. Exemplary diseases or conditions associated with cachexia include, but are not limited to, cancer, chronic heart failure, acquired immunodeficiency syndrome (AIDS), chronic obstructive pulmonary disease (COPD), and chronic kidney disease (CKD).

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

[0329] Another aspect of this disclosure includes methods for treating a subject suffering from a disease or condition related to 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, other diseases or conditions related to metabolic disorders and / or body composition are apparent to those skilled in the art and are within the scope of this disclosure.

[0330] Another aspect of this disclosure includes methods for treating a subject suffering from a disease or condition associated with congenital myopathy. Exemplary congenital myopathy includes, but is not limited to, X-linked myotubular myopathy, autosomal dominant central nucleus myopathy, autosomal recessive central nucleus myopathy, linear myopathy, and congenital fibrous asymmetric myopathy.

[0331] Another aspect of this disclosure includes methods for treating subjects suffering from diseases or conditions associated with muscular dystrophy. Exemplary muscular dystrophys include, but are not limited to, Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy (FSH), and limb-girdle muscular dystrophy.

[0332] Another aspect of this disclosure includes methods for treating subjects suffering from gynecological urinary-related diseases or conditions, speech disorders (stenosis), extraocular myopathy, carpal tunnel syndrome, Guillain-Barré syndrome, or osteosarcoma.

[0333] To implement the methods disclosed herein, an effective amount of the above-described pharmaceutical composition may be administered to a subject requiring treatment (e.g., a human) via a suitable route, such as intravenous administration, for example, by concentrated injection or continuous infusion over a period of time, via intramuscular, intraperitoneal, intraspinal, subcutaneous, intra-articular, intrasynovial, intrabursal, oral, inhalation, or local routes. Commercially available nebulizers, including jet nebulizers and ultrasonic nebulizers, may be used for administration of the liquid formulation. The liquid formulation may be directly nebulized, and the lyophilized powder may be nebulized after reconstitution. Alternatively, the anti-myosin / latent myosin antibody may be nebulized using a fluorocarbon formulation and a metered-dose inhaler, or inhaled as a lyophilized and ground powder.

[0334] Subjects to be treated by the methods described herein may be mammals, more preferably humans. Mammals include, but are not limited to, livestock, animals, pets, primates, horses, dogs, cats, mice, and rats. Human subjects requiring treatment may be human patients suffering from myopathy-related diseases / disorders (as described above), at risk of developing such diseases / disorders, or suspected of having such diseases / disorders. Subjects with tropomyostatin / latent myostatin-related diseases or disorders can be identified by routine medical examinations, such as laboratory tests, organ function tests, CT scans, or ultrasound. Subjects suspected of having any of these diseases / disorders may exhibit one or more symptoms of the disease / disorder. Subjects at risk of a disease / disorder may be subjects with one or more risk factors for that disease / disorder.

[0335] As used herein, “effective amount” means the amount of each active agent required to impart a therapeutic effect to a subject, alone or in combination with one or more other active agents. As is recognized by those skilled in the art, effective amount varies depending on the specific condition being treated, the severity of the condition, individual patient parameters (including age, physical condition, body shape, sex, and weight), duration of treatment, the nature of concurrent treatments (if any), the specific route of administration, and similar factors within the knowledge and skills of the healthcare provider. These factors are well known to those skilled in the art and can be addressed using only routine testing. Generally, the maximum dose of a single component or combination thereof is preferred, that is, the highest safe dose based on reasonable medical judgment. However, those skilled in the art will understand that patients may adhere to lower or tolerable doses for medical, physiological, or virtually any other reason. In some embodiments, effective amount refers to the amount of an antibody or its antigen-binding portion sufficient to reduce or alleviate the severity and / or duration of the disorder or one or more of its symptoms, prevent the progression of the disorder, induce the remission of the disorder, prevent the recurrence, development, onset, or progression of one or more symptoms associated with the disorder, detect the disorder, or enhance or improve the prophylactic or therapeutic effect of another treatment (e.g., a prophylactic or therapeutic agent).

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

[0337] As used herein, the term "control muscle mass" refers to a reference standard that can be used to assess the effect of a condition (e.g., treatment with tropomyostatin / latent myostatin antibody) on target muscle mass in a subject. In some embodiments, control muscle mass is a predetermined value. In some embodiments, control muscle mass is determined experimentally. In some embodiments, control muscle mass is the mass of target muscle in subjects who have not received tropomyostatin / latent myostatin antibody. In some embodiments, control muscle mass is the mass (e.g., mean mass) of target muscle in a population of subjects who have not received tropomyostatin / latent myostatin antibody. In some embodiments, control muscle mass is the mass of target muscle in subjects prior to (e.g., immediately before) the administration of tropomyostatin / latent myostatin antibody. In some embodiments, control muscle mass is the mass of target muscle in subjects who have been given a normal antibody (e.g., an antibody of the same isotype as the troponin / latent myostatin antibody) obtained from animals that have never been exposed to the antigen targeted by the troponin / latent myostatin antibody, instead of the troponin / latent myostatin antibody. In some embodiments, control muscle mass is the mass of target muscle in subjects who have been given a medium (e.g., saline) instead of the troponin / latent myostatin antibody.

[0338] In some embodiments, when administering tropomyostatin / latent myostatin antibodies to subjects, the effective amount is an amount that effectively increases the force generation capacity of the target muscle in the subject (e.g., maximum force generation as measured in vitro using a muscle lever system adapted for a horizontal perfusion bath) compared to the control force generation capacity. In some embodiments, the increase in force generation capacity is an increase of 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 generation capacity. In some embodiments, the increase in force generation capacity is an increase in the range of 1-fold to 5-fold, 2-fold to 10-fold, 1-fold to 1.5-fold, 1-fold to 2-fold, etc., compared to the control force generation capacity.

[0339] As used herein, the term "control regenerative capacity" refers to a reference standard that can be used to assess the effect of a condition (e.g., treatment with tropomyostatin / latent myostatin antibody) on the regenerative capacity of muscles in a subject. In some embodiments, control regenerative capacity is a predetermined value. In some embodiments, control regenerative capacity is determined experimentally. In some embodiments, control regenerative capacity is the regenerative capacity of target muscles in subjects who have not received tropomyostatin / latent myostatin antibody. In some embodiments, control regenerative capacity is the regenerative capacity of target muscles (e.g., mean regenerative capacity) in a group of subjects who have not received tropomyostatin / latent myostatin antibody. In some embodiments, control regenerative capacity is the regenerative capacity of target muscles in subjects prior to (e.g., immediately before) the administration of tropomyostatin / latent myostatin antibody. In some embodiments, control regenerative capacity is the regenerative capacity of the target muscle in subjects who have been administered a normal antibody (e.g., an antibody of the same isotype as the troponin / latent myostatin antibody) in place of the troponin / latent myostatin antibody, obtained from animals that have never been exposed to the antigen targeted by the troponin / latent myostatin antibody. In some embodiments, control regenerative capacity is the regenerative capacity of the target muscle in subjects who have been administered a medium (e.g., saline) in place of the troponin / latent myostatin antibody.

[0340] Empirical considerations, such as half-life, generally aid in dosage determination. For example, antibodies compatible with the human immune system, such as humanized or fully human antibodies, can be used to prolong the antibody's half-life and prevent it from being attacked by the host's immune system. The frequency of administration can be determined and adjusted during treatment, and generally, but not necessarily, based on the treatment and / or inhibition and / or relief and / or delay of the disease / disorder associated with myopathy. Alternatively, a sustained-release formulation of anti-myosostatin / latent myosostatin may be suitable. Various formulations and devices for achieving sustained release are apparent to those skilled in the art and are within the scope of this disclosure.

[0341] In one instance, the dosage of anti-myosin / latent myosin antibody, as described herein, can be empirically determined in individuals who have received one or more antibody administrations. Individuals are given escalating doses of the antagonist. To assess the efficacy of the antagonist, indicators of the disease / disorder can be tracked.

[0342] Generally, for the administration of any antibody described herein, the initial candidate dose may be about 2 mg / kg. For the purposes of this disclosure, typical daily dose ranges may be any one of approximately 0.1 μg / kg to 3 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg, to 30 mg / kg to 100 mg / kg or higher, depending on the factors mentioned above. For repeated administration over several days or longer, treatment continues, depending on the condition, until the desired symptom suppression occurs or until a sufficient therapeutic level is achieved to alleviate the disease or disorder or its symptoms associated with tropomyostatin / latent myostatin. Exemplary dosing regimens include an initial dose of about 2 mg / kg, followed by a maintenance dose of about 1 mg / kg of the antibody, or a maintenance dose of about 1 mg / kg every other week. However, other dosing regimens may be useful, depending on the pharmacokinetic decay pattern desired by the practitioner. For example, dosing 1-4 times per week is envisioned. In some embodiments, dosing may be used in the range of 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). In some embodiments, the dosing frequency is once weekly, once every 2 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, or once every 10 weeks; or once monthly, once every 2 months, or once every 3 months, once every 4 months, once every 5 months, once every 6 months, once every 8 months, once every 10 months, annually, or longer. Progression of this treatment can be easily monitored using routine techniques and analyses. The dosing regimen (including the antibodies used) may vary over time.

[0343] In some implementations, for adult patients of normal weight, a dose ranging from approximately 0.3 to 5.00 mg / kg may be administered. Specific dosing regimens, such as dose, time, and repetition, depend on the individual and their medical history, as well as the nature of the individual drug (such as the drug's half-life and other relevant considerations).

[0344] For the purposes of this disclosure, the appropriate dose of anti-troponin / latent myostatin antibody depends on the specific antibody (or combination thereof) used, the type and severity of the disease / disorder, whether the antibody is administered for prophylactic or therapeutic purposes, prior treatment, the patient's clinical history and response to antagonists, and the judgment of the attending physician. In some embodiments, the clinician administers the anti-troponin / latent myostatin antibody until a dose is reached to achieve the desired outcome. Administration of the anti-troponin / latent myostatin antibody can be continuous or intermittent, for example, depending on the recipient's physiological condition, whether the purpose of administration is therapeutic or prophylactic, and other factors known to the experienced practitioner. Administration of the anti-troponin / latent myostatin antibody can be substantially continuous over a pre-selected time period or can be a series of intermittent doses, for example, before, during, or after the onset of a troponin / latent myostatin-related disease or disorder.

[0345] As used herein, the term “treatment” means the application or administration of a composition comprising one or more active agents to a subject having a myopathy-related disorder, symptom of a disorder, or tendency to develop a disorder, for the purpose of curing, healing, relieving, alleviating, altering, treating, improving, enhancing, or influencing the symptoms of a disorder, symptom of a disease, or tendency to develop a disease / disorder.

[0346] Reducing disease / disorders associated with tropomyostatin / latent myostatin includes delaying the development or progression of the disease, or lessening its severity. Disease reduction does not necessarily require a curative outcome. As used herein, “delaying” the development of disease / disorders associated with tropomyostatin / latent myostatin means postponing, hindering, slowing, stabilizing, stabilizing, and / or slowing the progression of the disease. This delay can have varying lengths of time, depending on the individual’s medical history and / or treatment. Methods of “delaying” or reducing the development of the disease or delaying the onset of the disease are those that reduce the likelihood of one or more symptoms of the disease occurring within a given time period and / or reduce the degree of symptom relief within a given time period, compared to when the method was not used. Such comparisons are typically based on clinical studies using multiple subjects with sufficient evidence to yield statistically significant results.

[0347] The term "development" or "progression" of a disease refers to the initial presentation and / or subsequent progression of the disease. Disease development can be detectable and evaluated using standard clinical techniques. However, development also refers to progression that may be undetectable. For the purposes of this disclosure, development or progression refers to the biological process of symptoms. "Development" includes occurrence, relapse, and onset. As used herein, an "onset" or "occurrence" of a myopathy-related disease / disorder includes initial onset and / or relapse.

[0348] In some embodiments, the anti-tropomyostatin / latent myostatin antibody described herein is administered to the subject in need of treatment in an amount sufficient to inhibit the proteolytic activation of tropomyostatin / 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 antibody is administered in an amount that effectively reduces tropomyostatin / latent myostatin or latent myostatin levels by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more).

[0349] Conventional methods known to those skilled in the medical field can be used to administer the pharmaceutical composition to a subject, depending on the type or site of the disease to be treated. This composition can also be administered via other conventional routes, such as oral, parenteral, inhalation spray, topical, rectal, nasal, oral, vaginal, or via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Additionally, it can be administered to a subject via a cumulative injection route, such as using 1-, 3-, or 6-month cumulative injections or biodegradable materials and methods.

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

[0351] In one embodiment, the anti-myosin / 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 storage sources or local delivery catheters for the anti-myosin / latent myostatin antibody, such as perfusion catheters, indwelling catheters or intraneedle catheters, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site-specific carriers, direct injection, or direct application. See, for example, PCT Publication No. WO 00 / 53211 and US Pat. No. 5,981,568.

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

[0353] Therapeutic compositions containing polynucleotides (e.g., those encoding anti-myosin / latent myosin antibodies as described herein) are administered in gene therapy regimens in the range of about 100 ng to about 200 mg of DNA for topical application. In some embodiments, DNA concentrations 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 higher, may also be used during gene therapy regimens.

[0354] The therapeutic polynucleotides and peptides described herein can be delivered using gene delivery media. These media can be viral or non-viral in origin (see 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 promoters and / or enhancers or heterologous promoters and / or enhancers. Expression of the coding sequences can be constitutive or regulated.

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

[0356] Non-viral delivery media and methods may also be used, including, but not limited to, multi-cationically aggregated DNA linked to or unlinked with a single inactivated adenovirus (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery media (see, e.g., US Pat. No. 5,814,482; PCT Publication No. WO 95 / 07994; WO 96 / 17072; WO 95 / 30763; and WO97 / 42338) and nuclear charge neutralization or fusion with the cell membrane. Naked DNA may also be used. Exemplary methods for introducing naked DNA are described in PCT Publication No. WO 90 / 11092 and US Pat. No. 5,580,859. Liposomes that can be used as gene delivery media are described in US Patent No. 5,422,120; PCT Publication Nos. WO 95 / 13796; WO 94 / 23697; WO 91 / 14445; and EP Patent No. 0524968. Other pathways are described in Philip, Mol. Cell. Biol. (1994) 14:2411 and Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0357] The specific dosing regimens used in the methods described herein, such as dosage, timing, and repetition, depend on the specific subject and that subject's medical history.

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

[0359] The efficacy of treatment for myopathy-related diseases / disorders can be evaluated using any appropriate method. For example, the efficacy of treatment for myopathy-related diseases / disorders can be evaluated by assessing myasthenia (e.g., evaluating the pattern and severity of myasthenia), electromyography, assessing blood chemistry (e.g., evaluating electrolytes, assessing endocrine causes, measuring creatinine kinase levels, determining erythrocyte sedimentation rate, and performing antinuclear antibody analysis), and biopsy evaluation (e.g., through histological, histochemical, electron microscopy, biochemical, and genetic analysis).

[0360] Kits for alleviating myopathy-related diseases / disorders

[0361] This disclosure also provides kits for alleviating myopathy-related diseases / disorders. Such kits may include one or more containers containing anti-myosin / latent myosin antibodies, such as any of those described herein.

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

[0363] Instructions for use related to anti-myosin / latent myosin antibody generally include information about the dosage, dosing schedule, and route of administration for the intended treatment. Containers may be single-dose, bulk (e.g., multi-dose packs), or subunit doses. Instructions provided with kits disclosed herein are typically written instructions on a label or packaging insert (e.g., paper pages included in the kit), but machine-readable instructions (e.g., instructions carried on a disk or optical storage disc) are also acceptable.

[0364] The label or packaging insert indicates that the composition is intended for the treatment of, delaying of, and / or relief of myopathy-related diseases or disorders. The instructions may provide information for implementing any of the methods described herein.

[0365] The kit disclosed herein is packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. Packaging for use with specific devices such as inhalers, nasal administration devices (e.g., nebulizers), or infusion devices such as micropumps is also envisioned. The kit may have a sterile entry point (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). The container may also have a sterile entry point (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is an anti-myosin / latent myosin antibody as described herein.

[0366] The kit may optionally include additional components such as buffers and explanatory information. Normally, the kit includes a container and a label or insert on or attached to the container. In some embodiments, this disclosure provides an article of manufacture containing the contents of the kit described herein.

[0367] Analysis for detecting tropomyostatin / latent myostatin

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

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

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

[0371] The antigen present in the binding complex may or may not be in its original in-situ conformation. In some embodiments, the binding complex is formed between an antibody and a purified protein antigen or a separated protein containing the antigen, wherein the antigen is not in its original in-situ conformation. In some embodiments, the binding complex is formed between an antibody and a purified protein antigen, wherein the antigen is not in its original in-situ conformation and is immobilized on a solid support (e.g., a PVDF membrane). In some embodiments, the binding complex is formed with an antibody and, for example, a cell surface protein present in situ in its original conformation (e.g., on the cell surface).

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

[0373] In any detection, diagnostic, and monitoring method, antibodies (including antigen-binding fragments) or antigens can be directly or indirectly coupled to the surface of a solid support. Methods for coupling with solid supports are standard and can be accomplished through covalent and non-covalent interactions. Non-limiting examples of coupling methods include adsorption, cross-linking, protein A / G-antibody interactions, and streptavidin-biotin interactions. Other coupling methods will be readily apparent to those skilled in the art.

[0374] In some aspects, detection, diagnostic, and monitoring methods include comparing the level of antibodies (including antigen-binding fragments) that bind to an antigen (e.g., tropomyostatin / latent myostatin) with one or more reference criteria. Reference criteria may be, for example, the level of a corresponding tropomyostatin / latent myostatin in a subject with or without tropomyostatin / latent myostatin. In one embodiment, the reference criterion is the level of tropomyostatin / latent myostatin detected in a sample that does not contain tropomyostatin / latent myostatin (e.g., background level). Alternatively, the background level can be determined from a sample containing a specific tropomyostatin / latent myostatin by contacting the sample with a nonspecific antibody (e.g., an antibody obtained from non-immune serum). Then again, the reference criterion may be the level of tropomyostatin / latent myostatin detected in a sample containing tropomyostatin / latent myostatin (e.g., a positive control). In some cases, the reference standard may be a series of levels related to the changing concentrations of tropomyostatin / latent myostatin in the sample and usable for quantitatively measuring the concentration of tropomyostatin / latent myostatin in the sample. The foregoing examples of reference standards are not limiting, and other suitable reference standards will be readily apparent to those skilled in the art. In some embodiments, the level of antibodies binding to tropomyostatin / latent myostatin is compared to the level of mature myostatin. In some cases, the level of tropomyostatin / latent myostatin is compared to mature myostatin to determine the ratio of inactive to active myostatin in the sample.

[0375] The level of tropomyostatin / latent myostatin can be measured from a biological sample as provided herein. A biological sample refers to any biological material that can be obtained from a subject or cells. For example, a biological sample can be whole blood, plasma, serum, saliva, cerebrospinal fluid, urine, cells (or cell lysis products), or tissue (e.g., normal tissue or tumor tissue). In some embodiments, the biological sample is a fluid sample. In some embodiments, the biological sample is a solid tissue sample. For example, 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, solid tissue samples can be prepared into fluid samples using methods conventional in the art.

[0376] Biological samples may also include one or more cells from a cell line. In some embodiments, the cell line includes 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 exist, including but not limited to human embryonic kidney (HEK) cells, HeLa cells, cancer cells from the National Cancer Institute's 60 cancer cell line (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 (from a myeloma clone), and Saos-2 (bone cancer) cells.

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

[0378] Another embodiment relates to diagnostic compositions comprising any one of the aforementioned antibodies, antigen-binding fragments, polynucleotides, carriers, or cells, and optionally, suitable methods for detection. For example, antibodies are suitable for use in immunoassays where they can be used in a liquid phase or bound to a solid-phase support. Examples of immunoassays utilizing such antibodies are competitive and non-competitive immunoassays in direct or indirect forms. Examples of such immunoassays include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), sandwich (immunoassay) assays, flow cytometry, Western blotting, immunoprecipitation, immunohistochemistry, immunomicroscopy, cross-flow immunochromatography, and proteomics arrays. Antigens and antibodies can be bound to many different solid supports (e.g., carriers, membranes, columns, proteomics arrays, etc.). Examples of solid support materials include glass, polystyrene, polyvinyl chloride, polyvinylidene fluoride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified cellulose such as nitrocellulose, polyacrylamide, agarose, and magnetite. The nature of the support can be fixed or suspended in solution (e.g., beads).

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

[0380] In some embodiments, the antibody is immobilized on a solid support before contacting the antigen. In other embodiments, antibody immobilization occurs after the binding complex has formed. In still other embodiments, the antigen is immobilized on a solid support before the binding complex has formed. A detection reagent is added to the reaction chamber to detect the immobilized binding complex. In some embodiments, the detection reagent comprises a second antibody that is detectably labeled against the antigen. In some embodiments, the first antibody is itself detectably labeled and is therefore the detection reagent.

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

[0382] In some embodiments, the antigen is immobilized on a solid support, such as a PVDF membrane, in a reaction chamber (e.g., a membrane bath) before contact with the antibody. A detection reagent is added to the reaction chamber to detect the immobilized binding complex. In some embodiments, the detection reagent comprises a second antibody detectably labeled against the antigen. In some embodiments, the detection reagent comprises a second antibody detectably labeled against a first antibody. As disclosed herein, detectable labels may be, for example, radioisotopes, fluorophores, luminescent molecules, enzymes, biotin moieties, epitope tags, or dye molecules. In some embodiments, the first 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.

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

[0384] Therefore, the following detailed description is to be understood as illustrative only, and the remainder of this disclosure is not in any way limiting. All publications cited herein are incorporated by reference for the purposes or subjects described herein.

[0385] Example

[0386] Example 1: Antibody generation and selection

[0387] Antibody Summary

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

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

[0390]

[0391] Platform and identification of parental antibodies

[0392] Parental Ab1 antibodies were identified using proto-myostatin and latent myostatin as the primary antigens for selection via selection from a primary phage display library. Phage selection and initial screening were performed using a library displaying conventional scFv in a manner similar to that described by McCafferty et al. (McCafferty et al., 1990). Each round of selection consisted of pre-cleansing (to remove non-specific phage antibodies), incubation with antigen, washing, elution, and amplification. Selection employed a panning strategy using both a solid phase (biotinylated antigen coated on immunotubes) and a solution phase (biotinylated antigen captured using streptavidin-coated beads) in multiple rounds.

[0393] A total of 10,000 individual scFv clones were screened for binding to either tropomyostatin or latent myostatin through two separate actions. The first procedure used tropomyostatin / latent myostatin as the antigen, while the second action used latent myostatin as the antigen. The DNA of the target scFv clones was sequenced and 216 unique clones were identified. Positive-binding scFv clones were reverse-screened for binding to proGDF11 and a group of unrelated proteins to confirm specificity for tropomyostatin / latent myostatin. From this group of unique scFv clones (134 GDF8-specific clones), 101 were converted to full-length IgG (IgG1 isotype) for further characterization.

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

[0395] Two forms of epitope panning were used. First, chimeric constructs were designed and generated, exchanging portions of the pre-domains of myostatin and GDF11. The interactions of these chimeric proteins with the screening antibodies were determined by ELISA. Epitope binning was performed using a ForteBio BLI instrument, in which biotinylated tropomyostatin / latent myostatin antibodies were immobilized on a streptavidin-coated biosensor chip, and cross-blocking of the antibodies was assessed by sensor responses. These epitope binning experiments, along with data from ELISA binding experiments, allowed our functionally active leader antibodies (see below) to be segregated into three distinct epitope groups (see Table 3).

[0396] Table 3: Classification of five anti-myosin / latent myosin IgG1 antibodies

[0397]

[0398] *Statistical significance was determined by one-way ANOVA and Dunnett's method.

[0399] Ab8 does not bind to latent myostatin, but only to tropomyostatin. The murine tropomyostatin / latent myostatin preparation contains approximately 40% latent substance, which significantly reduces efficacy in functional analyses.

[0400] ND: Not determined.

[0401] To evaluate the antibody's ability to bind to and inhibit tropomyostatin / latent myostatin activation, a variety of biochemical and cellular analyses were established. Binding kinetics with tropomyostatin and latent myostatin were measured using a ForteBio Octet, where biotinylated substrate proteins were immobilized on a streptavidin-coated sensor chip. The equilibrium dissociation constants from the screened candidates are shown in Table 3.

[0402] To measure the ability of IgG to inhibit myostatin signaling, a myostatin activation assay was developed. Conditioned media were generated from cells overexpressing mTll2 (a tolloid protease required for myostatin activation) or furin (a precursor protein convertase that cleaves the anterior domain of mature growth factor). After pre-incubation with the test antibody, tropomyostatin / latent myostatin or latent myostatin was incubated with a mixture of mTll2 and furin conditioned medium (tropomyostatin) or mTll2 conditioned medium (latent myostatin). Following overnight proteolysis, the release of mature growth factor was measured using a CAGA-based reporter assay in 293T cells. The antibody was further validated by dose-response in the same assay, and the results are shown in Table 3.

[0403] The five parental antibodies (Table 3) consistently exhibited strong selectivity and activity in all the above analyses and were further selected for further in vivo characterization (discussed in Example 2). For consistency, the binding and activity of these antibodies to tropomyostatin / latent myostatin were summarized, such as Ab8 not recognizing latent myostatin.

[0404] To determine the mechanism of action of the antibody candidates, samples were analyzed by Western blot using polyclonal antibodies generated against the pre-domain of myostatin, as shown in Figure 3. This allowed for the tracking of a fragment (boxed) of the pre-domain of myostatin, which is generated after mTll2 cleavage. A dose-dependent decrease in the generation of this fragment was observed with increasing Ab1 concentration. This experiment demonstrates that the antibody in epitope box 1 acts by blocking the cleavage of tropomyostatin and latent myostatin by the tolloid family of proteases.

[0405] Based on the in vitro and in vivo activity of the active anti-myosin / latent myosin antibody, Ab1 was selected as a lead for further characterization, including affinity maturation, germlining, and manufacturability analysis.

[0406] Optimization of Ab1

[0407] The Ab1 antibody was selected for further characterization. Affinity for tropomyostatin / latent myostatin was optimized using yeast display. Additionally, the Ab1 sequence was germinated to reduce the potential immunogenicity at non-germ-type amino acid positions within the human variable region framework.

[0408] Ab1 optimizes affinity demonstrated by yeast.

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

[0410] Many high-affinity scFv clones identified in yeast action contain a substitution at position 28 of the heavy chain. For some clones, the threonine-to-asparagine substitution results in the addition of an irregular N-glycosylation motif within CDRH1. Since N-glycosylation within the antibody variable region can be undesirable, any clone containing the glycosylation motif undergoes further substitution to include alanine at that position.

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

[0412] Primary sequence and backbone of anti-myosin / latent myostatin antibody

[0413] Sequence alignments of the variable regions of parental Ab1 with its affinity-optimized variants are shown below. Complementarity-determining regions (CDRs) are defined using Kabat (underlined) and IMGT nomenclature (bold). Permutations from parental Ab1 are shown in lowercase (hereinafter and...). Figures 24A-24B ).

[0414] A. Heavy chain variable region

[0415]

[0416]

[0417] B. Light chain variable region

[0418]

[0419] Basic principles of antibody engineering and allotype selection

[0420] In some implementations, antibodies that can be used to block myostatin will lack effector function. Therefore, for humanized constructs, the IgG4-Fc region is selected. Antibodies of the IgG4 isotype bind poorly to complement C1q and therefore do not significantly activate complement. These antibodies also bind weakly to the Fcγ receptor, resulting in insufficient or absent antibody-dependent cell-mediated cytotoxicity (ADCC).

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

[0422] Germination and immunogenicity risk assessment

[0423] The Ab1 parental antibody and its variants are phage-displayed fully human IgG4 (S228P)λ antibodies. The Fc moiety of the antibody contains a single stable mutation to prevent Fab arm exchange (as described above). This IgG4 Fc is not expected to have measurable binding to the Fcγ receptor (see Example 2).

[0424] For example, the variable frame region of Ab1, isolated from fully human primitive phages, contains five non-species amino acids (see below and...). Figure 22 Complementarity-determining regions (CDRs) are defined using Kabat nomenclature and underlined. Non-germ residues are shown in lowercase.

[0425] A. Heavy chain variable region

[0426]

[0427] B. Light chain variable region

[0428]

[0429] To mitigate the potential for immunogenicity, alternative variants of the Ab1 molecule are generated, which replace non-germ framework residues with their corresponding germline amino acids. In some embodiments, substitutions involving Ab1 can be similarly applied to Ab3 and Ab4 or germinate any antibody disclosed herein for its suitability.

[0430] Sequence alignments of the Ab1 variable region with 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). Frame region permutations present in parental Ab1 are shown in lowercase.

[0431] A· Heavy chain variable region

[0432]

[0433] B·Light Chain Variable Region

[0434]

[0435]

[0436] Three of the five substitutions were found to be far from the CDR region and therefore had no effect on binding. The proline at position 2 of the light chain targets CDRL3 packaging, and the substitution with germline serine actually improves binding with tropomyostatin / latent myostatin by stabilizing the CDR conformation.

[0437] Overall, the antibody prevalence was higher than 99% in individuals (calculated as 100% minus % non-species AA, excluding CDRH3). No chemical conjugation was observed. The heavy chain CDRH2 sequence contains a potential isomerization tendency (Asp-Gly), which is also present in the germline IgHV3-30 sequence.

[0438] Example 2: Pharmacological Characterization

[0439] In vitro pharmacological analysis

[0440] A total of 24 optimized Ab1 variants were expressed and purified as IgG4, and their improved binding and functional activities were determined. Modifications to these molecules included germline mutations in the parental variable region and mutations in the CDR, which conferred enhanced binding to tropomyostatin / latent myostatin during affinity maturation screening (see Example 1).

[0441] The Ab1 variant was screened in several different ELISA-based analyses, where binding to tropomyosin and latent myostatin proteins (human, mouse, and cynomolgus monkey) was reassessed along with extensive screening of negative control proteins to verify that nonspecific binding was not introduced due to affinity maturation. Negative controls included GDF11 proteins (proGDF11, latent GDF11, and mature GDF11), TGFβ proteins, and activin proteins (proactivin). Additionally, the antibody was evaluated for multispecificity (which leads to rapid clearance) in a screening similar to that previously published (Hotzel et al., 2012). Any antibody exhibiting significant interactions with negative control proteins or with baculovirus particles in the multispecific screening was not further considered as a candidate for the development program.

[0442] Twenty-four optimized Ab1 variants were also evaluated in a tropomyostatin activation assay to determine their functional potency, with EC50 values ​​from dose-response curves compared to the parental Ab1 antibody. Most antibodies showed equivalent or improved EC50 values, while a few showed reduced potency in this assay. Those showing reduced potency in the activity assay were excluded from further analysis.

[0443] Three variants were identified that exhibited improved binding to tropomyostatin and latent myostatin while maintaining specificity for both. Binding and activity data of these three variants to the parental Ab1 molecule are summarized in Tables 4-7, and their sequences are shown in Example 1.

[0444] Table 4: Binding characteristics of antibodies against human / cynomolgus monkey / mouse tropomyostatin to parental Ab1IgG4.

[0445]

[0446] Table 5: Binding characteristics of antibodies against human / cynomolgus monkey / mouse tropomyostatin to the correct germline residue Ab1 IgG4 (Ab2) with non-germinated residues.

[0447]

[0448] Table 6: Binding characteristics of antibodies against human / cynomolgus monkey / mouse tropomyostatin to Ab3IgG4 (Ab4) containing corrected germline residues.

[0449]

[0450]

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

[0452]

[0453] Based on in vitro and in vivo bioactivity analysis of cells

[0454] In dose-response studies, the optimized Ab1 variant was evaluated in GDF8 activation assays. In these experiments, 0.5 μM tropomyosin was pre-incubated with increased amounts of the test sample. Following this pre-incubation step, conditioned medium from HEK293 cells overexpressing mT112 and furin was added to release mature growth factor from tropomyosin. After overnight incubation at 30°C, the substance was added to 293T cells carrying a SMAD-based luciferase reporter plasmid, and the activity of the released substance was recorded. Data from the screening showed… Figure 4 middle.

[0455] Compared to other TGFβ family members, it exhibits selectivity for myostatin.

[0456] The selectivity of the candidate antibody was also assessed through both binding and functional analyses to verify the lack 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 pre-domain region. Epitope mapping studies determined that the parental Ab1 molecule recognizes epitopes on the pre-domains of both tropomyostatin and latent myostatin, as ELISA analysis has shown that this antibody binds to the construct composed of the pre-domain of myostatin. Although myostatin and GDF11 share less than 50% identity in their pre-domains, and we did not expect significant cross-reactivity, the specificity of the lead antibody was carefully evaluated.

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

[0458] Table 8: Interactions between candidate molecules and proGDF11 at high concentrations

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

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

[0461] The binding affinity of antibody candidates was determined using the FortéBio Octet QKe dip-read label-free assay system employing biolayer interferometry. Antigens were immobilized in each assay with biosensors (streptavidin-coated biosensors for proGDF8, proGDF11, and proactivin; direct amine conjugation for all other antigens) and antibodies / constructs were present in solution at high concentrations (50 μg / mL) to measure binding interactions.

[0462] Antibody binding affinity was determined using a FortéBio Octet QKe dip-read label-free assay system employing biolayer interferometry. Human proGDF8, latent GDF8, proGDF11, and proactivin were biotinylated and immobilized on streptavidin-coated biosensors (FortéBio). Mature growth factor was immobilized on an amine-reactive tip via direct amine coupling according to the manufacturer's instructions (FortéBio). In each assay, the antibody / construct was present in solution at a single high concentration (50 μg / mL) to measure binding interactions. Growth factors were purchased from R&D systems, and biotinylated proteins were produced as described.

[0463] Table 9a: Comparison of antibody binding to different forms of several TGFβ family members.

[0464]

[0465] *Non-specific binding.

[0466] Results from antigen binding studies are summarized in Table 9a. Experiments with no detectable binding are indicated by a negative sign (-). Some calculated Kd values ​​exist that correspond to data with poor binding responses, which are represented in the table as weak nonspecific binding (*).

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

[0468] The binding affinity analyses for mouse proteins were also evaluated and reported in Table 9b. Mouse proGDF8 protein was generated by negative selection to remove all mature and latent mouse GDF8 from the sample using an antibody (AbMyo2) that tightly recognizes latent and mature GDF8. 50 nM of antibody was used to saturate the anti-human Fc capture tip (FortéBio). Initially, all antibodies were tested against a single 200 nM concentration of mouse proGDF8, mouse latent GDF8, and mature GDF8. If binding was observed, the Kd value was determined by immobilizing the antibody as previously described and using analyte titrated with 200–0.82 nM via a 3-fold dilution. Kd was determined using the FortéBio data analysis software 8.2 with a global fit. For binding to mature myostatin, 5 μg / mL of growth factor (R&D systems) was conjugated to the amine reactive sensor tip (FortéBio) in acetate buffer at pH 5. All antibodies were initially tested for binding to this myostatin-coupled sensor at 333 nM. The bound antibodies were then tested at concentrations ranging from 333 to 1.37 nM via 3-fold dilution. Overall fit was used to determine the Kd of the interaction using FortéBio Data Analysis 8.2.

[0469] Table 9b: Comparison of antibody binding to different forms of human and mouse GDF8.

[0470]

[0471]

[0472] Results from antigen binding studies are summarized in Table 9b. Experiments with no detectable binding are indicated by a negative sign (-). Some values, marked <1E-12, have very low dissociation rates, making it impossible to quantify high affinity. Surprisingly, AbMyo failed to recognize recombinant proGDF8, unlike the results reported by Latres et al. (2015), in which they reported binding of AbMyo to proGDF8 in immunoprecipitation assays from serum of antibody-administered mice, which could produce artifacts. Another surprising result is the interaction between Ab2 and activated GDF8 (a complex of GDF8 with a tolloid-cleaved predomain). This result is unexpected because Ab2 blocks tolloid cleavage of the predomain and indicates that the interaction of Ab2 with both proGDF8 and latent GDF8 does not require an intact tolloid cleavage site.

[0473] Evaluation of Fc-zone functionality

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

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

[0476] Table 10: Relative binding affinity of Ab2 and related antibodies to CD64.

[0477]

[0478] Undetermined

[0479] Ab1 and its related variants exhibit epigenetic binding affinity for CD64 and C1q similar to other IgG4 clinical candidate antibodies, but significantly lower than that of antibodies against the IgG1 isotype. Based on the biology of IgG4 antibodies, it is concluded that anti-myosin / latent myosin antibodies do not induce significant effector functions in vivo.

[0480] efficacy in animal models

[0481] Based on in vitro characterization, four antibodies (Ab7, Ab1, Ab8, and Ab9) were selected for testing in vivo. The aim of this study was to evaluate the ability of these four candidate antibodies to modulate mouse muscle mass. Ten (10) female SCID mice were divided into five (5) groups and administered the test substance weekly via intraperitoneal (IP) injection on days 0, 7, 14, 21, 28, and 35. All animals underwent grip strength assessment prior to test substance administration (day 0). Grip strength assessment was also performed on the last day of the study (day 42). On day 0, blood was collected via retroocular sampling for evaluation of complete blood count (CBC). Animals were assessed for body weight and general health observation daily after administration. On day 42, following the grip strength assessment, animals were euthanized by CO2 overdose and blood was collected via cardiac puncture for CBC evaluation. Additional blood was collected for plasma preparation. Various tissues were isolated and weighed. The muscles collected were: gastrocnemius, pectoralis major, soleus, triceps surae, tibialis anterior, quadriceps (rectus femoris), and diaphragm. The organs collected were: heart, kidneys, spleen, liver, and inguinal white adipose tissue. All tissues, except for the gastrocnemius (which was fixed in formalin (leg 1) and OCT (leg 2) for histological analysis), were weighed and flash-frozen.

[0482] Summarize

[0483] The data on the average daily percentage weight change of animals in the SCH-02 study showed that... Figure 6 In all five groups, animals showed weekly weight gain. Animals treated with antibody Ab1 showed the following results: Figure 6 The animals shown had the largest weight gain (14.6%). Animals treated with Ab1 alone had a statistically significant increase in mean daily percentage change in body weight compared to the solvent (PBS) control group. Figure 6 ).

[0484] The weight of the removed muscle is plotted in Figures 7 and 8. Animals treated with Ab1 showed greater reduction in gastrocnemius muscle mass compared to the control group treated with PBS. Figure 7A ) and diaphragm ( Figure 8BThe weight increases were statistically significant, by 27.6% and 49.8%, respectively. Additional muscle from the Ab1-treated animals showed weight increases compared to the PBS control, but these differences were not statistically significant. There were no statistically significant differences in mean tissue weight between the treatment groups for the heart, spleen, kidneys, liver, and adipose tissue.

[0485] SCID dose-response study

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

[0487] Ten (10) female SCID mice were divided into ten (10) groups and administered the test product twice weekly via intraperitoneal (IP) injection (10 ml / kg) on ​​days 0, 3, 7, 10, 14, 17, 21, and 24. The test product doses 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). The control group was administered PBS and IgG-control (30 mg / kg). The treatment groups are described in Table 11. The animals were 10 weeks old at the start of the study. Body weight was measured on day -4 and twice weekly throughout the study (corresponding to the day of administration). Body mass composition parameters (fat mass, lean body mass, and water content) were measured by Echo MRI (QNMR) on days -4, 7, 14, 21, and 28. Thirty (30) days after the first antibody dose, the animals were euthanized by CO2 overdose and their blood was collected via cardiac puncture for CBC evaluation and plasma preparation. Additionally, at the end of the study, various tissues were isolated and weighed. The muscles collected were: gastrocnemius, soleus, tibialis anterior, quadriceps (rectus femoris), and diaphragm. The muscles were excised from both the right and left legs of the study mice. For analysis, the weights of the individual muscles from both legs were combined and the average muscle weight in grams was calculated. Other tissues collected were: heart, kidney, spleen, liver, and adipose tissue. All tissues, except for the gastrocnemius (which was fixed in formalin (left leg) and OCT (right leg) for histological analysis), were weighed and then flash-frozen.

[0488] Table 11: Research Design

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

[0490] The mean percentage changes in body weight and mean percentage changes in lean body weight from animals treated with the solvent (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 significant weight gains on day 28 of the study, at 15.3% and 14.4%, respectively, compared to the IgG control. Figure 9A Animals treated with Ab1 (at doses of 60, 20, 6, and 2 mg / kg / week) showed statistically significant increases in lean body mass on day 28 compared to animals treated with IgG control, at 14.1%, 12.4%, 17.1%, and 15.5%, respectively. Figure 9B ).

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

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

[0493] Given the known ability of the anti-myosin antibody Ab1 to increase muscle mass in healthy SCID mice, it was necessary to determine whether Ab1 treatment could also protect animals against treatments that induce muscle atrophy. A model of corticosteroid-induced muscle atrophy was established by treating animals with dexamethasone in their drinking water for two weeks. The selected dose (2.5 mg / kg / day) induced a significant decrease in lean body mass and the mass of individual hind limb muscles. In the following experiments, animals were treated with different doses of Ab1 to determine whether it could protect them from the effects of this dexamethasone-induced muscle atrophy.

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

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

[0496]

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

[0498] At the end of the two-week treatment period with dexamethasone and the test sample, individual muscles were removed and weighed. Weight data for both muscles (gastrocnemius and quadriceps) were collected at... Figure 12A-12B Figure 1 shows the results. Animals receiving dexamethasone via drinking water and also receiving either PBS or IgG control antibody showed significant atrophy of the gastrocnemius and quadriceps muscles compared to the non-disease control group (Group 1) (Groups 5 and 6). Animals treated with both dexamethasone and 20 mg / kg / wk (instead of 2 mg / kg / wk) of Ab1 (Group 7) showed a significant difference in muscle weight compared to either the dexamethasone-treated control group (Groups 5 and 6). Additionally, mice treated with both dexamethasone and 20 mg / kg / wk of antibody Ab1 (Group 7) did not show a significant difference in gastrocnemius and quadriceps muscle weight compared to the non-disease control group (Group 1). The mean percentage difference in muscle weight between each group and the control group (Group 1, PBS, and water) is shown in the figure. Figure 12C-12D In the PBS and IgG Ctl groups, dexamethasone treatment induced a 16.5% and 18.9% reduction in gastrocnemius muscle mass, respectively. Conversely, animals treated with both dexamethasone and 20 mg / kg / wk Ab1 showed only a 4.0% reduction in gastrocnemius muscle mass, which was not significantly different from the non-disease control group (group 1). Although animals treated with both dexamethasone and 2 mg / kg / wk Ab1 (group 8) showed only a 10% reduction in gastrocnemius muscle mass, this reduction was not significantly different from the reductions observed in the PBS and IgG control groups (groups 5 and 6). Similar results were observed for the quadriceps muscle. Figure 12D ).

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

[0500] Known for its ability to increase muscle mass in healthy SCID mice, the anti-myosin antibody Ab1 was investigated to determine whether Ab1 treatment could also protect animals from treatment-induced muscle atrophy. A disuse atrophy model was established in mice by applying a cast to their right leg for two weeks. Maintaining the foot in a plantar flexed position while the right leg was in a cast for this duration induced a significant reduction in muscle mass in the individual hind limb. In the following experiments, animals were treated with different doses of Ab1 to determine the extent to which it protected animals from this cast-induced muscle atrophy.

[0501] Table 13: Treatment groups used in the study of the gypsum-induced shrinkage model

[0502]

[0503] In this study, ten (10) male mice (C57BL / 6) were enrolled in eight (8) groups at 14.5 weeks of age. Starting on day 0 of the study, mice were anesthetized and had a plaster cast applied to the right hind limb to plantarflex the paw (groups 5–8). Control groups (groups 1–4) were also anesthetized but without a plaster cast on the hind limb. Tests were administered twice weekly via intraperitoneal (IP) injection (10 ml / kg) on ​​days 0, 3, 7, and 10. Tests and dosages were as follows: PBS (groups 1 and 5), 10 mg / kg IgGCtl (groups 2 and 6), 10 mg / kg Ab1 (groups 3 and 7), and 1 mg / kg Ab1 (groups 4 and 8). Treatment groups are described in Table 13. Body weight was measured at least twice weekly throughout the study. Body mass composition parameters (fat mass, lean mass, and water content) were measured by EchoMRI (QNMR) on days -1, 7, and 14. Fourteen (14) days after the first antibody dose, the animals were euthanized by CO2 overdose and their blood was collected by cardiac puncture for plasma preparation.

[0504] In addition, various tissues were separated and weighed. The muscles collected were: gastrocnemius, soleus, plantar fasciae brevis, tibialis anterior, and quadriceps femoris. For analysis, the weight of individual muscles from the animal's right hind limb was collected. Other tissues collected were: heart, adipose tissue, and spleen. All tissues, except for the gastrocnemius (which was fixed in formalin for histological analysis), were weighed and then flash-frozen.

[0505] Summarize

[0506] In this experiment, we tested whether treatment with the anti-myosin antibody Ab1 could protect mice from disuse muscle atrophy induced by a cast on the right hind limb. During the study, body weight was measured twice weekly, and lean body mass was measured by QNMR on days -1, 7, and 14. The mean percentage changes in body weight and mean percentage changes in lean body mass for the non-disease control group (Group 1) and the groups with casts for two weeks (Groups 5–8) are shown in Figure 13. A cast on the right hind limb had no negative effect on body weight gain. Figure 13A And any difference in lean body mass between the groups was not significant. Figure 13B ).

[0507] At the end of the two-week study, individual muscles were removed and weighed. Weight data for both muscles (gastrocnemius and quadriceps) were collected. Figures 14A-14B Figure 1 shows the results. Animals with leg casts who also received either PBS or IgG control antibodies showed significant atrophy of the gastrocnemius and quadriceps muscles compared to the control group without casts (Group 1) (Groups 5 and 6). Animals with casts who also received Ab1 at 20 mg / kg / wk instead of 2 mg / kg / wk (Group 7) showed a significant difference in muscle weight compared to either the cast-treated control group (Groups 5 and 6). Additionally, cast-treated mice treated with 20 mg / kg / wk of antibody Ab1 (Group 7) did not show a significant difference in gastrocnemius and quadriceps muscle weight compared to the control group without casts (Group 1). The mean percentage difference in muscle weight between each group and the mean muscle weight of the control group without casts (Group 1) is shown in the figure. Figure 14C-14D In the PBS and IgG Ctl groups, the percentage reduction in gastrocnemius muscle mass induced by cast application was 22.8% and 23.5%, respectively. Conversely, cast-treated mice treated with 20 mg / kg / wk Ab1 showed only a 10.0% reduction in gastrocnemius muscle mass. This difference was statistically significant compared to the cast-treated controls (groups 5 and 6) receiving PBS and IgG Ctl antibodies. The reduction in muscle mass in cast-treated mice treated with 2 mg / kg / wk Ab1 was not statistically different from that in the PBS and IgG controls (groups 5 and 6). Similar results were observed for the quadriceps muscle. Figure 14D ).

[0508] The structural structures of the domains of tropomyostatin and latent myostatin (indicating protease cleavage sites) are shown in Figure 16A An example of tropomyostatin partially cleaved by precursor protein convertase running on an SDS-PAGE gel is shown in [the image]. Figure 16B In the reducing conditions, the protein band consists of tropomyostatin monomer (~50kD), pre-domain (~37kD), and growth factor (12.5kD).

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

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

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

[0512] Eight (8) female SCID mice were divided into fourteen (14) groups and administered the test product twice weekly via intraperitoneal (IP) injection (10 ml / kg) on ​​days 0, 3, 7, 10, 14, 17, 21, and 24. The test product was administered at three different doses (10 mg / kg, 1 mg / kg, and 0.25 mg / kg) for Ab1, Ab2, Ab4, and Ab6, and at 10 mg / kg for IgG-Ctl antibody. The treatment groups are described in Table 14. The animals were 10 weeks old at the start of the study. Body weight was measured twice weekly throughout the study (corresponding to the day of administration). Body mass composition parameters (fat mass, lean mass, and water content) were measured by Echo MRI (QNMR) on days 0, 7, 14, 21, and 28. Twenty-eight (28) days after the first antibody dose, the animals were euthanized by CO2 overdose and their blood was collected by cardiac puncture for plasma preparation.

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

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

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

[0516] The mean percentage change in lean body mass (relative to day 0) of animals treated with solvent (PBS), IgG control, and different doses of Ab1, Ab2, Ab4, and Ab6 was shown in the data. Figure 15 Animals treated with Ab1, Ab2, Ab4, and Ab6 at dose levels of 20 mg / kg / wk showed a significant increase in lean body mass on days 21 and 28 compared to animals treated with IgG control and solvent (PBS). Animals treated with Ab1 and Ab2 at dose levels of 2 mg / kg / wk also showed significant changes in lean body mass on days 21 and 28 of the study. Animals treated with Ab1, Ab2, Ab4, and Ab6 at dose levels of 0.5 mg / kg / wk did not show a significant change in lean body mass relative to the control group.

[0517] At the end of the study (day 28), muscle samples were collected and weighed. The quadriceps (rectus femoris) and gastrocnemius muscles weighed [weight missing]. Figure 18A and 18B Figure 1 shows the results. Animals treated with Ab1, Ab2, Ab4, and Ab6 at dose levels of 20 mg / kg / wk showed a significant increase in gastrocnemius and quadriceps (rectus femoris) muscle mass compared to animals treated with the solvent (PBS). Animals treated with Ab2 and Ab4 at dose levels of 2 mg / kg / wk also showed a significant change in gastrocnemius muscle mass. Animals treated with Ab2 at dose levels of 2 mg / kg / wk also showed a significant change in quadriceps (rectus femoris) muscle mass. For animals treated with Ab1, Ab2, Ab4, and Ab6 at dose levels of 0.5 mg / kg / wk, there was no significant change in muscle mass compared to the control group. The percentage differences in gastrocnemius and quadriceps (rectus femoris) muscle mass (when compared to the solvent control) of animals treated with different doses of Ab1, Ab2, Ab4, and Ab6 are listed below. Figure 18C middle.

[0518] Duration of the study on the role of Ab1 in SCID mice

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

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

[0521]

[0522]

[0523] Data on the change in mean lean body mass of animals treated with solvent (PBS), IgG control, Ab1, and AbMyo showed that... Figure 19 Data are presented as changes in lean body mass relative to day 0 of the study. At day 21 following a single dose of the test substance, animals treated with Ab1 (group 3) showed a significant increase in lean body mass (compared to IgG control animals – group 1), which was indistinguishable from the changes in lean body mass after three doses of Ab1 (group 6). These changes in lean body mass were also comparable to those seen in animals treated with a single dose of AbMyo (group 4) or animals treated with three weekly doses of AbMyo (group 7).

[0524] Example 3: Chemistry / Pharmacology

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

[0526] Molecular modeling of Ab1 identified several potential sites for post-translational modifications. Two asparagine residues in the light chain and seven asparagine residues in the heavy chain are readily deamidated. Two of these residues are located within the CDR region of the heavy chain.

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

[0528] Summarize

[0529] This article presents a tropomyostatin / latent myostatin-specific antibody that blocks the activation of tropomyostatin and / or latent myostatin. This activation-blocking antibody, administered to healthy mice, increased lean body mass and muscle size, with only a single dose required to maintain muscle enhancement over a period of one month. Furthermore, antibody administration protected healthy mice from muscle atrophy in two independent models of muscle atrophy. The data demonstrate that blocking myostatin activation promotes robust muscle growth and prevents muscle atrophy in vivo, representing an alternative mechanism for therapeutic interventions against muscle atrophy.

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

[0531] Western blotting was performed to determine the presence of tropomyosin and latent myostatin in muscle tissue and circulation during muscle atrophy, as well as under normal conditions. A standard model of muscle atrophy involved treating mice with dexamethasone at 2.5 mg / kg / week (administered in drinking water), with muscle and plasma collected after 2 weeks of treatment. This model frequently resulted in a 15-25% reduction in muscle mass during treatment. Control muscle and plasma were collected from mice never treated with dexamethasone. The rectus femoris, tibialis anterior, and soleus muscles were excised, flash-frozen in liquid nitrogen, and stored at -80°C until use. Muscle lysis products were generated by grinding followed by dissolution in T-PER buffer supplemented with protease and phosphatase inhibitors. Plasma was collected using standard methods and stored at -80°C.

[0532] Multiple samples containing the same protein concentration were separated onto PVDF membranes by PAGE gel electrophoresis and Western blotting. For myolysis products, 10–50 ng of total protein was loaded onto the gel. Plasma was diluted 1:10 in PBS, and 10 μl of each sample was loaded onto the gel. As size standards, 0.1–1 ng of recombinant tropomyostatin and / or latent myostatin were also loaded onto the gel. Identification of myostatin protein was performed using an antibody (AF1539, R&D Systems) that recognizes the pre-domain of myostatin. This analysis showed that tropomyostatin is the dominant form in muscle, while latent myostatin is the predominant form in plasma. Figure 25 Furthermore, it has been shown that in mice with dexamethasone-induced muscle atrophy, tropomyostatin is increased in muscle tissue, while latent myostatin is decreased in plasma.

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

[0534] Based on these data, a model of myostatin "flow" in normal and diseased muscles is presented. As demonstrated, in normal muscle ( Figure 28A Tropostatin is produced in muscle and converted into latent myostatin via cleavage by furin, a protease that may be present intracellularly or extracellularly (Anderson et al., 2008). Latent myostatin in certain parts of the muscle is then released into the circulation, forming a circulating pool of latent myostatin. In muscle atrophy, an increase in the production of active myostatin-growth factor drives muscle atrophy. This increase is thought to be caused by the upregulation of tropomyostatin levels in muscle and the increased conversion of latent myostatin into active growth factor. Figure 28B The data outlined here directly support the first step of this model, indicating increased tropomyostatin in muscle. The data also support the second step, as the observed reduced muscle mass in dexamethasone-treated mice suggests increased production of mature myostatin without a corresponding increase in latent myostatin in muscle. Therefore, the decreased plasma myostatin levels indicate increased conversion to mature myostatin.

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

[0536] Immunoprecipitation was performed to determine the presence of circulating tropomyostatin and to investigate the binding of Ab2 and AbMyo to endogenous myostatin precursors in serum and muscle. Ab2 recognizes the major form of myostatin in muscle. Figure 27 The results shown demonstrate that serum tropomyostatin pools are precipitated with Ab2, indicating the presence of extracellular tropomyostatin in vivo. Ab2 immunoprecipitates tropomyostatin from muscle extracts, in addition to binding to serum tropomyostatin, latent myostatin, and other partially processed forms of myostatin. Conversely, AbMyo effectively binds to serum latent myostatin and partially processed precursors without detectable interactions with tropomyostatin in muscle. Since muscle is the site where myostatin signaling occurs, this provides a significant advantage for understanding the mechanism of action of Ab2.

[0537] The homogenized muscle lysate was prepared as follows: Frozen mouse quadriceps muscle was ground using a CryoPrep grinder (Covaris, Woburn MA). The ground muscle was then homogenized with 1x Halt... TM The tissue was resuspended to a concentration of 50 mg / mL in M-Per buffer (ThermoFisher Scientific) without EDTA (ThermoFisher Scientific) containing a mixture of protease and phosphatase inhibitors. The tissue was then homogenized using a plastic pestle (Bio-Plas Cat#4030-PB) and further homogenized by repeated aspiration with a flat-tipped pipette tip. The muscle samples were then incubated at 4°C with end-to-end rotation for 30 min. Finally, the samples were centrifuged at 16,100 g for 10 min to precipitate the insoluble fraction. The soluble fraction was aspirated and used in downstream experiments.

[0538] For immunoprecipitation, Ab2, IgG Ctl, or AbMyo antibodies were used with Thermo Scientific Pierce. TMThe Co-Immunoprecipitation kit was covalently conjugated with agarose beads according to the manufacturer's instructions. 75 μg of each antibody was conjugated with 50 μL of bead slurry, and 30 μg of antibody was used in each immunoprecipitation. Immunoprecipitation was performed against 3 mL of bioreclamation (combined with normal mouse serum) 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-to-end rotation. After incubation, the beads were recovered using a QIAvac 24Plus vacuum manifold (Qiagen) by passing the entire sample volume through a rotary filter included in the co-immunoprecipitation kit. The beads were then washed 3x with 200 μL of IP lysis / wash buffer and once with 100 μL of 1x condition buffer according to the kit instructions. Elution was performed with 50 μL of elution buffer for 5 minutes and then mixed with 5 μL of 1M Tris, pH 9.5 in a collection tube.

[0539] By testing the myostatin substance pulled down by antibodies, AF1539 (R&D systems) ab124721 and (Abcam) Alexa were used. 680 AffiniPure donkey anti-sheep IgG (H+L), (Jackson ImmunoResearch) and 800CW donkey anti-rabbit IgG (H+L) (LI-COR Biosciences) Visualized by Western blot by Thermo Scientific. SEA BLOCK blocking buffer was used for blocking and incubation with the primary antibody.

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

[0541] Research Design

[0542] Female Sprague-Dawley rats aged seven to eight 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 was collected from three rats / group at 4 hours, 48 ​​hours, 7 days, 14 days, 21 days, and 28 days post-administration. Collection was performed using standard methods, and samples were stored at -80°C. Lean body mass was measured by quantitative nuclear magnetic resonance (qNMR) at baseline (before administration on day 0) and at days 7, 14, 21, and 28 (eight rats / group), and skeletal muscle (rectus femoris, tibialis anterior, and soleus) was collected, weighed, and flash-frozen in liquid nitrogen at the end of the study (day 28) for storage at -80°C.

[0543] result

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

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

[0546] The relative levels of tropomyostatin and latent myostatin were determined by quantitative Western blotting of myolysis products or serum samples. Myolysis products were generated from flash-frozen muscle samples by grinding followed by dissolution in T-PER buffer supplemented with protease and phosphatase inhibitors. After dissolution, samples containing the same protein concentration were separated by PAGE gel electrophoresis and Western blotted onto low-fluorescence PVDF membranes. For myolysis products, 10–50 ng of total protein was loaded onto the gel. Plasma was diluted 1:10 in PBS, and 10 μl of each sample was loaded onto the gel. As size standards, 0.1–1 ng of recombinant tropomyostatin and / or latent myostatin were also loaded onto the gel. Identification of myostatin protein was performed using an antibody (AF1539, R&D Systems) recognizing the predomain of latent myostatin, followed by detection with a fluorescently labeled secondary antibody. For all Western blot analyses, at least three samples / groups were measured.

[0547] Ab2 treatment increased latent myostatin levels in rat serum by approximately 20-fold compared to IgG control rats. Figure 31AThese data are consistent with effects observed with other antibody drugs, thus reflecting the binding of the drug target in circulation. In rat muscle (rectus femoris), Ab2 treatment resulted in a 1.9x increase in the latent form of myostatin (vs. IgG control rats). No statistically significant change in tropomyostatin was observed. These data suggest that Ab2 binds to its target, tropomyostatin / latent myostatin, and alters myostatin treatment in both muscle and circulation. An increase in latent tropomyostatin in rat muscle was also observed with Ab2 treatment, but no increase in tropomyostatin (…). Figure 31B ).

[0548] Example 7: Increased muscle mass and altered expression of myostatin protein in mice treated with Ab2, and comparison with anti-myostatin antibody.

[0549] Research Design

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

[0551] result

[0552] The pharmacodynamic effects of the Ab2 treatment were evaluated by measuring lean body mass (by qNMR) during the study. Figure 32 The study showed lean body mass measurements during the course of the study, with mice treated with Ab2 exhibiting a significant increase in lean body mass compared to mice treated with the human IgG control antibody. For the first three weeks of the study, mice treated with the comparative antibody (AbMyo) showed equivalent lean body mass increases to the Ab2 group. However, by day 28 post-administration, the AbMyo-treated mice did not maintain their increased lean body mass. Conversely, the mice in the Ab2-treated group maintained their increased lean body mass throughout the entire duration of the study (56 days). These data suggest that Ab2 has a longer duration of action than AbMyo.

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

[0554] The relative levels of tropomyostatin and latent myostatin were determined by quantitative Western blotting of myolysis products or serum samples. Myolysis products were generated from flash-frozen muscle samples by grinding followed by dissolution in T-PER buffer supplemented with protease and phosphatase inhibitors. After dissolution, samples containing the same protein concentration were separated by PAGE gel electrophoresis and Western blotted onto low-fluorescence PVDF membranes. For myolysis products, 10–50 ng of total protein was loaded onto the gel. Plasma was diluted 1:10 in PBS, and 10 μl of each sample was loaded onto the gel. As size standards, 0.1–1 ng of recombinant tropomyostatin and / or latent myostatin were also loaded onto the gel. Identification of myostatin protein was performed using an antibody (AF1539, R&D Systems) recognizing the predomain of latent myostatin, followed by detection with a fluorescently labeled secondary antibody. For all Western blot analyses, at least three samples / groups were measured.

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

[0556] Myostatin levels in muscle (rectus femoris) were also assessed by Western blotting. The relative levels of latent myostatin and tropomyostatin were measured in mouse muscle lysate products by Western blotting. Latent myostatin was elevated in both Ab2 and AbMyo-treated muscles. Figure 35A However, in AbMyo-treated muscle, the increase in latent myostatin returned to baseline by day 28, while in Ab2-treated muscle, the increase in latent myostatin remained elevated until at least this time (P < 0.003 vs. AbMyo treatment). A similar trend was observed with tropomyostatin. Figure 35BAlthough the difference was not statistically significant (P = 0.068), these data suggest that Ab2 has a longer duration of action at the site of drug action (skeletal muscle).

[0557] Example 8: Ab2 enhances muscle strength production.

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

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

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

[0561] Tic-twitch tension was monitored with a 1ms pulse and the voltage was increased until maximum force was achieved. A series of stimuli (1ms pulse, 250ms training duration) were then performed at increasing stimulation frequencies: 1, 10, 20, 40, 60, 80, 100, 150 Hz, followed by a final stimulus of 1 Hz.

[0562] like Figure 36A As shown, muscle mass and function improved after 4 weeks of treatment with Ab2. Average EDL weight increased by 33%, and average gastrocnemius and quadriceps weight increased by 19%.

[0563] like Figure 36B As described, the maximum force increases by 30% after 4 weeks of Ab2 administration.

[0564] Although several embodiments of this disclosure have been described and illustrated herein, those skilled in the art will readily envision various other ways and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and such variations and / or modifications are each considered to be within the scope of this disclosure. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and constructions described herein are meant to be exemplary and that actual parameters, dimensions, materials, and / or constructions will depend on one or more specific applications used in the teachings of this disclosure. Those skilled in the art will recognize or be able to determine many equivalents to the specific embodiments of this disclosure described herein using only conventional experimentation. Therefore, it should be understood that the foregoing embodiments are given by way of example only, and that this disclosure may be practiced in ways other than those specifically described and claimed within the scope of the appended claims and their equivalents. This disclosure relates 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 included within the scope of this disclosure if such features, systems, articles, materials, and / or methods are not contradictory.

[0565] The indefinite articles “a” and “an” used in the description and claims herein shall be understood to mean “at least one (a)” unless the contrary is clearly indicated.

[0566] As used herein, the phrase “and / or” in the specification and claims should be understood to mean “any one or both” of the elements so linked (i.e., elements presented together in some cases and separately in others). Unless the contrary is explicitly stated, elements other than those specifically specified by the “and / or” clause may optionally be mentioned, whether or not they are related to these specifically specified elements. Thus, as a non-limiting example, the reference to “A and / or B” when used in conjunction with open-ended terms such as “comprising” may, in one embodiment, refer to A without B (optionally including elements other than B); in another embodiment, refer to B without A (optionally including elements other than A); in yet another embodiment, refer to both A and B (optionally including other elements); and so on.

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

[0568] As used herein, in the specification and claims, the phrase "at least one" relating to a series of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include each and every element specifically listed in the list of elements, nor exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically specified in the list of elements referred to by the phrase "at least one," whether or not they are related to those specifically specified elements. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B," or equivalently, "at least one of A and / or B"), in one embodiment, may refer to at least one A, optionally including more than one A, without B (and optionally including elements other than B); in another embodiment, may refer to at least one B, optionally including more than one B, without A (and optionally including elements other than A); in yet another embodiment, may refer to at least one A, optionally including more than one A, and at least one B, optionally including more than one B (and optionally including other elements); and so on.

[0569] In the claims and in the foregoing description, all conjunctions such as “comprising,” “including,” “with,” “having,” “containing,” “involving,” “accommodating,” etc., are understood to be open-ended, meaning including but not limited to. Only the conjunctions “consisting of…” and “consisting substantially of…” should be closed or semi-closed conjunctions, respectively, as shown in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0570] The use of ordinal terms such as “first,” “second,” “third,” etc., used to modify claim elements in claims does not imply any priority, precedence, or order of one claim element relative to another, or chronological order of actions of a method of execution. Rather, they are merely used as markers to distinguish one claim element with a specific name from another element with the same name (only for the use of ordinal terms) to differentiate claim elements.

Claims

1. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof for use in improving body composition and / or improving metabolic status in a subject having a myopathy, wherein the antibody or antigen-binding fragment thereof specifically binds to pro / latent myostatin but not to mature myostatin, wherein the antibody or antigen-binding fragment thereof comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein: a) CDRH1 consists of the amino acid sequence of SEQ ID NO: 1, CDRH2 consists of the amino acid sequence of SEQ ID NO: 4, CDRH3 consists of the amino acid sequence of SEQ ID NO: 10, CDRL1 consists of the amino acid sequence of SEQ ID NO: 12, CDRL2 consists of the amino acid sequence of SEQ ID NO: 18, and CDRL3 consists of the amino acid sequence of SEQ ID NO: 22, wherein the CDR sequences are numbered according to the Kabat numbering system; or b) CDRH1 consists of the amino acid sequence of SEQ ID NO: 2, CDRH2 consists of the amino acid sequence of SEQ ID NO: 5, CDRH3 consists of the amino acid sequence of SEQ ID NO: 10, CDRL1 consists of the amino acid sequence of SEQ ID NO: 13, CDRL2 consists of the amino acid sequence of SEQ ID NO: 19, and CDRL3 consists of the amino acid sequence of SEQ ID NO: 22, wherein the CDR sequences are numbered according to the Kabat numbering system.

2. The pharmaceutical composition of claim 1, wherein the antibody is a human or humanized antibody.

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

4. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment thereof inhibits the proteolytic formation of mature myostatin by BMP / tolloid proteases.

5. The pharmaceutical composition of claim 1, wherein the myopathy is a neuromuscular disorder.

6. The pharmaceutical composition of claim 1, wherein the myopathy is spinal muscular atrophy (SMA).

7. The pharmaceutical composition of claim 1, wherein the myopathy is amyotrophic lateral sclerosis (ALS).

8. The pharmaceutical composition of claim 1, wherein the myopathy is a muscular dystrophy.

9. The pharmaceutical composition of claim 8, wherein the muscular dystrophy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or facioscapulohumeral muscular dystrophy (FSHD).

10. The pharmaceutical composition of claim 1, wherein the composition is formulated for subcutaneous or intravenous administration. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 11. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment thereof has reduced affinity for myostatin / pro-myostatin at acidic pH as compared to physiological pH.

12. The pharmaceutical composition of claim 11, wherein the acidic pH ranges from 4.0-6.5 and the physiological pH ranges from 7.0-7.

4.

13. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment thereof comprises 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.

14. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 50 and a light chain comprising the amino acid sequence of SEQ ID NO:

51.

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

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