Anti-pro / latent type myostatin antibody, and method for using the same

Abstract

To provide an improved anti-myostatin therapeutic drug.SOLUTION: Provided is a pharmaceutical composition to be used in a method for treating the state of a muscle in a subject, where the pharmaceutical composition specifically binds to pro / latent type myostatin, contains antibody which inhibits release of mature myostatin or a fragment thereof, and the pharmaceutical composition causes two or more of (a) an increase in the amount of a muscle tissue and / or function in the subject; (b) a rise of the metabolic rate of the subject; (c) an increase in the insulin sensitivity of the subject; (d) a rise of the brown fat tissue level in the subject, and the like.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 276,698, filed January 8, 2016, U.S. Provisional Patent Application No. 62 / 328,597, filed April 27, 2016, U.S. Provisional Patent Application No. 62 / 333,816, filed May 9, 2016, U.S. Provisional Patent Application No. 62 / 333,810, filed May 9, 2016, U.S. Provisional Patent Application No. 62 / 413,278, filed October 26, 2016, and International Application No. PCT / US2016 / 052014, filed September 15, 2016, the entire contents of each of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on January 6, 2017, is named 127036-00320_SL.txt and is 108,683 bytes in size. [Background technology]

[0003] Myostatin, also known as GDF8, is a member of the TGFβ superfamily and belongs to a subfamily that includes two members: myostatin and GDF11. Like other members of the TGFβ superfamily, both myostatin and GDF11 are initially expressed as inactive precursor polypeptides (termed promyostatin and proGDF11, respectively).

[0004] Myostatin is a well-known negative regulator of skeletal muscle mass and is released from its autoinhibitory N-terminal prodomain by two separate protease cleavage steps. These cleavage events, which lead to the local release of "mature" myostatin from its inactive complex, can be referred to as supracellular activation. Following activation, mature myostatin signals by binding to a complex of type I and type II cell surface receptors (Alk4 / 5 and ActRIIB), whose downstream signaling induces muscle atrophy.

[0005] Myostatin is an interesting target for the treatment of muscle wasting. Several therapeutic agents targeting the ActRIIB signaling pathway have completed early-to-mid-stage clinical trials for muscle-wasting conditions, including sarcopenia, muscular dystrophy, cachexia, and hip replacement / hip fracture. However, to date, primary clinical strategies have focused on directly blocking the interaction between mature myostatin and cell surface receptors, and several therapeutic programs have been discontinued due to lack of specificity (leading to unacceptable toxicity) and / or lack of efficacy. Therefore, improved anti-myostatin therapeutic agents are needed. Summary of the Invention [Means for solving the problem]

[0006] The present invention encompasses the recognition that blocking the activation step of myostatin, rather than targeting already active myostatin, may provide an advantageous way to selectively inhibit myostatin signaling in vivo. The invention may have therapeutic utility for any condition in which selective reduction of myostatin signaling in vivo is beneficial. More particularly, the invention relates to the treatment of myostatin activation This includes the surprising finding that by specifically inhibiting myostatin, not only can an increase in muscle mass be achieved, but also enhanced muscle function and the prevention of muscle atrophy and metabolic dysregulation. Unexpectedly, beneficial therapeutic effects can also be achieved even under pathology in subjects with impaired, but not complete, signal transduction between neurons and their target tissues, such as target muscles. To the best of the inventors' knowledge, this is the first such demonstration achieved in vivo by inhibiting myostatin signaling, despite the fact that several antagonists have been developed to inhibit myostatin since its discovery some 20 years ago. Thus, in one aspect, the present invention also includes methods for treating conditions involving impaired signal transduction between motor neurons and their target muscles, including under pathology, by using myostatin inhibitors, such that a therapeutic benefit is achieved in a subject.

[0007] Thus, in one aspect, a method for inhibiting myostatin activation in a subject is provided, comprising administering to the subject a composition comprising a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin and blocks the release of mature myostatin, and the method results in the following in the subject: (a) an increase in muscle tissue mass and / or function in the subject; (b) an increase in the metabolic rate of the subject; (c) an increase in insulin sensitivity in the subject; (d) an increase in the level of brown adipose tissue in the subject; (e) an increase in the level of beige adipose tissue in the subject; (f) an increase in the level of erythrocytes in the subject; (g) a reduction in the level of white adipose tissue in the subject; (h) a reduction in the ratio of adipose tissue to muscle tissue in the subject; (i) an increase in glucose uptake by brown adipose tissue, beige adipose tissue, or muscle tissue in the subject; (j) a decrease in glucose uptake by white adipose tissue or hepatic tissue; (k) a decrease in muscle catabolism of protein and / or release of amino acids from muscle in the subject; (l) an increase in insulin-dependent blood glucose regulation in the subject; (m) a decrease in intramuscular fat infiltration in the subject; (n) a clinically meaningful improvement in a standardized quality of life test score; (o) prevention of muscle loss or muscle atrophy in the subject; and / or (p) prevention of the development of metabolic dysregulation associated with muscle dysfunction in the subject, wherein the subject is a human subject, e.g., a human subject who would benefit from reduced myostatin signaling.

[0008] In one embodiment, the method further comprises selecting a subject suffering from a muscle condition or disorder, hi another embodiment, the method further comprises selecting a subject suffering from or at risk of developing a metabolic disorder.

[0009] In one embodiment, administration of an effective amount of a myostatin inhibitor, e.g., an antibody or its antigen-binding fragment that specifically binds to pro / latent myostatin and blocks the activation of mature myostatin, results in an increase in the level of latent myostatin in the serum of a subject compared to a control.

[0010] In one embodiment, the subject has a muscle condition selected from the group consisting of myopathy, muscle atrophy, muscular dystrophy, and nerve damage. In one embodiment, the muscle atrophy is associated with a loss of motor neurons. In one embodiment, the loss comprises a genetic mutation. In another embodiment, the muscle atrophy is associated with spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), or myasthenia gravis. In one embodiment, the nerve damage comprises partial denervation of neurons that innervate muscles, or defective signaling between motor neurons and target muscles. In one embodiment, the nerve damage is a spinal cord injury (SCI). In another embodiment, the SCI is a partial / incomplete SCI. The location of the lesion in an SCI can be anywhere along the spinal cord. In one embodiment, the SCI is: i) T1-T6; ii) T7-L5; iii) C6-C7; iv) C5-C6; or or v) lesions between C3 and C8. In one embodiment, the subject is in the acute phase of SCI; the subacute phase of SCI; or the chronic phase of SCI. In one embodiment, the subject has or is at risk of developing a metabolic disorder associated with SCI. In one embodiment, the metabolic disorder is or includes insulin resistance, inflammation, abnormal lipid metabolism, or increased intramuscular fat infiltration. In one embodiment, the muscle atrophy includes glucocorticoid-induced muscle atrophy.

[0011] In one embodiment, the subject has a metabolic disease selected from the group consisting of type I diabetes, type II diabetes, obesity, metabolic syndrome / prediabetes, cardiovascular disease, nonalcoholic steatohepatitis (NASH), spinal cord injury (SCI), hypometabolic states, double diabetes, Cushing's disease, and obesity syndrome.

[0012] In one embodiment, the subject is treated with a second therapy. In one embodiment, the second therapy comprises a neuroprotective therapy. In another embodiment, the neuroprotective therapy comprises a stem cell therapy. In some embodiments, the neuroprotective therapy is an agent that promotes motor neuron survival. In some embodiments, the agent is olesoxime, riluzole, or thyrotropin-releasing hormone.

[0013] In another embodiment, the second treatment comprises splice-correction therapy. In one embodiment, the splice-correction therapy is a survival motor neuron (SMN) corrector. In some embodiments, the SMN corrector is an SMN2 splice corrector that increases expression of functional SMN protein in the subject.

[0014] In some embodiments, the SMN2 splicing corrector is an antisense molecule. In some embodiments, the SMN2 splicing corrector is Spinraza® (Nusinersen). In some embodiments, the antisense molecule is administered by intravenous or intrathecal injection. However, other suitable routes of administration may be used. In some embodiments, the SMN2 splicing corrector is a small molecule. In some embodiments, the SMN2 splicing corrector is RG7800 (Roche), RG7916 (Roche), or LMI070 (Novartis). In some embodiments, the small molecule is administered orally or by another suitable method.

[0015] In another aspect, the present specification discloses a method for treating or preventing a disease associated with defective neural signaling between neurons and a target tissue, e.g., a target muscle, in a human subject, the method comprising the steps of selecting a human subject suffering from a disease associated with defective neural signaling between neurons and the target tissue; and administering to the human subject a composition comprising a myostatin inhibitor, e.g., an antibody or its antigen-binding fragment that specifically binds to pro / latent myostatin and blocks the release of mature myostatin, in an amount effective to treat or prevent the disease, thereby treating or preventing the disease associated with defective neural signaling in the human subject.

[0016] In some embodiments, the target tissue expresses myostatin (e.g., myostatin precursor and / or mature myostatin). In one embodiment, the target tissue is selected from the group consisting of muscle tissue, adipose tissue, brain tissue, liver tissue, and vascular tissue. In one embodiment, the target tissue is muscle.

[0017] In another aspect, disclosed herein are methods for treating a pathology that causes impaired but not complete loss of signal transduction between neurons and target muscles in a subject. Such methods include administering to the subject a myostatin inhibitor, e.g., an anti-pro / latent myostatin inhibitor. The method includes administering a composition comprising the body in an amount effective to treat the muscle located under the lesion in a subject. In some embodiments, the amount is effective to prevent muscle loss or muscle atrophy under the lesion in the subject. In some embodiments, the amount is effective to increase the muscle mass and / or function under the lesion in the subject.

[0018] In some embodiments, the pathology is associated with an incomplete spinal cord injury.

[0019] In one embodiment, the muscle contains fast-twitch muscle fibers. In another embodiment, the muscle underlying the lesion is selected from the group consisting of soleus, gastrocnemius, biceps, and triceps. In one embodiment, the amount is effective to increase the mass and / or function of the muscle above the lesion in the subject. In another embodiment, the myostatin inhibitor is an agent that blocks, antagonizes, or inhibits myostatin signaling in vivo. In some embodiments, such an agent is an antibody or antigen-binding portion thereof, a small molecule, or gene therapy. In some embodiments, the antibody specifically binds to pro / latent myostatin in vivo and blocks the release of mature myostatin. In some embodiments, the antibody binds to mature myostatin. In some embodiments, the antibody selectively (e.g., preferentially) binds to mature myostatin over mature GDF11. In some embodiments, the antibody specifically binds to mature myostatin but does not bind to mature GDF11. In some embodiments, the antibody binds to and / or blocks the myostatin receptor.

[0020] In some embodiments, the lesion is a spinal cord lesion. In one embodiment, the subject has an incomplete spinal cord injury (SCI). In one embodiment, the incomplete SCI comprises a lesion between i) T1-T6; ii) T7-L5; iii) C6-C7; iv) C5-C6; or v) C3-C8.

[0021] In one embodiment, the amount is effective to treat a metabolic condition in the subject. In one embodiment, the amount is effective to: (a) increase the mass and / or function of muscle tissue in the subject; (b) increase the metabolic rate of the subject; (c) increase insulin sensitivity in the subject; (d) increase the level of brown adipose tissue in the subject; (e) increase the level of beige adipose tissue in the subject; (f) decrease the level of white adipose tissue in the subject; (g) decrease the level of visceral adipose tissue in the subject; (h) decrease the ratio of adipose tissue to muscle tissue in the subject; (i) decrease glucose uptake by brown adipose tissue, beige adipose tissue, or muscle tissue in the subject. (j) increasing glucose uptake by white adipose tissue or hepatic tissue; (k) decreasing muscle catabolism of protein and / or muscle release of amino acids in a subject; (l) increasing insulin-dependent blood glucose regulation in a subject; (m) decreasing intramuscular fat infiltration in a subject; (n) producing a clinically meaningful improvement in a standardized quality of life test score; (o) preventing muscle loss or muscle atrophy in a subject; and / or (p) preventing the development of metabolic dysregulation associated with muscle dysfunction in a subject.

[0022] In another aspect, the present disclosure provides a method for treating or preventing a metabolic disease in a human subject, comprising the steps of selecting a human subject suffering from a metabolic disease; and administering to the human subject an effective amount of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, thereby treating or preventing the metabolic disease in the human subject.

[0023] In one embodiment, the metabolic disease is selected from the group consisting of type I diabetes, type II diabetes, obesity, metabolic syndrome / prediabetes, cardiovascular disease, nonalcoholic steatohepatitis (NASH), spinal cord injury (SCI), hypometabolic states, double diabetes, Cushing's disease, and obesity syndrome. In one embodiment, the obesity is sarcopenic obesity. In one embodiment, the hypometabolic condition is selected from the group consisting of conditions associated with prolonged immobilization, conditions associated with bed rest, conditions associated with casting, conditions associated with stroke, conditions associated with amputation, and post-surgical conditions. In one embodiment, the Cushing's disease is selected from the group consisting of corticosteroid-induced Cushing's disease and tumor-induced Cushing's disease. In one embodiment, the obesity syndrome is selected from the group consisting of Prader-Willi syndrome, obesity syndrome associated with a genetic disorder, and obesity syndrome associated with a hypothalamic disorder.

[0024] In another aspect, the present disclosure provides a method for treating or preventing a disease associated with defective neural signaling between neurons and a target tissue in a human subject, the method comprising the steps of selecting a human subject suffering from a disease associated with defective neural signaling between neurons and a target tissue; and administering to the human subject an effective amount of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, thereby treating or preventing the disease associated with defective neural signaling in the human subject. In some embodiments, the target tissue expresses myostatin (e.g., myostatin precursor and / or mature myostatin).

[0025] In one embodiment, the disease associated with defective neural signaling between neurons and target tissues is selected from the group consisting of spinal cord injury (SCI), myasthenia gravis, amyotrophic lateral sclerosis (ALS), and spinal muscular atrophy (SMA). In one embodiment, the disease associated with defective neural signaling between neurons and target tissues is spinal cord injury (SCI). In one embodiment, the human subject is in an acute spinal cord injury (SCI) stage. In one embodiment, the human subject is in a subacute spinal cord injury (SCI) stage. In one embodiment, the human subject is in a chronic spinal cord injury (SCI) stage.

[0026] In one aspect, the disclosure provides a method for promoting fiber type switching in a subject, comprising administering to the subject a composition comprising a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin and blocks release of mature myostatin, in an amount effective to promote fiber type switching, thereby promoting fiber type switching in the subject.

[0027] In another aspect, the disclosure provides a method for preferentially increasing type II or fast-twitch muscle fibers over type I or slow-twitch muscle fibers in a subject. The method includes administering to the subject a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin and blocks release of mature myostatin, in an amount effective to preferentially increase type II or fast-twitch muscle fibers over type I or slow-twitch muscle fibers, thereby preferentially increasing type II or fast-twitch muscle fibers over type I or slow-twitch muscle fibers in the subject.

[0028] In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases the mass and / or function of muscle tissue in a human subject. In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases the mass and / or function of fast-twitch muscle tissue in a human subject. In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases the mass and / or function of slow-twitch muscle tissue in a human subject.

[0029] In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases the metabolic rate of a human subject. In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases insulin sensitivity in a human subject. In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases insulin sensitivity in a human subject. Administration of the myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases the level of brown adipose tissue in a human subject. In one embodiment, administration of the myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases the level of beige adipose tissue in a human subject. In one embodiment, administration of the myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, decreases the level of white adipose tissue in a human subject. In one embodiment, administration of the myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, decreases the level of visceral adipose tissue in a human subject. In one embodiment, administration of the myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, decreases the ratio of adipose tissue to muscle tissue in a human subject.

[0030] In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases glucose uptake by muscle tissue in a human subject. In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, decreases glucose uptake by a target tissue, wherein the target tissue is selected from the group consisting of white adipose tissue, liver tissue, and vascular tissue.

[0031] In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, reduces muscle catabolism of proteins and / or release of amino acids from muscles in a human subject. In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases insulin-dependent blood glucose regulation in a human subject.

[0032] In another aspect, disclosed herein is a method for increasing metabolic rate in a human subject, comprising the steps of selecting a human subject who is likely to benefit from an increased metabolic rate; and administering to the human subject an effective amount of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, thereby increasing the metabolic rate in the human subject.

[0033] In another aspect, disclosed herein is a method for increasing the level of brown adipose tissue in a human subject, comprising the steps of selecting a human subject who would benefit from increasing the level of brown adipose tissue; and administering to the human subject an effective amount of a myostatin inhibitor, for example, an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, thereby increasing the level of brown adipose tissue in the human subject.

[0034] In another aspect, disclosed herein is a method for increasing the level of beige adipose tissue in a human subject, comprising the steps of selecting a human subject who would benefit from increasing the level of beige adipose tissue; and administering to the human subject an effective amount of a myostatin inhibitor, for example, an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, thereby increasing the level of beige adipose tissue in the human subject.

[0035] In another aspect, disclosed herein is a method for increasing insulin-dependent blood glucose control in a human subject, comprising the steps of selecting a human subject who would benefit from increased insulin-dependent blood glucose control; and administering to the human subject an effective amount of a myostatin inhibitor, for example, an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, thereby increasing insulin-dependent blood glucose control in the human subject.

[0036] In another aspect, a method for reducing muscle protein catabolism and / or muscle amino acid release in a human subject comprises the steps of selecting a human subject who would benefit from reduced muscle protein catabolism and / or muscle amino acid release; and administering to the human subject a myostatin inhibitor, e.g., a compound that inhibits pro / latent myostatin. Disclosed herein are methods comprising administering an effective amount of an antibody or antigen-binding fragment thereof that specifically binds to a human subject, thereby reducing muscle catabolism of protein and / or muscle release of amino acids.

[0037] In another aspect, disclosed herein is a method for reducing glucose uptake by a target tissue in a human subject, comprising the steps of selecting a human subject who would benefit from reduced glucose uptake by a target tissue selected from the group consisting of white adipose tissue, hepatic tissue, and vascular tissue; and administering to the human subject an effective amount of a myostatin inhibitor, for example, an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, thereby reducing glucose uptake by the target tissue in the human subject.

[0038] In one embodiment, the target tissue comprises macrophages, smooth muscle cells and foam cells.

[0039] In another aspect, a method of treating or preventing a metabolic disease in a human subject includes the steps of selecting a human subject suffering from a metabolic disease; and administering to the human subject a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, to the human subject, resulting in: (a) an increase in muscle tissue mass and / or function in the human subject; (b) an increase in metabolic rate in the human subject; (c) an increase in insulin sensitivity in the human subject; (d) an increase in the level of brown adipose tissue in the human subject; (e) an increase in the level of beige adipose tissue in the human subject; or (f) an increase in white adipose tissue in the human subject. (g) a decrease in the level of visceral adipose tissue in the human subject; (h) a decrease in the ratio of adipose tissue to muscle tissue in the human subject; (i) an increase in glucose uptake by white adipose tissue, hepatic tissue, or vascular tissue in the human subject; (j) a decrease in muscle catabolism of protein and / or release of amino acids from muscle in the human subject; and / or (k) an increase in insulin-dependent blood glucose regulation in the human subject, thereby treating or preventing a metabolic disease in the human subject.

[0040] In another aspect, disclosed herein is a method for preventing sub-lesional muscle atrophy in a subject suffering from a pathology, the method comprising the steps of selecting a subject suffering from a pathology and administering to the human subject an effective amount of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin. Such a subject may be at risk of developing muscle dysfunction, e.g., atrophy.

[0041] In one embodiment, the lesion results from a spinal cord injury (SCI). In one embodiment, the human subject is in an acute spinal cord injury (SCI) phase. In one embodiment, the human subject is in a subacute spinal cord injury (SCI) phase. In some embodiments, the SCI is an incomplete SCI, characterized by partial preservation of function of the affected nerve and / or target muscle(s). In some embodiments, the subject suffers from an incomplete SCI with partial denervation of motor neurons and is in the acute or subacute phase of SCI. In some embodiments, the subject is within 6 months of SCI, e.g., within 5 months, 4 months, 3 months, 2 months, 4 weeks, or 2 weeks after SCI. In some embodiments, the subject does not exhibit atrophy and / or muscle loss beneath the lesion.

[0042] In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, further increases the mass and / or function of muscle above the lesion. In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases the mass and / or function of fast switch muscles. In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases the mass and / or function of slow switch muscles. The amount and / or function of the slow switch muscle increases.

[0043] In some embodiments, the amount of muscle tissue is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the amount of muscle tissue is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0044] In some embodiments, muscle tissue function is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, muscle tissue function is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0045] In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases locomotor function in a human subject. In some embodiments, the locomotor function of the human subject is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the locomotor function of the human subject is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0046] In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases motor coordination and balance in a human subject. In some embodiments, the motor coordination and balance of a human subject is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the motor coordination and balance of a human subject is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0047] In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases muscle strength in a human subject. In some embodiments, the muscle strength of a human subject increases by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the muscle strength of a human subject increases by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0048] In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases grip strength in a human subject. In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, reduces white adipose tissue levels in a human subject. In some embodiments, white adipose tissue levels are reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 82 In other embodiments, the level of white adipose tissue is reduced by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0049] In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, results in an increase in total body weight in a human subject. In some embodiments, the level of total body weight is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the level of total body weight is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0050] In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases the metabolic rate of a human subject. In some embodiments, the metabolic rate is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the metabolic rate is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0051] In one embodiment, the muscle is selected from the group of soleus, gastrocnemius, biceps and triceps.

[0052] In one embodiment, the myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, is administered to the human subject within less than 5, 10, 20, 30, 40, 50, or 60 minutes after the human subject has acquired a lesion. In one embodiment, the myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, is administered to the human subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 24 hours after the human subject has acquired a lesion. In one embodiment, the myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, is administered to the human subject within at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, or within at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 24 months, 48 ​​months, or 60 months of the human subject developing a lesion. In one embodiment, the myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, is administered to the human subject for about 1 to 30 days, about 1 to 50 days, about 1 to 100 days, about 1 to 200 days, or about 1 to 300 days.

[0053] In one embodiment, the myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, is administered chronically to a human subject. In one embodiment, the myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, is administered to a human subject at a dose ranging from 0.01 mg / kg to 100 mg / kg. In one embodiment, the myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, is administered to a human subject intraperitoneally, intravenously, intramuscularly, topically, or subcutaneously.

[0054] In one embodiment, the methods disclosed herein further comprise administering a second treatment to the human subject. In one embodiment, the second treatment is insulin, an insulin-sensitive Sex enhancers, alpha-glucosidase inhibitors, biguanides, sulfonylureas, insulin secretagogues, amylin agonists, phosphotyrosine phosphatase inhibitors, aldose reductase inhibitors, neurotrophic factors, PKC inhibitors, advanced glycation endproduct (AGE) inhibitors, active oxygen quenching agents, statins, squalene synthetase inhibitors, fibrates, niacin, PCSK9 inhibitors, triglyceride-lowering agents, cholesterol sequestrants, angiotensin-converting enzyme inhibitors, angiotensin II antagonists, calcium channel blockers, ursodiol, pioglitazone, orlistat, betaine, rosiglitazone, central anti-obesity agents agent), gastrointestinal lipase inhibitor, beta3-adrenergic receptor agonist, peptide-based appetite suppressant, cholecystokinin agonist, dopamine agonist, DPP-4 inhibitor, glucagon-like peptide, meglitinide, sulfonylurea, sodium glucose transporter (SGLT) 2 inhibitor, cyclooxygenase inhibitor, progesterone derivative, metoclopramide-based agent, tetrahydrocannabinol-based agent, and lipid metabolism improver.

[0055]

[0010] In another aspect, disclosed herein is a method of treating a subject, the method comprising the steps of: selecting a human subject who exhibits either i) elevated levels of promyostatin in target muscles compared to control levels of promyostatin, or ii) decreased levels of circulating latent myostatin compared to control levels of latent myostatin; and administering to the human subject a therapeutically effective amount of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin and blocks the release of mature myostatin, thereby treating the subject. In one embodiment, the subject exhibits both i) and ii).

[0056] In one embodiment, the administering step results in an increase in the level of latent myostatin in the target muscle after the administering step. In one embodiment, the increase in latent myostatin in the target muscle after the administering step is detectable within 48 hours after the administering step. In one embodiment, the increase in latent myostatin in the target muscle after the administering step is detectable for at least 28 days after the administering step. In one embodiment, the level of latent myostatin in the target muscle after the administering step is increased by at least 1.2-fold compared to the level of latent myostatin in the target muscle before the administering step.

[0057] In one embodiment, the administering step results in an increase in the level of latent myostatin in the subject's circulation after the administering step. In one embodiment, the increase in latent myostatin in the subject's circulation after the administering step is detectable within 48 hours after the administering step. In one embodiment, the increase in latent myostatin in the subject's circulation after the administering step is detectable for at least 28 days after the administering step. In one embodiment, the level of latent myostatin in the subject's circulation after the administering step is at least two-fold higher than the level of latent myostatin in the circulation before the administering step.

[0058] In one embodiment, the selecting step comprises determining the level of promyostatin in the target muscle. In one embodiment, the selecting step comprises determining the level of latent myostatin in the circulation.

[0059] In one embodiment, the method further comprises determining the level of promyostatin in the target muscle after the administering step. In one embodiment, the method further comprises determining the level of circulating latent myostatin after the administering step. In one embodiment, the level of promyostatin in the target muscle is determined by obtaining a muscle tissue sample from the subject and determining the level of promyostatin in the muscle tissue sample. In one embodiment, the level of circulating latent myostatin is determined by obtaining a blood sample from the subject and determining the level of latent myostatin in the blood sample. This is determined by determining the level of eostatin.

[0060] In one embodiment, the target muscle is a fast-twitch muscle. In one embodiment, the target muscle comprises fast oxidative fibers.

[0061] In one embodiment, the administering step comprises a single dose of the myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, hi one embodiment, the administering step comprises at least two doses of the myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof.

[0062] In one embodiment, a loss of muscle mass in the subject is prevented. In one embodiment, the subject exhibits an increase in muscle mass after the administering step. In one embodiment, the subject exhibits an increase in muscle function after the administering step.

[0063] In one embodiment, the subject is a human subject.

[0064] In another aspect, disclosed herein is a method for preventing muscle mass loss and / or increasing muscle mass in a human subject, comprising administering to the human subject a single dose of a therapeutically effective amount of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin and blocks the release of mature myostatin, wherein the subject exhibits a sustained increase in muscle mass for at least 8 weeks after administration of the dose, thereby preventing muscle mass loss and / or increasing muscle mass in a human subject. In one embodiment, the subject exhibits a progressive increase in muscle mass for at least 12 weeks after administration. In one embodiment, the subject exhibits a sustained increase in muscle mass for at least 16 weeks after administration.

[0065] In another aspect, disclosed herein is a method of preventing muscle mass loss and / or increasing muscle mass in a human subject, comprising administering to the human subject more than two doses, including at least a first and a second dose, of a therapeutically effective amount of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin and blocks the release of mature myostatin, wherein the first and second doses are administered to the subject at least about four weeks apart, and the subject exhibits a sustained increase in muscle mass for at least eight weeks after the first dose, thereby preventing muscle mass loss and / or increasing muscle mass in the human subject. In one embodiment, the first and second doses are administered to the subject at least about eight weeks apart.

[0066] In one embodiment, the subject is one who would benefit from increased muscle mass and / or increased muscle function. In one embodiment, the subject has or is at risk of developing a myopathy. In one embodiment, the myopathy is spinal cord injury. In one embodiment, the myopathy is a secondary myopathy. In one embodiment, the secondary myopathy is denervation, genetic muscle weakness, or cachexia.

[0067] In one embodiment, the subject has muscle atrophy or is at risk of developing muscle atrophy. In one embodiment, the muscle atrophy is glucocorticoid-induced muscle atrophy. In one embodiment, the glucocorticoid-induced muscle atrophy is muscle atrophy induced by cortisol (hydrocortisone), cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate (doca), or aldosterone.

[0068] In one embodiment, the subject receives a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof. Administration of the fragment is followed by administration of a glucocorticoid, hi one embodiment, the glucocorticoid is administered at a dose sufficient to induce a significant decrease in lean body mass in a control subject who has not received the myostatin inhibitor, e.g., an antibody or antigen-binding portion thereof.

[0069] In one embodiment, the antibody or antigen-binding fragment thereof is administered at a dose of about 0.01 mg / kg to about 30 mg / kg. In one embodiment, the myostatin inhibitor, e.g., antibody or antigen-binding fragment thereof, is administered intraperitoneally, intravenously, intramuscularly, or subcutaneously.

[0070] In one embodiment, the myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, inhibits myostatin signaling in a subject. In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to pro / latent myostatin and does not bind to mature myostatin. In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to pro / latent myostatin and does not bind to another member of the transforming growth factor beta family. In one embodiment, the member of the transforming growth factor beta family is GDF11 or activin. In one embodiment, the antibody or antigen-binding fragment thereof does not bind to GDF11.

[0071] In one embodiment, the antibody or antigen-binding fragment thereof cross-reacts with human and murine pro / latent myostatin. In one embodiment, the antibody or antigen-binding fragment thereof inhibits the formation of mature myostatin via proteolysis by thrombopoietin. In one embodiment, the antibody or antigen-binding fragment thereof inhibits the formation of mature myostatin via proteolysis by thrombopoietin with an IC50 of less than 1 μM.

[0072] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising a complementarity determining region 3 (CDRH3) comprising the sequence set forth in any one of SEQ ID NOs: 10-11 and 66. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising a complementarity determining region 3 (CDRL3) comprising the sequence set forth in any one of SEQ ID NOs: 22-23 and 67. In one embodiment, the antibody or antigen-binding fragment thereof comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises the sequence set forth in any one of SEQ ID NOs: 1 to 3, CDRH2 comprises the sequence set forth in any one of SEQ ID NOs: 4 to 9, CDRH3 comprises the sequence set forth in any one of SEQ ID NOs: 10 to 11 and 66, CDRL1 comprises the sequence set forth in any one of SEQ ID NOs: 12 to 17, CDRL2 comprises the sequence set forth in any one of SEQ ID NOs: 18 to 21, and CDRL3 comprises the sequence set forth in any one of SEQ ID NOs: 22 to 23 and 67.

[0073] In one embodiment, CDRH1 comprises the sequence set forth in SEQ ID NO: 1 or 2, CDRH2 comprises the sequence set forth in SEQ ID NO: 4 or 5, CDRH3 comprises the sequence set forth in SEQ ID NO: 10, CDRL1 comprises the sequence set forth in SEQ ID NO: 12 or 13, CDRL2 comprises the sequence set forth in SEQ ID NO: 18 or 19, and CDRL3 comprises the sequence set forth in SEQ ID NO: 22.

[0074] In one embodiment, CDRH1 comprises the sequence set forth in SEQ ID NO: 1 or 3, CDRH2 comprises the sequence set forth in SEQ ID NO: 6 or 7, CDRH3 comprises the sequence set forth in SEQ ID NO: 11, CDRL1 comprises the sequence set forth in SEQ ID NO: 14 or 15, CDRL2 comprises the sequence set forth in SEQ ID NO: 20 or 21, and CDRL3 comprises the sequence set forth in SEQ ID NO: 23.

[0075] In one embodiment, CDRH1 comprises the sequence set forth in SEQ ID NO: 1 or 3, and CDRH2 comprises the sequence set forth in SEQ ID NO: 8 or 9, CDRH3 comprises the sequence set forth in SEQ ID NO: 11, CDRL1 comprises the sequence set forth in SEQ ID NO: 16 or 17, CDRL2 comprises the sequence set forth in SEQ ID NO: 20 or 21, and CDRL3 comprises the sequence set forth in SEQ ID NO: 23.

[0076] In one embodiment, CDRH1 comprises the sequence set forth in SEQ ID NO: 1 or 2, CDRH2 comprises the sequence set forth in SEQ ID NO: 4 or 5, CDRH3 comprises the sequence set forth in SEQ ID NO: 66, CDRL1 comprises the sequence set forth in SEQ ID NO: 12 or 13, CDRL2 comprises the sequence set forth in SEQ ID NO: 18 or 19, and CDRL3 comprises the sequence set forth in SEQ ID NO: 67.

[0077] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain sequence set forth in any one of SEQ ID NOs: 24 to 29. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable domain sequence set forth in any one of SEQ ID NOs: 30 to 35. In one embodiment, 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.

[0078] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 50. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 51.

[0079] In one embodiment, the antibody or antigen-binding fragment thereof competes with any other antibody described herein for binding to pro / latent myostatin, hi one embodiment, the antibody or antigen-binding fragment thereof binds to pro / latent myostatin at the same epitope as the antibodies described herein.

[0080] In one embodiment, the antibody or antigen-binding fragment thereof is -6The antibody competes for binding to pro / latent myostatin with an equilibrium dissociation constant, Kd, ​​between the antibody and pro / latent myostatin of less than M. In one embodiment, the Kd is 10 -11 M~10 -6 It is in the range of M.

[0081] In one embodiment, the antibody or antigen-binding fragment thereof is a human antibody, a humanized antibody, a diabody, a chimeric antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. In one embodiment, the antibody is a humanized antibody. In one embodiment, the antibody is a human antibody. In one embodiment, the antibody or antigen-binding fragment thereof comprises a framework having human germline sequences.

[0082] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant domain selected from the group consisting of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE constant domains. In 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, creating an IgG1-like hinge and allowing interchain disulfide bond formation. In one embodiment, the antibody or antigen-binding portion thereof does not bind to GDF11. [Brief explanation of the drawings]

[0083] [Figure 1] Figures 1A-1B show the domain structure and promyostatin assembly of myostatin. Figure 1A shows myostatin secreted as a proprotein, with an inhibitory prodomain followed by a C-terminal growth factor domain that exists as a disulfide-linked dimer. Figure 1B shows the assembled precursor protein in an inactive conformation, in which the prodomain (dark gray) encapsulates the growth factor (light gray) via a "straight jacket" assembly. This figure is an adaptation of the structure of latent TGFβ1 (Shi et al., Nature, 2011).

[0084] [Figure 2] Figure 2 shows that myostatin activation involves two distinct protease events, generating three major myostatin species. The biosynthetic precursor protein, promyostatin, is processed by two distinct proteases. Cleavage of promyostatin (and proGDF11) is carried out by proprotein convertases, such as furin / PACE3 (Paired Basic Amino Acid Cleaving Enzyme 3) or PCSK5 (Proprotein Convertase Subtilisin / Kexin type 5), which cleave at the conserved RXXR site between the prodomain and the mature growth factor. This cleavage generates a latent complex in which the mature growth factor is shielded from binding to its receptor by the prodomain. Activation and release of the active growth factor are achieved after cleavage by additional proteases of the BMP / tolloid family, such as TLL-2 (tolloid-like protein 2) or BMP1 (bone morphogenetic protein 1). These cleavage events produce the mature form of myostatin, which may be referred to as active or mature myostatin.

[0085] [Figure 3AB]Figures 3A-3C show that Ab1 blocks promyostatin cleavage by members of the tolloid family of proteases. Latent myostatin samples preincubated with increasing amounts of Ab1 were analyzed in a myostatin activation assay. After analysis of myostatin release by reporter assay (Figure 3A), samples were then run under reducing conditions and probed by Western blot with an antibody raised against the myostatin prodomain (Figure 3B). The approximately 18 kDa band (boxed), corresponding to the ARM portion of the prodomain generated after tolloid cleavage, decreased proportionally with increasing Ab1 doses. Latent and promyostatin standards (45 ng loaded) show that promyostatin migrated at approximately 50 kDa and the prodomain at approximately 37 kDa. Figure 3C shows that myostatin activation involves two distinct protease events, generating three major myostatin species. The biosynthetic precursor protein, promyostatin, is processed by two distinct proteases. Cleavage of promyostatin (and proGDF11) is carried out by proprotein convertases such as furin / PACE3 (Paired Basic Amino acid Cleaving Enzyme 3) or PCSK5 (Proprotein Convertase Subtilisin / Kexin type 5), which cleave at a conserved RXXR site between the prodomain and the mature growth factor. This cleavage generates a latent complex in which the mature growth factor is shielded from binding to its receptor by the prodomain. See Figure 3B, which illustrates the possible inhibition of the tolloid protease and blocks further cleavage of promyostatin. Activation and release of the active growth factor are achieved after cleavage by additional proteases of the BMP / tolloid family, such as TLL-2 (tolloid-like protein 2) or BMP1 (bone morphogenetic protein 1). [Figure 3C]Figures 3A-3C show that Ab1 blocks promyostatin cleavage by members of the tolloid family of proteases. Latent myostatin samples preincubated with increasing amounts of Ab1 were analyzed in a myostatin activation assay. After analysis of myostatin release by reporter assay (Figure 3A), samples were then run under reducing conditions and probed by Western blot with an antibody raised against the myostatin prodomain (Figure 3B). The approximately 18 kDa band (boxed), corresponding to the ARM portion of the prodomain generated after tolloid cleavage, decreased proportionally with increasing Ab1 doses. Latent and promyostatin standards (45 ng loaded) show that promyostatin migrated at approximately 50 kDa and the prodomain at approximately 37 kDa. Figure 3C shows that myostatin activation involves two distinct protease events, generating three major myostatin species. The biosynthetic precursor protein, promyostatin, is processed by two distinct proteases. Cleavage of promyostatin (and proGDF11) is carried out by proprotein convertases such as furin / PACE3 (Paired Basic Amino acid Cleaving Enzyme 3) or PCSK5 (Proprotein Convertase Subtilisin / Kexin type 5), which cleave at a conserved RXXR site between the prodomain and the mature growth factor. This cleavage generates a latent complex in which the mature growth factor is shielded from binding to its receptor by the prodomain. See Figure 3B, which illustrates the possible inhibition of the tolloid protease and blocks further cleavage of promyostatin. Activation and release of the active growth factor are achieved after cleavage by additional proteases of the BMP / tolloid family, such as TLL-2 (tolloid-like protein 2) or BMP1 (bone morphogenetic protein 1).

[0086] [Figure 4]Figure 4 shows the performance of the parent Ab1 antibody and other candidates in a cell-based reporter assay. After overnight proteolytic reactions with enzymes from both the proprotein convertase and thrombin protease families, the release of mature growth factors was measured by a CAGA-based reporter assay in 293T cells. Results were compared to control reactions, and the percentage of pro-myostatin or pro-GDF11 released during the assay was calculated. The standard deviations of the means of three replicates are shown; however, due to small values, most data points are not visible on the graph.

[0087] [Figure 5] FIG. 5 graphically shows that Ab1, Ab2, Ab4, and Ab6 antibodies do not inhibit pro-GDF11 activation.

[0088] [Figure 6] Figure 6 shows the results of an assay assessing mean percent weight change. Animals were weighed daily to calculate percent weight change from day 0. Data represent group mean ± SEM. Mean percent change data for each group on study day 42 were analyzed using one-way ANOVA followed by a Holm-Sidak post-hoc test in comparison to the PBS control group. **p<0.01.

[0089] [Figure 7] Figures 7A-7D show the results of assays assessing tissue weight. Figure 7A shows the mean gastrocnemius muscle weight. Figure 7B shows the mean pectoralis muscle weight. Figure 7C shows the mean soleus muscle weight. Figure 7D shows the mean triceps muscle weight. Statistical evaluation was performed using one-way ANOVA followed by a Holm-Sidak post-hoc test in comparison with the vehicle control group (Group 1). Data represent group means ± SEM. **p<0.01. Bar graphs show Groups 1-5 from left to right.

[0090] [Figure 8]Figures 8A-8C show the results of assays assessing tissue weight. Figure 8A shows the mean tibialis anterior muscle weight. Figure 8B shows the mean diaphragm weight. Figure 8C shows the mean quadriceps muscle weight. Statistical evaluation was performed using one-way ANOVA followed by a Holm-Sidak post-hoc test in comparison with the vehicle control group (Group 1). Data represent group means ± SEM. *p<0.05. Bar graphs show Groups 1-5 from left to right.

[0091] [Figure 9] Figures 9A-9B show the results of assays assessing mean percent body weight change and mean percent lean mass change. Figure 9A is a graph showing the calculated percent body weight change from day 0 for animals weighed twice weekly throughout the study. In Figure 9B, animals underwent EchoMRI (QNMR) to measure body composition on days -4, 7, 14, 21, and 28, and percent lean mass change from day -1 was calculated. Data represent group means ± SEM. For both body weight and lean mass, mean percent change data for each group at day 28 of the study were analyzed using one-way ANOVA followed by a Holm-Sidak post-hoc test in comparison to the IgG control group (Group 2). ***p<0.0005, **p<0.005, *p<0.05, ns (not significant).

[0092] [Figure 10AB] Figures 10A-10D are graphs showing the results of an assay assessing muscle weight. Figure 10A shows the mean quadriceps (rectus femoris) weight, Figure 10B shows the mean gastrocnemius weight, Figure 10C shows the mean tibialis anterior weight, and Figure 10D shows the mean diaphragm weight. The percentage difference in mean muscle weight for the Ab1-treated group compared to the IgG control group is indicated above each bar. Statistical evaluation was performed using one-way ANOVA followed by a Holm-Sidak post-hoc test in comparison with the IgG control group (Group 2). Data represent group means ± SEM. ****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05, ns (not significant). [Figure 10CD]Figures 10A-10D are graphs showing the results of an assay assessing muscle weight. Figure 10A shows the mean quadriceps (rectus femoris) weight, Figure 10B shows the mean gastrocnemius weight, Figure 10C shows the mean tibialis anterior weight, and Figure 10D shows the mean diaphragm weight. The percentage difference in mean muscle weight for the Ab1-treated group compared to the IgG control group is indicated above each bar. Statistical evaluation was performed using one-way ANOVA followed by a Holm-Sidak post-hoc test in comparison with the IgG control group (Group 2). Data represent group means ± SEM. ****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05, ns (not significant).

[0093] [Figure 11] Figures 11A-11B show the results of assays assessing mean percent body weight change and mean percent lean mass change. Figure 11A shows percent body weight change from day 0 calculated from animals weighed twice weekly throughout the study. (Figure 11B) Animals underwent EchoMRI (QNMR) to measure body composition on days -1, 6, and 13, and percent lean mass change from day -1 was calculated. PBS = phosphate-buffered saline; Dex = dexamethasone; IgG(20) = IgG control antibody administered at 20 mg / kg / week; Ab1(20) = Ab1 antibody administered at 20 mg / kg / week; and Ab1(2) = Ab1 antibody administered at 2 mg / kg / week. Data represent group means ± SEM. The mean percent change data for each group on day 14 (for body weight) and day 13 (for lean mass) were analyzed using one-way ANOVA followed by Dunnett's multiple comparison test for Group 1 (****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05) and Group 5 (++++p<0.0001, +++p<0.0005, ++p<0.005, +p<0.05). ns (not significant).

[0094] [Figure 12AB]Figures 12A-12D are graphs showing the results of an assay assessing different muscle weights. Figure 12A shows the mean gastrocnemius muscle weight (grams), Figure 12B shows the mean quadriceps (rectus femoris) muscle weight (grams), Figure 12C shows the mean gastrocnemius muscle weight change compared to control animals treated with PBS (IP) and regular drinking water (Group 1), and Figure 12D shows the mean quadriceps (rectus femoris) muscle weight change compared to control animals treated with PBS (IP) and regular drinking water (Group 1). PBS = phosphate-buffered saline, Dex = dexamethasone, IgG(20) = IgG control antibody administered at 20 mg / kg / week, Ab1(20) = Ab1 antibody administered at 20 mg / kg / week, and Ab1(2) = Ab1 antibody administered at 2 mg / kg / week. For Figures 12A-12B, error bars represent standard deviation (SD). For Figures 12C-12D, error bars represent the standard error of the mean (SEM). Statistical evaluation was performed using one-way ANOVA followed by Dunnett's multiple comparison test for Group 1 (****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05) and Group 5 (++++p<0.0001, +++p<0.0005, ++p<0.005, +p<0.05). ns (not significant). Bars, from left to right, show PBS, water; PBS, dex; IgG control; Ab1(20); and Ab1(2). [Figure 12CD]Figures 12A-12D are graphs showing the results of an assay assessing different muscle weights. Figure 12A shows the mean gastrocnemius muscle weight (grams), Figure 12B shows the mean quadriceps (rectus femoris) muscle weight (grams), Figure 12C shows the mean gastrocnemius muscle weight change compared to control animals treated with PBS (IP) and regular drinking water (Group 1), and Figure 12D shows the mean quadriceps (rectus femoris) muscle weight change compared to control animals treated with PBS (IP) and regular drinking water (Group 1). PBS = phosphate-buffered saline, Dex = dexamethasone, IgG(20) = IgG control antibody administered at 20 mg / kg / week, Ab1(20) = Ab1 antibody administered at 20 mg / kg / week, and Ab1(2) = Ab1 antibody administered at 2 mg / kg / week. For Figures 12A-12B, error bars represent standard deviation (SD). For Figures 12C-12D, error bars represent the standard error of the mean (SEM). Statistical evaluation was performed using one-way ANOVA followed by Dunnett's multiple comparison test for Group 1 (****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05) and Group 5 (++++p<0.0001, +++p<0.0005, ++p<0.005, +p<0.05). ns (not significant). Bars, from left to right, show PBS, water; PBS, dex; IgG control; Ab1(20); and Ab1(2).

[0095] [Figure 13] Figures 13A-13B show the results of assays assessing mean percent body weight change and mean percent lean mass change. Figure 13A shows the percent body weight change from day 0 calculated for animals weighed twice weekly throughout the study. Figure 13B shows the percent lean mass change from day -1 calculated for animals that underwent EchoMRI (QNMR) to measure body composition on days -1, 7, and 14. PBS = phosphate-buffered saline; IgG(20) = IgG control antibody administered at 20 mg / kg / week; Ab1(20) = Ab1 antibody administered at 20 mg / kg / week; and Ab1(2) = Ab1 antibody administered at 2 mg / kg / week. Data represent group means ± SEM.

[0096] [Figure 14AB]Figures 14A-14D show the results of an assay assessing muscle weight. Figure 14A shows the average gastrocnemius muscle weight (grams) from the casted leg, Figure 14B shows the average quadriceps (rectus femoris) weight (grams) from the casted leg, Figure 14C shows the average percent change in gastrocnemius muscle weight compared to non-casted control animals (Group 1) treated with PBS (IP), and Figure 14D shows the average percent change in quadriceps (rectus femoris) weight compared to non-casted control animals (Group 1) treated with PBS (IP). PBS = phosphate-buffered saline; IgG(20) = IgG control antibody administered at 20 mg / kg / week; Ab1(20) = Ab1 antibody administered at 20 mg / kg / week; Ab1(2) = Ab1 antibody administered at 2 mg / kg / week. For Figures 14A-14B, error bars represent standard deviation (SD). For Figures 14C-14D, error bars represent the standard error of the mean (SEM). Statistical evaluation was performed using one-way ANOVA followed by Dunnett's multiple comparison test for Group 1 (****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05) and Group 5 (++++p<0.0001, +++p<0.0005, ++p<0.005, +p<0.05). ns (not significant). Bars from left to right indicate: PBS, non-immobilized; PBS, immobilized; IgG control (1), immobilized; Ab1 (20), immobilized; Ab1 (2), immobilized. [Figure 14CD]Figures 14A-14D show the results of an assay assessing muscle weight. Figure 14A shows the average gastrocnemius muscle weight (grams) from the casted leg, Figure 14B shows the average quadriceps (rectus femoris) weight (grams) from the casted leg, Figure 14C shows the average percent change in gastrocnemius muscle weight compared to non-casted control animals (Group 1) treated with PBS (IP), and Figure 14D shows the average percent change in quadriceps (rectus femoris) weight compared to non-casted control animals (Group 1) treated with PBS (IP). PBS = phosphate-buffered saline; IgG(20) = IgG control antibody administered at 20 mg / kg / week; Ab1(20) = Ab1 antibody administered at 20 mg / kg / week; Ab1(2) = Ab1 antibody administered at 2 mg / kg / week. For Figures 14A-14B, error bars represent standard deviation (SD). For Figures 14C-14D, error bars represent the standard error of the mean (SEM). Statistical evaluation was performed using one-way ANOVA followed by Dunnett's multiple comparison test for Group 1 (****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05) and Group 5 (++++p<0.0001, +++p<0.0005, ++p<0.005, +p<0.05). ns (not significant). Bars from left to right indicate: PBS, non-immobilized; PBS, immobilized; IgG control (1), immobilized; Ab1 (20), immobilized; Ab1 (2), immobilized.

[0097] [Figure 15]Figure 15 shows the results of an assay assessing lean mass change at day 21 (top right) and day 28 (top left). The figure also shows the percent lean mass change for the three different doses of antibody tested: 20 mg / kg / week (bottom left), 2 mg / kg / week (bottom center), and 0.5 mg / kg / week (bottom right), a PBS control, and an IgG control. Statistical evaluation was performed using one-way ANOVA followed by Dunnett's multiple comparison test for group 1 (****p<0.0001, ***p<0.005, **p<0.01, *p<0.05) and the IgG control. For the top two panels, the bar graphs are, from left to right, PBS; IgG control, 20 mg / kg / week; Ab1 20 mg / kg / week; Ab1 2 mg / kg / week; Ab1 0.5 mg / kg / week; Ab2 20 mg / kg / week; Ab2 2 mg / kg / week; Ab2 0.5 mg / kg / week; Ab4 20 mg / kg / week; Ab4 2 mg / kg / week; Ab4 0.5 mg / kg / week; Ab6 20 mg / kg / week; Ab6 2 mg / kg / week; and Ab6 0.5 mg / kg / week. For the bottom left panel (20 mg / kg / week), the data points correspond to 28 days after dosing, from top to bottom: Ab1, Ab4, Ab2, Ab6, IgG control, and PBS. For the lower middle panel (2 mg / kg / week), data points correspond to 28 days post-dose, from top to bottom: Ab2, Ab1, Ab6, Ab4, IgG control, and PBS. For the lower right panel (0.5 mg / kg / week), data points correspond to 28 days post-dose, from top to bottom: IgG control, Ab1, Ab2, PBS, Ab4, and Ab6.

[0098] [Figure 16] Figures 16A-16B show the domain structure and characterization of myostatin precursor forms. Figure 16A shows the domain structure of promyostatin and latent myostatin, indicating protease cleavage sites. Figure 16B shows partially proprotein convertase-cleaved promyostatin run on an SDS PAGE gel. Under reducing conditions, the protein bands consisted of the promyostatin monomer (approximately 50 kD), prodomain (approximately 37 kD), and growth factor (12.5 kD).

[0099] [Figure 17] Figures 17A-17B show that Ab1 is specific for myostatin. Figure 17A shows that Ab1 specifically binds to promyostatin and latent myostatin; no binding was observed to other members of the TGFB superfamily, particularly the corresponding forms of GDF11. Ab1 was administered at high concentrations (50 μg / mL) to Forte-Bio BLI chips coated with the indicated antigens, and on- and off-rates were measured to obtain approximate Kd values. The magnitude of the biosensor response, indicating binding events, is graphically displayed by black bars, with the calculated Kd shown in orange. Figure 17B shows that Ab1 blocks the activation of promyostatin, but not pro-GDF11. Following overnight proteolysis with enzymes from both the proprotein convertase and thrombin protease families, the release of mature growth factors was measured using a CAGA-based reporter assay in 293T cells. Results were compared to control reactions to calculate the percentage of promyostatin or proGDF11 released in the assay.

[0100] [Figure 18AB] Figures 18A-18C show SCID dose responses with candidate antibodies. Figure 18A shows gastrocnemius muscle weight, and Figure 18B shows quadriceps (rectus femoris) muscle weight. Figure 18C shows the mean percent muscle weight change compared to the PBS control. The bar graphs in Figures 18A-18B, from left to right, are PBS; IgG control 20 mg / kg / week; Ab1 20 mg / kg / week; Ab1 2 mg / kg / week; Ab1 0.5 mg / kg / week; Ab2 20 mg / kg / week; Ab2 2 mg / kg / week; Ab2 0.5 mg / kg / week; Ab4 20 mg / kg / week; Ab4 2 mg / kg / week; Ab4 0.5 mg / kg / week; Ab6 20 mg / kg / week; Ab6 2 mg / kg / week; and Ab6 0.5 mg / kg / week. [Figure 18C]Figures 18A-18C show SCID dose responses with candidate antibodies. Figure 18A shows gastrocnemius muscle weight, and Figure 18B shows quadriceps (rectus femoris) muscle weight. Figure 18C shows the mean percent muscle weight change compared to the PBS control. The bar graphs in Figures 18A-18B, from left to right, are PBS; IgG control 20 mg / kg / week; Ab1 20 mg / kg / week; Ab1 2 mg / kg / week; Ab1 0.5 mg / kg / week; Ab2 20 mg / kg / week; Ab2 2 mg / kg / week; Ab2 0.5 mg / kg / week; Ab4 20 mg / kg / week; Ab4 2 mg / kg / week; Ab4 0.5 mg / kg / week; Ab6 20 mg / kg / week; Ab6 2 mg / kg / week; and Ab6 0.5 mg / kg / week.

[0101] [Figure 19] Figure 19 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 mass were examined after 21 days following different dosing protocols.

[0102] [Figure 20] FIG. 20 is a schematic diagram illustrating an assay to reconstitute myostatin activation in vitro.

[0103] [Figure 21]Figures 21A-21B show the heavy chain (Figure 21A, SEQ ID NO: 50) and light chain (Figure 21B, SEQ ID NO: 51) of a humanized monoclonal antibody (Ab2) of the IgG4 subtype in which serine has been substituted with proline. This creates an IgG1-like hinge sequence and minimizes the incomplete formation of interchain disulfide bridges characteristic of IgG4. The complementarity-determining regions (CDRs) are underlined. The NST sequence, in bold, is an N-linked glycosylation consensus sequence site. The DP sequence, in bold, is a potential cleavage site. The NX sequence, in bold, where X can be S, T, or G, is a potential deamidation site. The DX sequence, in bold, where X can be G, S, T, or SDG, is a potential isomerization site. The methionine, in bold, is a potential methionine oxidation site. The Q, in bold, is a predicted N-terminal pyroglutamic acid.

[0104] [Figure 22] Figure 22 is a schematic diagram showing the reduction of immunogenicity risk by germlining. 24H4(WT) contains five non-germlined amino acids in the framework regions as shown in the schematic.

[0105] [Figure 23AB] Figures 23A-23C show the optimization of Ab1. Optimized candidates that specifically bind to promyostatin were selected, thereby yielding dozens of clones with increased affinity. FACS was performed to demonstrate increased binding of the yeast clones (Figure 23B) compared to Ab1 (Figure 23A). Figure 23C shows that the affinity-matured variants also have slower off-rates by octets. [Figure 23C] Figures 23A-23C show the optimization of Ab1. Optimized candidates that specifically bind to promyostatin were selected, thereby yielding dozens of clones with increased affinity. FACS was performed to demonstrate increased binding of the yeast clones (Figure 23B) compared to Ab1 (Figure 23A). Figure 23C shows that the affinity-matured variants also have slower off-rates by octets.

[0106] [Figure 24] Figures 24A-24B show sequence alignments of the variable heavy chain region (Figure 24A) and variable light chain region (Figure 24B) of parent Ab1 with affinity-optimized variants Ab3 and Ab5. Sequence identifiers, from top to bottom, correspond to SEQ ID NOS: 24, 26, and 28 (Figure 24A). Sequence identifiers, from top to bottom, correspond to SEQ ID NOS: 30, 32, and 34 (Figure 24B). Complementarity-determining regions (CDRs) are defined using Kabat (underlined) and IMGT nomenclature (bold). Substitutions from parent Ab1 are shown in light gray.

[0107] [Figure 25] Figure 25 shows the expression of promyostatin and latent myostatin in muscle and plasma from normal and atrophic mice.

[0108] [Figure 26] Figure 26 shows quantification of changes in promyostatin and latent myostatin in muscle and plasma. Bars from left to right show promyostatin in control muscle, latent myostatin in control muscle, promyostatin in dexamethasone (DEX)-treated muscle, latent myostatin in dexamethasone (DEX)-treated muscle, and latent myostatin in serum from control and DEX-treated mice.

[0109] [Figure 27]Figure 27 shows that Ab2 specifically recognizes promyostatin and latent myostatin and binds to the major forms of myostatin in both serum and muscle. Non-reducing Western blot for the prodomain (dark gray) and mature growth factor (light gray). Recombinant promyostatin (rpromyostatin) shows the migration of promyostatin and the myostatin prodomain (latent myostatin) on the gel, highlighted by arrows. In serum, both Ab2 and AbMyo bind to latent myostatin (prodomain band) and the multipartite processed precursor, but only Ab2 recognizes promyostatin (upper band). In muscle, Ab2 precipitates promyostatin, and there is no interaction between AbMyo and promyostatin in muscle tissue.

[0110] [Figure 28] Figures 28A-28B provide a model of myostatin flux in normal and atrophic muscle. In normal muscle (Figure 28A), promyostatin is produced in the muscle and converted to latent myostatin via cleavage by furin protease, which can occur either inside or outside the cell. A percentage of the latent myostatin in the muscle is then released into the circulation, forming a circulating pool of latent myostatin. In muscle atrophy (Figure 28B), an increase in active myostatin growth factor is caused by upregulation of promyostatin in the muscle and increased conversion of latent myostatin to active growth factor. As a result, the muscle pool of latent myostatin is redirected to form mature myostatin via mTLL2 cleavage, and circulating latent myostatin is reduced.

[0111] [Figure 29] Figure 29 shows the detection of Ab2 (top line) and IgG control (bottom line) antibodies in the serum of treated rats. Ab2 showed increased levels in the circulation compared to the IgG control, with an average of 17.1 μg / ml of Ab2 in serum at the end of the study. Ab2 levels were determined by human IgG-specific ELISA, with known amounts of each antibody used as a reference standard.

[0112] [Figure 30] Figures 30A-30B show the pharmacodynamic effects of Ab2 in treated rats. Figure 30A shows that rats treated with Ab2 exhibited increased lean mass compared to animals treated with PBS or IgG control. Ab2 and IgG were administered intravenously at a dose of 10 mg / kg on day 0. Lean mass was measured by qNMR (N=8 per group) at baseline (day 0) and 7, 14, 21, and 28 days after treatment. Figure 30B shows that rectus femoris and tibialis anterior muscles were harvested from all groups (N=8 per group) at the end of the study and weighed to measure muscle mass. Rats treated with Ab2 exhibited a 14% and 11% increase in rectus femoris and tibialis anterior muscle mass, respectively.

[0113] [Figure 31] Figures 31A-31B show the levels of pro / latent myostatin in rats treated with Ab2. Figure 31A shows that treatment with Ab2 (top line) increases latent myostatin levels in rat serum by approximately 20-fold. Figure 31B shows that treatment with Ab2 increases the latent form of myostatin in rat muscle (rectus femoris) by 1.9-fold. The bars from left to right correspond to promyostatin, latent myostatin, promyostatin, and latent myostatin. No statistically significant changes in promyostatin are observed in rat muscle. These data are from quantitative Western analysis with n=3 samples per group.

[0114] [Figure 32] Figure 32 shows that treatment with Ab2 (Ab2) or comparator antibody (AbMyo) increases lean mass in mice as early as 7 days after antibody administration. The increase in lean mass is comparable for Ab2 and AbMyo up to 21 days after administration. However, by 28 days after administration, the increase in lean mass is lost in the AbMyo-treated group, while the increase in Ab2-treated group is maintained throughout the course of the study. The top line corresponds to Ab2, the middle line corresponds to AbMyo, and the bottom line corresponds to the IgG control (5 mg / kg).

[0115] [Figure 33] Figure 33 shows that serum levels of drug were measured using an anti-human IgG ELISA after a single 5 mg / kg dose of Ab2 (top line) or comparator antibody (AbMyo; bottom line). Drug was detected in serum as early as 1 hour after administration, and levels of greater than 1 μg / ml for both antibodies were detectable throughout the study. However, Ab2 exhibited a significantly longer half-life and estimated area under the curve (AUCINF) than AbMyo, suggesting that Ab2 exhibits significantly greater exposure than AbMyo at similar doses.

[0116] [Figure 34] Figure 34 shows that serum myostatin was measured using fluorescent Western blot in drug-treated mice and controls. Despite increased serum exposure of Ab2, serum latent myostatin levels were similar in both Ab2- and AbMyo-treated mice. These data suggest that circulating levels of free drug are sufficiently in excess of target levels that increased serum exposure of Ab2 does not result in a greater increase in circulating latent myostatin than observed in the AbMyo group. Data groups, from left to right, correspond to IgG, Ab2, AbMyo, IgG, Ab2, and AbMyo.

[0117] [Figure 35] Figures 35A-35B show the relative levels of latent and pro-myostatin measured by fluorescent Western blot in mouse muscle lysates. Figure 35A shows that latent myostatin is elevated in Ab2- and AbMyo-treated muscles. However, while the increase in latent myostatin in AbMyo-treated muscles returns to baseline by day 28, latent myostatin in Ab2-treated muscles remains elevated at least until this time (P<0.003 vs. AbMyo treatment). Figure 35B shows that a similar trend is observed for promyostatin, although the difference between the Ab2- and AbMyo-treated groups at day 28 is not statistically significant (P=0.068).

[0118] [Figure 36A] Figure 36A shows the myostatin binding and blocking activity of a panel of antibodies.

[0119] [Figure 36B] FIG. 36B relates myostatin binding and blocking activity with effects on muscle atrophy.

[0120] [Figure 37A-D] Figures 37A-37H show that administration of muAb1 in healthy animals enhances muscle function. Figure 37A shows the functional performance of the plantarflexor in vivo after 4 weeks of treatment with the murine version of Ab1. Maximum force was normalized to limb length. Force measurements were obtained from electrical stimulation of the plantarflexor muscles via the sciatic nerve. On average, there was a 19% increase in maximum force between 40 and 150 Hz (p=0.003). Specifically, an 18% increase in maximum force was measured at 60 Hz (p=0.083). Figure 37B shows the increase in gastrocnemius muscle weight after administration of muAb1. Figure 37C shows that when normalized to gastrocnemius muscle weight, there was no change in maximum force, indicating no change in muscle quality. Figure 37D shows the muscle function of the extensor digitorum longus in vitro after 4 weeks of treatment with muAb1. EDL force measurements over increasing stimulation frequencies were normalized to EDL length. Increases in muscle force were 24% at 80 Hz (p=0.024), 28% at 100 Hz (p=0.010), and 27% at 150 Hz (p=0.011). Figure 37E shows that EDL weight increased after administration of muAb1. Figure 37F shows that, when normalized to EDL weight, there was no change in maximum force, indicating no change in muscle quality. Figure 37G shows the average type IIB fiber area for muAb1 and PBS. Figure 37H shows the four muscle fiber types (%) in the PBS and muAb samples. [Figure 37E-H]Figures 37A-37H show that administration of muAb1 in healthy animals enhances muscle function. Figure 37A shows the functional performance of the plantarflexor in vivo after 4 weeks of treatment with the murine version of Ab1. Maximum force was normalized to limb length. Force measurements were obtained from electrical stimulation of the plantarflexor muscles via the sciatic nerve. On average, there was a 19% increase in maximum force between 40 and 150 Hz (p=0.003). Specifically, an 18% increase in maximum force was measured at 60 Hz (p=0.083). Figure 37B shows the increase in gastrocnemius muscle weight after administration of muAb1. Figure 37C shows that when normalized to gastrocnemius muscle weight, there was no change in maximum force, indicating no change in muscle quality. Figure 37D shows the muscle function of the extensor digitorum longus in vitro after 4 weeks of treatment with muAb1. EDL force measurements over increasing stimulation frequencies were normalized to EDL length. Increases in muscle force were 24% at 80 Hz (p=0.024), 28% at 100 Hz (p=0.010), and 27% at 150 Hz (p=0.011). Figure 37E shows that EDL weight increased after administration of muAb1. Figure 37F shows that, when normalized to EDL weight, there was no change in maximum force, indicating no change in muscle quality. Figure 37G shows the average type IIB fiber area for muAb1 and PBS. Figure 37H shows the four muscle fiber types (%) in the PBS and muAb samples.

[0121] [Figure 38AB] Figures 38A-38I show that administration of muAb1 in healthy animals enhances muscle function. Figures 38A-38B show the maximum velocity of relaxation and contraction and the force-frequency relationship according to the functional performance of the plantar flexor muscle in vivo after 4 weeks of treatment with muAb1. Figures 38C-38D show the maximum velocity of relaxation and contraction and the force-frequency relationship according to the muscle function of the extensor digitorum longus in vitro after 4 weeks of treatment with muAb1. Figures 38E-38I show quantification of the cross-sectional area of ​​muscle fibers from the plantar flexor muscle group. [Figure 38CD]Figures 38A-38I show that administration of muAb1 in healthy animals enhances muscle function. Figures 38A-38B show the maximum velocity of relaxation and contraction and the force-frequency relationship according to the functional performance of the plantar flexor muscle in vivo after 4 weeks of treatment with muAb1. Figures 38C-38D show the maximum velocity of relaxation and contraction and the force-frequency relationship according to the muscle function of the extensor digitorum longus in vitro after 4 weeks of treatment with muAb1. Figures 38E-38I show quantification of the cross-sectional area of ​​muscle fibers from the plantar flexor muscle group. [Figure 38E-I] Figures 38A-38I show that administration of muAb1 in healthy animals enhances muscle function. Figures 38A-38B show the maximum velocity of relaxation and contraction and the force-frequency relationship according to the functional performance of the plantar flexor muscle in vivo after 4 weeks of treatment with muAb1. Figures 38C-38D show the maximum velocity of relaxation and contraction and the force-frequency relationship according to the muscle function of the extensor digitorum longus in vitro after 4 weeks of treatment with muAb1. Figures 38E-38I show quantification of the cross-sectional area of ​​muscle fibers from the plantar flexor muscle group.

[0122] [Figure 39] Figures 39A-39B show cross sections of the tibialis anterior muscle probed with anti-pro / latent GDF8 antibodies, Ab10 or a nonspecific targeting antibody (Figure 39A), and HuNeg (Figure 39B), counterstained with DAPI. Scale bar: 0.01 cm.

[0123] [Figure 40]Figures 40A-40C show cross sections of tibialis anterior muscle probed with anti-pro / latent GDF8 antibody Ab10 incubated in blocking buffer alone (Figure 40A), anti-pro / latent GDF8 antibody Ab10 incubated in blocking buffer with a 10-fold molar excess of recombinant mouse GDF8 (Figure 40B), or anti-pro / latent GDF8 antibody Ab10 incubated in blocking buffer with a 10-fold molar excess of recombinant mouse GDF11 (Figure 40C). Figures 40A-40C are counterstained with DAPI.

[0124] [Figure 41] Figures 41A-41C show cross sections of tibialis anterior muscle probed with anti-pro / latent GDF8 antibody Ab10 and anti-laminin and counterstained with DAPI. Pro / latent GDF8 and laminin colocalize within the interstitial space at the apex of myofibers (arrows), between myofibers (arrowheads), and around interstitial nuclei (asterisks).

[0125] [Figure 42] Figures 42A-42B show the muscle weights of different groups at 1 hour, 4 weeks, and 8 weeks in a single-dose pharmacokinetic / pharmacodynamic duration study. Figure 44A shows the quadriceps (rectus femoris) weights of different groups, and Figure 44B shows the gastrocnemius weights of different groups.

[0126] [Figure 43]Figures 43A-43D show the effect of treatment with Ab2 on lean mass change in healthy cynomolgus monkeys. Healthy male cynomolgus monkeys were administered Ab2 at three different doses, 3 mg / kg, 10 mg / kg, and 30 mg / kg, by intravenous injection once weekly for 8 weeks, followed by a 4-week recovery period. Control animals received vehicle control (20 mM citric acid and 150 mM sodium chloride USP, pH 5.5). Lean mass was measured by dual-energy X-ray absorptiometry (DEXA). Figure 43A is a graph showing the mean % change in lean mass in muscle from all limbs of Ab2-treated and control animals measured at day 0, week 4, week 8, and week 12. Figure 43B is a graph showing the mean % change in lean mass in muscle from all limbs of Ab2-treated and vehicle-control animals measured at week 4. Figure 43C is a graph showing the mean % change in lean mass in limb muscle of Ab2-treated and vehicle-control animals measured at week 8. Figure 43D is a graph showing the mean % change in lean mass in limb muscle of Ab2-treated and vehicle-control animals measured at week 12.

[0127] [Figure 44] Figures 44A-44B are graphs showing the effect of treatment with Ab2 on muscle weight in biceps and gastrocnemius muscles from healthy cynomolgus monkeys. Healthy male cynomolgus monkeys were administered Ab2 at three different doses, 3 mg / kg, 10 mg / kg, and 30 mg / kg, by intravenous injection once weekly for 8 weeks, with a 4-week recovery period until week 12. Control animals received vehicle control (20 mM citric acid and 150 mM sodium chloride USP, pH 5.5). Muscle weight was measured by tissue weight at week 12.

[0128] [Figure 45] FIG. 45 shows the mean % change in lean mass from baseline (day 0) and the percentage difference in muscle weight compared to vehicle controls in healthy cynomolgus monkeys treated with Ab2.

[0129] [Figure 46] Figures 46A and 46B show latent myostatin levels in serum samples from healthy cynomolgus monkeys treated with Ab2 and control animals, as measured using quantitative fluorescent Western blot. Healthy male cynomolgus monkeys were administered three different doses, 3 mg / kg, 10 mg / kg, and 30 mg / kg, by intravenous injection once a week for 8 weeks, with a 4-week recovery period. Control animals were administered a vehicle control (20 mM citric acid and 150 mM sodium chloride USP, pH 5.5). Serum samples were collected on different test days, and the relative levels of latent myostatin in the serum samples were analyzed using quantitative fluorescent Western blot.

[0130] [Figure 47] Figure 47 shows the body weights of naive mice and sham, SCI-veh, SCI-IgG, and SCI-Ab1-treated groups 1 and 2 weeks after SCI. An asterisk * at the top of the bar indicates a significant difference from sham, and an asterisk * at the bottom of the bar indicates a significant difference from SCI-Ab1.

[0131] [Figure 48] Figure 48 shows the muscle wet weight (mass) of the sham, SCI-veh, SCI-IgG, and SCI-Abl treated groups 2 weeks after SCI. The excised muscles included the sublesional soleus and gastrocnemius and the supralesional biceps and triceps.

[0132] [Figure 49] FIG. 49 shows the analysis of total fat-free (lean) mass and fat mass in the sham, SCI-veh, SCI-IgG, and SCI-Abl treated groups 2 weeks after SCI.

[0133] [Figure 50]FIG. 50 shows lean mass as a percentage of body weight in sham, SCI-veh, SCI-IgG, SCI-Abl treated groups 2 weeks after SCI.

[0134] [Figure 51] Figure 51 shows the analysis of kcal / hour and TEE in the sham, SCI-veh, SCI-IgG, and SCI-Ab1 treated groups 2 weeks after SCI. In the bottom graph, the SCI / treated control group represents the combination of the SCI / veh + SCI / IgG groups from the top graph.

[0135] [Figure 52] Figure 52 shows BMS locomotor activity assessments in the sham, SCI-veh, SCI-IgG, and SCI-Ab1 groups at baseline (before survival surgery), 1 day after SCI, 1 week after SCI, and 2 weeks after SCI. Statistical comparisons at 1 week and 2 weeks after SCI reflect the combined SCI-veh + SCI-IgG data.

[0136] [Figure 53] FIG. 53 shows the rotarod time scores in the sham, SCI-veh, SCI-IgG, and SCI-Ab1 groups after pre-training (PT), 1 week after SCI, and 2 weeks after SCI.

[0137] [Figure 54] FIG. 54 shows grip strength in the sham, SCI-veh, SCI-IgG, and SCI-Ab1 groups after pre-training (PT), 1 week after SCI, and 2 weeks after SCI.

[0138] [Figure 55] FIG. 55 shows an immunofluorescence assay performed on frozen sections of tibialis anterior muscle from healthy mice, using Ab2 and co-staining with laminin. DETAILED DESCRIPTION OF THE INVENTION

[0139] The present disclosure relates to antibodies that can specifically bind to a myostatin precursor or precursor complex in vivo and block its proteolytic processing to mature myostatin, thereby inhibiting myostatin signaling to elicit beneficial effects in vivo. Such antibodies are useful for administration to subjects who may benefit from or be in need of reduced myostatin signaling.

[0140] In accordance with the present invention, administration of an effective amount of a myostatin inhibitor, such as an anti-pro / latent myostatin antibody described herein, to a subject can specifically inhibit myostatin activation in vivo, thereby producing a number of beneficial biological effects in the subject. Such biological effects include the following benefits: a) increasing muscle mass and / or function in a subject; b) an increase in the subject's metabolic rate; c) increasing the subject's insulin sensitivity; d) increasing the level of brown adipose tissue in the subject; e) increased levels of beige adipose tissue in the subject; f) a reduction in the level of white adipose tissue in the subject; g) a reduction in the level of visceral adipose tissue in the subject; h) a decrease in the ratio of adipose tissue to muscle tissue in the subject; i) increasing glucose uptake by white adipose tissue, viable tissue, or vascular tissue in a subject; j) a decrease in muscle catabolism of protein and / or muscle release of amino acids in a subject; k) increasing insulin-dependent blood glucose regulation in a subject; l) Reduction of intramuscular fat infiltration in subjects; m) clinically meaningful improvement in standardized quality of life test scores; (o) preventing muscle loss or muscle atrophy in a subject; and / or (p) preventing the development of metabolic dysregulation associated with muscle dysfunction in a subject; including two or more of, for example, three or more, four or more, five or more, six or more, 2-4, 2-5, 2-6, 3-5 or 3-6.

[0141] Thus, the present invention includes the use of myostatin inhibitors, e.g., antibodies or antigen-binding fragments thereof that specifically bind to promyostatin and / or latent myostatin in vivo and block activation of mature myostatin, in a subject, e.g., a human subject, who would benefit from reduced myostatin signaling. The present invention includes methods of treating or preventing conditions associated with myostatin dysregulation using antibodies or antigen-binding fragments thereof that specifically bind to promyostatin and / or latent myostatin and block activation of myostatin in an amount effective to treat or prevent such conditions. Conditions associated with myostatin dysregulation include muscle diseases and disorders and certain metabolic dysregulations.

[0142] In order that the present invention may be more readily understood, certain terms are first defined. Furthermore, it should be noted that whenever a value or range of values ​​for a parameter is listed, it is intended that values ​​and ranges intervening the listed values ​​are also part of the invention.

[0143] In the following description, for purposes of explanation, specific numbers, materials and configurations are set forth in order to provide a thorough understanding of the present invention. However, it is understood that the present invention may be practiced without these specific details. It will be apparent to those skilled in the art that in some instances well-known features may be omitted or simplified so as not to obscure the invention. Furthermore, references herein to phrases such as "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Appearances of phrases such as "in one embodiment" in various places in the specification do not necessarily all refer to the same embodiment.

[0144] definition The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0145] Unless otherwise indicated in the operating examples or otherwise, numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all cases by the term "about." When used in connection with percentages, the term "about" can mean ±1% of the average. Additionally, the term "about" can mean within ±1% of the value.

[0146] The terms "administer," "administering," or "administration" include any method of delivering an antibody or antigen-binding fragment thereof, e.g., a pharmaceutical composition comprising such an antibody or antigen-binding fragment, or a drug to a subject's system or to a specific area in or on a subject (systemic administration and local administration, respectively).

[0147] The term "antibody," as used herein, is intended to refer to an immunoglobulin molecule composed of four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The antibodies of the invention are described in further detail in International Patent Application WO2016073853A1 and International Application No. PCT / US2016 / 052014, filed September 15, 2016, the contents of each of which are incorporated by reference in their entirety. Antibody variants known in the art are also encompassed by the present invention.

[0148] The terms "antigen-binding fragment," "antigen-binding fragment," or "antigen-binding portion" of an antibody (or simply "antibody fragment" or "antibody portion"), as used herein, refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., pro / latent myostatin). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL domain, VH domain, CL domain, and CH1 domain; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH domain and CH1 domain; (iv) a Fv fragment consisting of the VL domain and VH domain of a single arm of an antibody; and (v) a d fragment consisting of the VH domain. and (vi) isolated complementarity-determining regions (CDRs). Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes but can be joined using recombinant methods by a synthetic linker that allows them to form a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv)); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains on another chain and generating two antigen-binding sites (see, e.g., Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ et al. (1994) Structure 2:1121-1123).

[0149] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective component(s) that are essential to the invention, but nevertheless embrace the inclusion of unspecified elements, whether essential or not.

[0150] The term "consisting of" refers to the compositions, methods, and their respective components described herein, and is exclusive of any element not recited in the description of the embodiment.

[0151] The term "control" or "control sample," as used herein, refers to any clinically or scientifically relevant comparison sample or counterpart, including, for example, a sample from a healthy subject, a sample from a subject having a deficiency that causes or predisposes the subject to a particular disease or condition, a subject with a disease or condition of interest, a sample from a subject treated with a pharmaceutical carrier, a sample from a subject prior to treatment, a sham- or buffer-treated subject or sample, an untreated subject or sample, etc.

[0152] The term "control level" refers to an accepted or predetermined level of a biological marker, e.g., the level of the marker obtained prior to treatment or onset of a disease or prior to administration of a drug, e.g., an antibody or antigen-binding portion thereof. The level of the biological marker present in a subject or population of subjects having one or more particular characteristics, e.g., the presence or absence of a particular disease or condition.

[0153] The term "decrease," as used herein, refers to a statistically significant reduction in such levels with respect to symptoms of a disease. A reduction can be, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% below the level of detection by a detection method. A reduction can also be, for example, about 1-10%, 10-20%, 1-30%, 20-50%, 30-60%, 40-70%, 50-80%, or 60-90% below the level of detection by a detection method. In certain embodiments, a reduction is to a level that is accepted as within the normal range for individuals without such a disorder, which can also be referred to as normalization of levels.

[0154] As used herein, the term "denervation" refers to the loss or disruption of nerve supply or neuronal input to its target tissue, such as muscle tissue. Causes of denervation include disease (e.g., genetic disorders of motor neurons), chemical toxicity, physical injury, or intentional surgical interruption of nerves. Denervation can be partial (also referred to as incomplete denervation) or complete denervation. Partial denervation can be, for example, the loss or disruption of at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the nerve supply or neuronal input to its target tissue. In some embodiments, partial denervation includes loss or disruption of about 1-10%, 10-20%, 1-30%, 20-50%, 30-60%, 40-70%, 50-80%, 60-90% of the nerve supply or neuronal input to the target tissue.

[0155] "Determining," as used herein, is understood to mean performing an assay or using a method to ascertain the state of someone or something, e.g., the presence, absence, level, or degree of a particular condition, biomarker, pathology, or physiological state.

[0156] "Onset" or "progression" of a disease refers to the initial manifestation of the disease and / or subsequent progression. Disease onset may be detectable and can be assessed using standard clinical techniques. However, onset also refers to progression, which may be undetectable. For purposes of this disclosure, onset or progression refers to the biological course of symptoms. "Onset" includes appearance, recurrence, and onset. As used herein, "onset" or "onset" of a myopathy-related disease / disorder includes initial onset and / or recurrence.

[0157] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example."

[0158] The term "epitope" includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitopic determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups, and in certain embodiments may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is a region of an antigen to which an antibody binds. In certain embodiments, an antibody is said to specifically bind an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. An epitope can be a linear epitope or a conformational epitope.

[0159] As used herein, the terms "effective amount" and "effective dose" refer to any amount or dose of a compound or composition sufficient to achieve its intended purpose(s), i.e., a desired biological or medical response in a tissue or subject, at an acceptable benefit / risk ratio. For example, in certain embodiments of the invention, the intended purpose may be to inhibit myostatin activation in vivo to achieve a clinically meaningful outcome associated with myostatin inhibition.

[0160] The relevant measure of the intended purpose can be objective (i.e., measurable by some assay or marker) or subjective (i.e., the subject gives an indication of or feels an effect). In some embodiments, a therapeutically effective amount is measured based on certain clinical criteria for the disease, disorder, or condition (e.g., manifested symptoms, progression / stage of disease, etc.). It is the amount that, when administered to a patient population that meets certain criteria (determined by age, genetic profile, etc.), produces a statistically significant therapeutic response in the population.

[0161] In some embodiments, an effective amount is an amount that, when administered according to a particular regimen, produces a positive clinical outcome with reasonably tolerable adverse effects (e.g., toxicity) at a level that is therefore well tolerated by the patient for continuing the treatment regimen, and the benefits of treatment outweigh the risks of toxicity. Those skilled in the art will understand that in some embodiments of the invention, a unit dosage may be considered to contain an effective amount if it contains an amount suitable for administration in relation to a dosage regimen that correlates with a positive outcome.

[0162] Therapeutically effective amount is generally administered in a dosage regimen that can comprise a number of unit doses.For any specific pharmaceutical product, therapeutically effective amount (and / or suitable unit dose in effective dosage regimen) can vary, for example, depending on administration route, depending on the combination with other pharmaceutical products.In some embodiments, the specific therapeutically effective amount (and / or unit dose) for any specific patient can depend on various factors, including the disorder to be treated and the severity of the disorder; the activity of the specific pharmaceutical product used; the specific composition used; the patient's age, weight, overall health, sex and diet; the administration time, administration route, and / or the rate of excretion or metabolism of the specific pharmaceutical product used; the duration of treatment; and similar factors well known in the medical field.

[0163] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences and fragments thereof. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences, for example in the CDRs, particularly CDR3 (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0164] For example, the term "elevated / increased" in reference to a symptom of a disease, e.g., a disease-related loss of function or amount, such as loss of muscle mass, refers to a statistically significant increase in such level. An increase can be, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% above the level of detection by a detection method. An increase can also be, for example, about 1-10%, 10-20%, 1-30%, 20-50%, 30-60%, 40-70%, 50-80%, or 60-90% above the level of detection by a detection method. In certain embodiments, an increase is to a level accepted as within the normal range for individuals without such a disorder, which can also be referred to as normalization of levels. In certain embodiments, the increase is a normalization of the level of a sign or symptom of the disease, an increase in the difference between the subject's level of a sign of the disease and the normal level of the sign of the disease. In certain embodiments, the method includes an increase in muscle tissue mass and / or function after treating a subject with an antibody that specifically binds pro / latent myostatin. In certain embodiments, the method includes an increase in the level of promyostatin in the target muscle compared to a control level of promyostatin.

[0165] The term "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds pro / latent myostatin is substantially free of antibodies that specifically bind to antigens other than pro / latent myostatin). However, an isolated antibody that specifically binds pro / latent myostatin may be substantially free of antibodies that specifically bind to antigens other than pro / latent myostatin, such as pro / latent myostatin molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0166] Unless otherwise specified, the term "mature myostatin" refers to the fully processed, biologically active form of myostatin, unless otherwise specified. A biologically active form of myostatin is capable of binding to and / or activating a myostatin receptor. The wild-type sequence of mature myostatin is provided as SEQ ID NO: 52. In some cases, mature myostatin may contain one or more mutations that may indicate altered structure / function or stability.

[0167] As used herein, the term "myostatin inhibitor" refers to any compound that inhibits or antagonizes the activity or expression level of myostatin, such as pro / latent myostatin. In some embodiments, the myostatin inhibitor can be an antibody (e.g., U.S. Pat. Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; and fragments thereof, such as domain antibodies (dAbs) described in U.S. Pat. Nos. 6,696,245), a small molecule inhibitor, an adnectin, an affibody, a DARPin, an anticalin, an avimer, a versabody, or gene therapy. The antibody or antigen-binding fragment thereof can bind to mature myostatin, myostatin receptor, and / or GDF11. In some embodiments, the myostatin inhibitor is a small molecule inhibitor. In other embodiments, myostatin inhibitors refer to gene therapy. In one embodiment, myostatin inhibitors specifically bind to myostatin and not to GDF11. In one embodiment, myostatin inhibitors may be used to treat metabolic diseases, muscle conditions or disorders, diseases or disorders associated with defective nerve signaling or partial denervation, or other conditions described herein. In another embodiment, myostatin inhibitors may be used to treat diseases involving fast-twitch muscle fibers as described herein. In another embodiment, myostatin inhibitors may be used to provide a therapeutic effect beneath a lesion as described herein.

[0168] As used herein, the phrase "circulating latent myostatin" or "circulating latent myostatin" refers to latent myostatin in the blood, plasma, or serum.

[0169] As used herein, the term "pro / latent myostatin" refers to promyostatin, latent myostatin, or both (i.e., the pro-form or precursor of myostatin).

[0170] "Specific" and "specificity" refer to the selective reactivity of an interaction with respect to interactions between members of a specific binding pair (e.g., a ligand and a binding site, an antibody and an antigen, biotin and avidin). The phrase "specifically binds to" and similar phrases refer to the ability of an antibody (or a fragment thereof reactive with an antigen) to specifically bind to an antigen (or a fragment thereof) and not to other entities. Specific binding is understood as a preference for binding to a particular antigen, epitope, receptor ligand, or binding partner, e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1,000-fold preference over a control nonspecific antigen, epitope, receptor ligand, or binding partner. "Specific binding," as used herein, refers to the binding of a specific antigen, epitope, receptor ligand, or binding partner to ... on , K. off , and K. D For example, the ligand may be a K off is 10 -2 sec -1 or less than 10 -3 sec -1 or less than 10 -4 sec -1 or less than 10 -5 sec -1 or less, or 10 -6 sec -1 or less; and / or K D is 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, and is 10 -11 M or less, or 10 -12M or less can be considered to specifically bind to the target site. It is understood that various proteins can share common epitopes or other binding sites (e.g., kinase reactive sites). In certain embodiments, a binding site can be bound by more than one ligand and still be considered specific based on binding preference compared to nonspecific antigens and / or by having certain binding kinetic parameters. Methods for selecting an appropriate nonspecific control are within the capabilities of those skilled in the art. Binding assays are generally performed under physiological conditions.

[0171] As used herein, the terms "slow-twitch muscle," "slow-twitch muscle," "type 1 muscle," or "type I muscle" refer to muscles rich in type I muscle fibers, which are frequently used, are more postural, and help enable long-term endurance, such as long-distance running. As used herein, the terms "fast-twitch muscle," "fast-twitch muscle," "type 2 muscle," or "type II muscle" provide higher energy output and strength and are used for powerful, explosive movements, such as sprinting, but such muscles fatigue quickly and cannot be used repeatedly. Fast-twitch muscle fibers are divided into two fiber type categories: intermediate-speed muscle fibers (type IIA) and fast-twitch muscle fibers (type IIB or IIx). Intermediate-speed muscle fibers are thicker, contract faster, and also wear out more quickly than slow-twitch muscle fibers. Fast-twitch muscle fibers, which are the most powerful and have the lowest endurance, are activated when the body approaches maximum exertion. While most muscles tend to be composed of a mixture of various fiber types, different muscles contain different ratios of fiber types. During development or in response to certain events (e.g., exercise, disease, injury, etc.), fiber types within a muscle or muscle group can undergo fiber type switching, resulting in phenotypic alterations in muscle physiology.

[0172] As used herein, the terms "subject" and "patient" can be used interchangeably. In one embodiment, a subject refers to a vertebrate, particularly a mammal in need of treatment, such as companion animals (e.g., dogs, cats, etc.), livestock (e.g., cows, pigs, horses, sheep, goats, poultry, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). In some embodiments, the subject is a human who will benefit from or is in need of treatment. In one embodiment, the subject is a human subject.

[0173] As used herein, the phrase "sustained increase," in reference to an increase in muscle mass, refers to an increase in muscle mass over a specified period of time following administration of a therapeutically effective amount of a myostatin inhibitor, such as an anti-pro / latent myostatin antibody described herein. The sustained increase may be continuous or discontinuous, but overall results in an increase in muscle mass over a specified period of time.

[0174] "Treating" or "preventing" a disease or disorder means delaying or preventing the onset of such disease or disorder, reversing, alleviating, ameliorating, inhibiting, slowing or halting the progress, worsening or aggravation, progression or severity of the conditions associated with such disease or disorder, but does not necessarily require complete treatment or prevention of the disease or disorder. In one embodiment, the symptoms of the disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0175] Myostatin Myostatin, also known as GDF8, is a member of the TGFβ superfamily and belongs to a subfamily that includes two members: myostatin and GDF11. Like other members of the TGFβ superfamily, both myostatin and GDF11 are initially formed as inactive precursor polypeptides (promyostatin and proG, respectively). The domain structure and nomenclature are shown in Figure 1A. Figure 1B illustrates a schematic model of the overall structure of promyostatin, in which the mature growth factor is trapped in a cage composed of two alpha helices connected by a loop termed the "latency lasso."

[0176] Myostatin is a well-characterized negative regulator of skeletal muscle mass that is released from an autoinhibitory N-terminal prodomain by two separate protease cleavage steps. These cleavage events in the muscle fiber microenvironment can be referred to as on-cell activation. Following activation, mature myostatin signals by binding to a complex of type I and type II cell surface receptors (Alk4 / 5 and ActRIIB), whose downstream signaling induces muscle atrophy. Interest in myostatin as a target for the treatment of muscle wasting has grown. Several therapeutic agents targeting the ActRIIB signaling pathway have completed early-to-mid-stage clinical trials for muscle-wasting conditions, including sarcopenia, muscular dystrophy, cachexia, and hip replacement / hip fracture. To date, primary clinical strategies have focused on blocking the interaction between mature myostatin and cell surface receptors. However, several therapeutic programs have been discontinued due to lack of specificity (leading to unacceptable toxicity) and / or lack of efficacy. In vivo, myostatin is primarily complexed with its inhibitory prodomain.

[0177] Aspects of the disclosure provided herein relate to the evaluation of the extent to which blocking the cellular activation of myostatin from these inhibitory prodomain complexes provides a means for specifically blocking myostatin pathway signaling. Another aspect of the disclosure relates to the evaluation of a panel of human monoclonal antibodies that selectively bind to myostatin precursor forms, including a subset that inhibits proteolytic activation in vitro. In some embodiments, activation-blocking antibodies have been found to be able to protect mice from dexamethasone-induced muscle atrophy. Evaluation of serum and muscle samples from healthy animals and animals undergoing dexamethasone-induced atrophy demonstrated altered biodistribution of precursor forms during atrophy, a unique finding with important implications for understanding muscle-wasting pathologies. Furthermore, treatment of healthy mice with a murine version of a potent activation-blocking antibody promoted robust muscle growth and resulted in significant increases in muscle function. The results provided herein provide insight into the importance of myostatin processing in skeletal muscle proteostasis. Furthermore, blocking cellular activation of growth factors from their precursor forms is a powerful method for preventing myostatin signaling, a technique that offers a novel therapeutic strategy that may also be applied to other members of the TGFβ superfamily.

[0178] Activation and release of mature myostatin are achieved through several distinct protease cleavage events. The first cleavage step of promyostatin and pro-GDF11 is carried out by a proprotein convertase, which cleaves at the conserved RXXR site between the prodomain and the mature growth factor. This cleavage produces "latent myostatin," in which mature myostatin is shielded from binding to its receptor by the prodomain. Activation and release of mature, active myostatin growth factor are achieved after latent myostatin is cleaved by additional proteases of the BMP / tolloid family, such as mTLL-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.

[0179] The term "promyostatin," also known as "proGDF8," refers to an inactive precursor of mature myostatin that comprises a disulfide-linked homodimer, each molecule of which contains an amino-terminal prodomain covalently linked to a carboxyl-terminal mature myostatin domain. In one embodiment, "promyostatin" has not been cleaved by either a proprotein convertase or a BMP / threonine family protease. Exemplary promyostatin sequences, variants thereof, and methods for producing promyostatin are well known in the art and are described in more detail herein.

[0180] As used herein, the term "latent myostatin" refers to an inactive precursor of mature myostatin comprising a disulfide-linked homodimer, each molecule of which contains an amino-terminal prodomain noncovalently linked to a carboxyl-terminal mature myostatin domain. In one embodiment, "latent myostatin" is produced from promyostatin that has been cleaved by a proprotein convertase but not by a BMP / tholomid family protease. In another embodiment, "latent myostatin" can be produced by combining the prodomain and the carboxy-terminal mature myostatin domain in vitro and allowing them to fold properly. See, e.g., Sengle et al., J. Biol. Chem., 286(7):5087-5099, 2011. Exemplary latent myostatin sequences, variants thereof, and methods for producing latent myostatin are well known in the art and are described in more detail herein.

[0181] Exemplary pro-GDF8 sequences for human, rat, mouse, and cynomolgus monkey are provided below. In these pro-GDF8 sequences, the proprotein convertase cleavage site is shown in bold and the toroid protease site is underlined. In some embodiments, the proprotein convertase cleavage site comprises amino acid residues 240 to 243 of SEQ ID NOs: 52-55. In some embodiments, the toroid protease site comprises amino acid residues 74-75 of SEQ ID NOs: 52-55. It should be understood that the exemplary pro-GDF8 sequences provided herein are not intended to be limiting, and additional pro-GDF8 sequences from other species, including any isoforms thereof, are within the scope of the present disclosure.

[0182] Pro-GDF8 (human):

[0183] [ka]

[0184] pro-GDF8 (rat):

[0185] [ka]

[0186] pro-GDF8 (mouse):

[0187] [ka]

[0188] Pro-GDF8 (cynomolgus monkey):

[0189] [ka]

[0190] Myostatin and GDF11 share a relatively high degree of conservation between their mature growth factor domains, with 90% identity, but are less well conserved in their prodomain regions, with less than 50% amino acid identity between the two. Myostatin and GDF11 bind to and signal through the same receptor, consisting of a type I receptor (ALK4 / 5) associated with a type II receptor (ACTRIIA / B). Myostatin engagement with both the type I and type II receptors initiates a signaling cascade that leads to SMAD phosphorylation and transcriptional activation of muscular atrophy genes. The relatively high degree of conservation in mature growth factors makes it difficult to identify reagents, such as monoclonal antibodies, that can distinguish between mature myostatin and GDF11.

[0191] In some embodiments, the growth factor domain and the N-terminal propeptide portion of GDF11 and a C-terminal portion of the propeptide of GDF8. This chimeric construct, described below, is referred to as GDF11Arm8.

[0192] GDF11Arm8 (SEQ ID NO: 65)

[0193] [ka]

[0194] The role of myostatin in muscle homeostasis and metabolic regulation Skeletal muscle is a dynamic organ that accounts for approximately 40% of body weight and turns over at a rate of 1–2% per day. Myostatin is thought to play a central role in maintaining muscle homeostasis in both healthy and disease states. Myostatin can induce muscle atrophy by inhibiting myoblast proliferation, increasing ubiquitin-proteasome activity, and downregulating the activity of the IGF-Akt pathway. These well-recognized effects are seen in numerous conditions that cause atrophy, including injury, diseases such as cachexia, disuse, and spaceflight, demonstrating the importance of the myostatin signaling mechanism. Based on this central role, significant research has been conducted to inhibit the action of myostatin in vivo. Indeed, antagonism of myostatin signaling has been shown to favor muscle growth / augmentation.

[0195] Furthermore, muscle is known to be the body's major protein reservoir and thus contributes to amino acid homeostasis. Along with glucose (mainly produced and stored as glycogen in the liver and muscle) and lipids (stored in adipose tissue), protein in muscle can serve as an energy source (i.e., broken down to produce energy). Defects or imbalances in the utilization or mobilization of these energy sinks within the body may underlie, at least in part, various types of metabolic dysregulation. Therefore, myostatin may play a direct role in regulating metabolism by coordinating the balance between the breakdown and synthesis / storage of glucose, fat, and / or muscle within the body. Indeed, while myostatin has primarily been considered a key regulator of muscle growth / loss since its discovery in 1997, findings presented in more detail herein suggest a broader role for myostatin as a metabolic regulator.

[0196] Myostatin pathway inhibition Several myostatin pathway inhibitors, such as small molecules, antibodies or their antigen-binding portions, and gene therapy, are in various stages of clinical development for the treatment of muscle-related conditions. Such pathway antagonists target either mature growth factors or their type II receptors. Notably, most of these antagonists are not myostatin specific and therefore 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 BMP10, which are regulators of reproductive biology, wound healing, erythropoiesis, and angiogenesis, respectively. Aspects of the present disclosure relate to the recognition that, due to the lack of specificity observed with these myostatin antagonists described elsewhere, these myostatin antagonists may pose greater risks to certain patient populations because they block additional biological pathways, such as those listed above, in addition to myostatin. This potentially limits the patient population that can be safely treated due to unacceptable adverse effects, such as abnormal bleeding, wound healing, or reproductive problems, caused by off-target antibody binding (Campbell et al., Muscle Nerve, (2016); David, L., Blood, Vol. 109, pp. 1953-1961 (2007)). For example, activin A is involved in both wound healing and reproductive biology, and therefore binding to activin A limits its use in patients who have recently undergone surgery or injury, or in women of reproductive age. Such increased risk of adverse effects or toxicity is of particular concern when: i) the patient population requires long-term treatment (e.g., chronic conditions); and / or ii) the patient population is or includes pediatric patients who may be susceptible to such adverse effects and / or toxicity. Thus, the present invention comprises a novel approach for inhibiting myostatin signaling in vivo with a potentially safer profile.

[0197] Thus, provided herein are myostatin inhibitors, such as antibodies or antigen-binding fragments thereof, that can bind to promyostatin and / or latent myostatin and thereby inhibit myostatin activation, and their use to treat diseases and disorders associated with myopathy. In some embodiments, provided herein are treatments that specifically target the more abundant and longer-lived myostatin precursors, such as promyostatin and latent myostatin, rather than the mature growth factor, given the predominance of latent complexes in the circulation. While not wishing to be bound by any particular theory, the myostatin inhibitors provided herein, such as antibodies or antigen-binding fragments thereof, may prevent the activation of promyostatin and / or latent myostatin via proteolysis to mature myostatin, which is considered the "active" form of myostatin that can activate the myostatin pathway, for example, by binding to type I (ALK4 / 5) and type II (ACTRIIA / B) receptors.

[0198] As used herein, the term "pro / latent myostatin" refers to promyostatin, latent myostatin, or both. In some embodiments, an anti-pro / latent myostatin antibody or antigen-binding fragment thereof specifically binds to promyostatin. In some embodiments, an anti-pro / latent myostatin antibody or antigen-binding fragment thereof specifically binds to latent myostatin. In some embodiments, an anti-pro / latent myostatin antibody or antigen-binding fragment thereof specifically binds to both latent and promyostatin. In preferred embodiments, an anti-pro / latent myostatin antibody or antigen-binding fragment thereof that specifically binds to promyostatin and / or latent myostatin does not bind to mature myostatin. In preferred embodiments, an anti-pro / latent myostatin antibody or antigen-binding fragment thereof that specifically binds to promyostatin and / or latent myostatin does not bind to pro / latent GDF11 or mature GDF11.

[0199] Anti-pro / latent myostatin antibodies or antigen-binding fragments thereof, and production thereof The present disclosure is based, at least in part, on the surprising discovery that blocking the activation step of myostatin, rather than targeting already active myostatin, may provide an advantageous way to selectively inhibit myostatin signaling in vivo. Thus, the present invention has therapeutic utility for any condition in which selective reduction of myostatin signaling in vivo is beneficial. More particularly, the present invention involves the surprising discovery that specifically inhibiting myostatin activation can achieve not only increased muscle mass but also enhanced muscle function and prevention of metabolic dysregulation. Unexpectedly, beneficial therapeutic effects can also be achieved even under pathology in subjects with impaired, but not complete, signal transduction between neurons and target tissues, such as target muscles.

[0200] Antibodies (used interchangeably with the plural) are immunoglobulin molecules capable of specifically binding to targets such as carbohydrates, polynucleotides, lipids, and polypeptides through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Antibodies include antibodies of any class (or subclass thereof), such as IgD, IgE, IgG, IgA, or IgM; antibodies need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to various classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known.

[0201] The antibodies or antigen-binding fragments thereof described herein can bind to pro / latent myostatin, thereby inhibiting the proteolytic activation of pro / latent myostatin to mature myostatin. In some cases, the antibodies or antigen-binding fragments thereof described herein can inhibit proteolytic activation of pro / latent myostatin by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some cases, the antibodies described herein can inhibit proteolytic cleavage of promyostatin by proprotein convertases (e.g., furin) by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some cases, the antibodies or antigen-binding fragments thereof described herein can inhibit proteolytic cleavage of promyostatin or latent myostatin by a toroidal protease (e.g., mTLL2) by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.

[0202] In some embodiments, inhibition of proteolytic cleavage of promyostatin or latent myostatin by toroid proteases results in a progressive increase in muscle mass. In some embodiments, subjects exhibit a progressive increase in muscle mass over at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, or 20 weeks (or any range bracketed by any of these values). The inhibitory activity of anti-pro / latent myostatin antibodies can be measured by routine methods, for example, by Western blot analysis as described in Example 1 and Figure 3 of WO2016 / 073853, the entire contents of which are expressly incorporated herein by reference. However, it should be understood that additional methods for measuring the inhibitory activity of anti-pro / latent myostatin antibodies against proteolytic cleavage of pro / latent myostatin can be used. In some embodiments, inhibition of pro / latent myostatin cleavage (e.g., by proprotein convertase and / or thrombin protease) provides a measure of inhibitor potency and can be reflected as an inhibition constant (Ki), which is the concentration of inhibitor (e.g., anti-pro / latent myostatin antibody) required to reduce the activity of the protease (e.g., of the proprotein convertase or thrombin protease) by half, and is independent of either the enzyme or the substrate concentration.

[0203] In some embodiments, the proprotein convertase comprises (i) a catalytic domain that hydrolyzes the peptide bond of a protein containing a proprotein convertase cleavage site, and (ii) a binding pocket that binds to rTGF, which also has a proprotein convertase cleavage site. Examples of proprotein convertases for use according to the present disclosure include, but are not limited to, PCSK5 / 6, PACE4, PACE7, and PACE3 (e.g., furin). In some embodiments, the proprotein convertase is obtained, e.g., purified, from any mammal, including, but not limited to, a human, monkey, or rodent (e.g., a mouse, rat, hamster). In another embodiment, the proprotein convertase is recombinantly produced.

[0204] In some embodiments, the proprotein convertase is homologous to a proprotein convertase selected from the group consisting of PCSK5 / 6, PACE4, PACE7, and PACE3 (e.g., furin). For example, the proprotein convertase may be at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least about 99.9% identical to PCSK5 / 6, PACE4, PACE7, or PACE3 (e.g., furin).

[0205] A proprotein convertase cleavage site, in some embodiments, is an amino acid sequence that can be cleaved by a proprotein convertase (e.g., PCSK5 / 6, PACE4, PACE7, and PACE3). In some embodiments, a proprotein convertase cleavage site comprises the amino acid sequence RXXR, where R is arginine and X is any amino acid. In some embodiments, a proprotein convertase cleavage site comprises the amino acid sequence RX-(K / R)-R, where R is arginine, K is lysine, and X is any amino acid. In some embodiments, a proprotein convertase cleavage site comprises the amino acid sequence RVRR (SEQ ID NO: 57), where R is arginine and V is valine. Exemplary proprotein convertase cleavage sites for human, rat, mouse, and cynomolgus monkey myostatin are shown in bold in SEQ ID NOs: 52-55. In some embodiments, a proprotein convertase cleavage site comprises the amino acid sequence RSRR (SEQ ID NO: 56).

[0206] In some embodiments, toroid proteases for use according to the present disclosure include, but are not limited to, BMP-1, mTLL-1, and mTLL-2. The toroid protease can be obtained from any mammal, including, but not limited to, a human, a monkey, or a rodent (e.g., a mouse, a rat, or a hamster). In some embodiments, the toroid protease is homologous to a toroid protease selected from the group consisting of BMP-1, mTLL-1, and mTLL-2. For example, the toroid protease may be at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least about 99.9% identical to BMP-1, mTLL-1, and mTLL-2.

[0207] A tolloid protease cleavage site, in some embodiments, is an amino acid sequence that can be cleaved by a tolloid (e.g., BMP-1, mTLL-1, and mTLL-2). Exemplary tolloid protease cleavage sites for human, rat, mouse, and cynomolgus monkey myostatin are underlined in SEQ ID NOs: 52-55. In some embodiments, a tolloid cleavage site comprises the amino acid sequence QR, where Q is glutamine and R is arginine.

[0208] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are The antibodies or antigen-binding fragments thereof described herein can bind to localized myostatin, thereby inhibiting myostatin activity. In some cases, the antibodies or antigen-binding fragments thereof described herein can inhibit myostatin signaling by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some embodiments, inhibition of myostatin signaling can be measured by routine methods, for example, using the myostatin activation assay described in Example 1 disclosed in WO2016 / 073853 (the entire contents of which are expressly incorporated herein by reference). However, it should be understood that additional methods can be used to measure myostatin signaling activity.

[0209] It should be understood that the extent of proteolytic cleavage of myostatin, for example, by proprotein convertases and / or thrombin proteases, can be measured and / or quantified using any suitable method. In some embodiments, the extent of proteolytic cleavage of myostatin is measured and / or quantified using an enzyme-linked immunosorbent assay (ELISA). For example, an ELISA can be used to measure the level of released growth factors (e.g., mature myostatin). As another example, an antibody or antigen-binding fragment thereof that specifically binds to promyostatin, latent myostatin, and / or mature myostatin can be used in an ELISA to measure the level of a particular form of myostatin (e.g., pro / latent / mature myostatin) and to quantitate the extent of proteolytic cleavage of myostatin. In some embodiments, the extent of proteolytic cleavage of myostatin is measured and / or quantified using immunoprecipitation followed by SDS-PAGE or mass spectrometry of tryptic peptides, fluorescence anisotropy-based techniques, FRET assays, hydrogen-deuterium exchange mass spectrometry, and / or NMR spectroscopy.

[0210] In some embodiments, antibodies, also known as immunoglobulins, are tetrameric glycosylated proteins composed of two light chains (L) of approximately 25 kDa each and two heavy chains (H) of approximately 50 kDa each. Two types of light chains, called lambda and kappa, can be found in antibodies. Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Each light chain contains an N-terminal variable (V) domain (V L ) and constant (C) domains (C L Each heavy chain typically comprises an N-terminal V domain (V H ), three or four C domains (C H 1-3) and a hinge region. H The most proximal C H The domain is C H It is named 1. V H and V L A domain consists of four regions of relatively conserved sequence called framework regions (FR1, FR2, FR3, and FR4) that form a scaffold for three regions of hypervariable sequence (complementarity determining regions, CDRs). The CDRs contain most of the residues involved in the specific interaction of the antibody with the antigen. The CDRs are referred to as CDR1, CDR2, and CDR3. Thus, the CDR components on the heavy chain are referred to as CDRH1, CDRH2, and CDRH3, while the CDR components on the light chain are referred to as CDRL1, CDRL2, and CDRL3. CDRs are used in the Sequences of Proteins of Immunological The CDRs typically refer to the Kabat CDRs as described in "Interest, US Department of Health and Human Services (1991)," edited by Kabat et al. Another standard for characterizing the antigen-binding site refers to the hypervariable loops described by Chothia. See, e.g., Chothia, D. et al. (1992) J. Mol. Biol. 227:799-817 and Tomlinson et al. (1995) EMBO J. 14:4628-4638. Yet another standard is the AbM definition used by Oxford Molecular's AbM antibody modeling software. Generally, the AbM definition is defined as described, e.g., in the Antibody Engineering Lab Manual (Duebel, S. and K. See Protein Sequence and Structure Analysis of Antibody Variable Domains, by Gottfried, R. (ed.), Springer-Verlag, Heidelberg. The embodiments described with respect to Kabat CDRs can alternatively be implemented using similarly described relationships with respect to Chothia hypervariable loops or with respect to AbM-defined loops, or with respect to any combination of these methods.

[0211] Anti-pro / latent myostatin antibodies or antigen-binding fragments thereof suitable for use in the methods of the present invention include those described in International Patent Application Nos. PCT / US15 / 59468 and PCT / US16 / 52014, the entire contents of each of which are incorporated herein by reference in their entirety.

[0212] In some embodiments, the anti-pro / latent myostatin antibodies or antigen-binding fragments thereof of the present disclosure, and the nucleic acid molecules of the present disclosure encoding the antibodies or antigen-binding fragments thereof, comprise the CDR amino acid sequences shown in Tables 1 to 3. [Table 1] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0213] In some embodiments, an anti-pro / latent myostatin antibody, or antigen-binding portion thereof, of the present disclosure includes any antibody or antigen-binding fragment thereof that includes a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or combinations thereof, as provided in any one of the antibodies shown in Tables 1-3. In some embodiments, an anti-pro / latent myostatin antibody, or antigen-binding portion thereof, includes a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of any one of the antibodies shown in Tables 1-3. The present disclosure also includes any nucleic acid sequence encoding a molecule that includes a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3 as provided in any one of the antibodies shown in Tables 1-3. The antibody heavy and light chain CDR3 domains may play a particularly important role in the binding specificity / affinity of an antibody to an antigen. Thus, the anti-pro / latent myostatin antibodies or antigen-binding portions thereof of the present disclosure, or their nucleic acid molecules, may comprise at least the heavy chain and / or light chain CDR3 of the antibodies shown in Tables 1-3.

[0214] Embodiments of the present disclosure relate to a monoclonal antibody or antigen-binding fragment that binds to pro / latent myostatin protein and comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3.

[0215] In some embodiments, CDRH1 comprises the sequence set forth in any one of SEQ ID NOs: 1-3. In some embodiments, CDRH2 comprises the sequence set forth in any one of SEQ ID NOs: 4-9. In some embodiments, CDRH3 comprises the sequence set forth in any one of SEQ ID NOs: 10-11, 66, 71, 76, 81, 86, 91, 96, 101, 106, and 111. CDRL1 comprises the sequence set forth in any one of SEQ ID NOs: 12-17. In some embodiments, CDRL2 comprises the sequence set forth in any one of SEQ ID NOs: 18-21. In some embodiments, CDRL3 comprises the sequence set forth in any one of SEQ ID NOs: 22-23, 67, 72, 77, 82, 87, 92, 97, 102, 107, and 112.

[0216] In some embodiments (e.g., for the anti-pro / latent myostatin antibody Ab1 shown in Table 1), CDRH1 comprises the sequence set forth in SEQ ID NO: 1 or 2, CDRH2 comprises the sequence set forth in SEQ ID NO: 4 or 5, CDRH3 comprises the sequence set forth in SEQ ID NO: 10, CDRL1 comprises the sequence set forth in SEQ ID NO: 12 or 13, CDRL2 comprises the sequence set forth in SEQ ID NO: 18 or 19, and CDRL3 comprises the sequence set forth in SEQ ID NO: 22, and the antibody binds to pro / latent myostatin.

[0217] In some embodiments (e.g., with respect to anti-pro / latent myostatin antibody Ab2 or its antigen-binding portion shown in Table 1), CDRH1 comprises the sequence set forth in SEQ ID NO: 1 or 2, CDRH2 comprises the sequence set forth in SEQ ID NO: 4 or 5, CDRH3 comprises the sequence set forth in SEQ ID NO: 66, CDRL1 comprises the sequence set forth in SEQ ID NO: 12 or 13, CDRL2 comprises the sequence set forth in SEQ ID NO: 18 or 19, and CDRL3 comprises the sequence set forth in SEQ ID NO: 67, and the antibody or its antigen-binding portion binds to pro / latent myostatin.

[0218] In some embodiments (e.g., for anti-pro / latent myostatin antibody Ab3 or its antigen-binding portion shown in Table 1), CDRH1 comprises the sequence set forth in SEQ ID NO: 1 or 3, CDRH2 comprises the sequence set forth in SEQ ID NO: 6 or 7, CDRH3 comprises the sequence set forth in SEQ ID NO: 11, CDRL1 comprises the sequence set forth in SEQ ID NO: 14 or 15, CDRL2 comprises the sequence set forth in SEQ ID NO: 20 or 21, and CDRL3 comprises the sequence set forth in SEQ ID NO: 23, and the antibody or its antigen-binding portion binds to pro / latent myostatin.

[0219] In some embodiments, the anti-pro / latent myostatin antibodies Abs shown in Table 1 5 or its antigen-binding portion) CDRH1 comprises the sequence set forth in SEQ ID NO: 1 or 3, CDRH2 comprises the sequence set forth in SEQ ID NO: 8 or 9, CDRH3 comprises the sequence set forth in SEQ ID NO: 11, CDRL1 comprises the sequence set forth in SEQ ID NO: 16 or 17, CDRL2 comprises the sequence set forth in SEQ ID NO: 20 or 21, and CDRL3 comprises the sequence set forth in SEQ ID NO: 23, and the antibody or its antigen-binding portion binds to pro / latent myostatin.

[0220] In some examples, any of the anti-pro / latent myostatin antibodies or antigen-binding portions thereof of the present disclosure include any antibody or antigen-binding fragment having one or more CDR (e.g., CDRH or CDRL) sequences substantially similar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and / or CDRL3. For example, an antibody may comprise one or more CDR sequences set forth in Tables 1-3 (SEQ ID NOS: 1-23, 66, 67, 71, 72, 76, 77, 81, 82, 86, 87, 91, 92, 96, 97, 101, 102, 106, 107, 111 and 112) containing up to 5, 4, 3, 2 or 1 amino acid residue variation compared to the corresponding CDR region of any one of SEQ ID NOS: 1-23, 66, 67, 71, 72, 76, 77, 81, 82, 86, 87, 91, 92, 96, 97, 101, 102, 106, 107, 111 and 112. The complete amino acid and nucleic acid sequences for the heavy and light chain variable regions of the antibodies listed in Table 1 are provided below.

[0221] In some embodiments, anti-pro / latent myostatin antibodies, or antigen-binding portions thereof, of the present disclosure include any antibody comprising a heavy chain variable domain of any one of SEQ ID NOs: 24-29, 73, 78, 83, 88, 93, 98, 103, 108, and 113, or a light chain variable domain of any one of SEQ ID NOs: 30-35, 74, 79, 84, 89, 94, 99, 104, 109, and 114. In some embodiments, anti-pro / latent myostatin antibodies, or antigen-binding portions thereof, of the present disclosure include any antibody comprising a heavy chain variable and light chain variable pair of SEQ ID NOs: 24 and 30; 25 and 31; 26 and 32; 27 and 33; 28 and 34; or 29 and 35).

[0222] Aspects of the present disclosure provide anti-pro / latent myostatin antibodies, or antigen-binding portions thereof, having heavy chain variable and / or light chain variable amino acid sequences homologous to any of those described herein. In some embodiments, the anti-pro / latent myostatin antibodies, or antigen-binding portions thereof, comprise a heavy chain variable sequence or light chain variable sequence that is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the heavy chain variable sequence of any of SEQ ID NOs: 24-29, 73, 78, 83, 88, 93, 98, 103, 108, and 113, or the light chain variable sequence of any one of SEQ ID NOs: 30-35, 74, 79, 84, 89, 94, 99, 104, 109, and 114. In some embodiments, the homologous heavy chain variable and / or light chain variable amino acid sequences are unchanged within any of the CDR sequences provided herein. For example, in some embodiments, a degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) may occur within the heavy and / or light chain variable sequences excluding any of the CDR sequences provided herein.

[0223] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such algorithms are described in Altschul et al., J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein molecule of interest. If gaps exist between the two sequences, the search is repeated. In this case, GAP BLAST is used as described by Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0224] In some embodiments, conservative mutations may be introduced into CDR or framework sequences at positions where the residues are unlikely to be involved in interactions with pro / latent myostatin as determined based on the crystal structure. As used herein, a "conservative amino acid substitution" refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants may be prepared according to methods for altering polypeptide sequences known to those skilled in the art, such as those found in references summarizing such methods, for example, Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al., 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, edited by F. M. Ausubel et al., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0225] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein contain mutations that confer desirable properties to the antibody or antigen-binding fragment thereof. For example, a naturally occurring IgG4 To avoid potential complications due to Fab arm exchange, which is known to occur in mAbs, the antibodies or antigen-binding portions thereof provided herein may contain a stabilizing "Adair" mutation in which serine 228 (EU numbering, residue 241 in Kabat numbering) is converted to proline to create an IgG1-like (CPPCP (SEQ ID NO: 58)) hinge sequence (Angal S. et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody," Mol Immunol 30:105-108; 1993). Thus, any of the antibodies may contain the stabilizing "Adair" mutation or the amino acid sequence CPPCP (SEQ ID NO: 58).

[0226] The anti-pro / latent myostatin antibodies, or antigen-binding portions thereof, of the present disclosure may optionally include an antibody constant region, or portion thereof. L The domain may be attached at its C-terminus to a light chain constant domain such as Cκ or Cλ. H The domain or a portion thereof can be attached to all or part of a heavy chain, such as IgA, IgD, IgE, IgG, and IgM, and any isotype subclass. The antibody can include a suitable constant region (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within this scope can include a V domain combined with any suitable constant region. H and V L domain, or an antigen-binding portion thereof.

[0227] In certain embodiments, V H and / or V LDomains may be reverted to germline sequences, e.g., the FRs of these domains are mutated using conventional molecular biology techniques to match those produced by germline cells. H and / or V L The domains may be reverted to the germline sequences of IgHV3-30 (SEQ ID NO: 36) and / or IgLV1-44 (SEQ ID NO: 37), respectively. H and / or V L domain It should be understood that any of the FR sequences may be reverted to any suitable germline sequence. In other embodiments, the FR sequences remain deviated from the consensus germline sequence.

[0228] IgHV3-30

[0229] [ka]

[0230] IgLV1-44

[0231] [ka]

[0232] In some embodiments, the anti-pro / latent myostatin antibody or antigen-binding fragment may or may not include the framework regions of the antibodies set forth in SEQ ID NOs: 24 to 35. In some embodiments, the anti-pro / latent myostatin antibody is a murine antibody and includes murine framework region sequences.

[0233] In some embodiments, the anti-pro / latent myostatin antibody or antigen-binding fragment thereof is capable of binding with relatively high affinity, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11The antibody or antigen-binding fragment thereof can bind to pro / latent myostatin with a Kd of less than 100 nM or less. For example, an anti-pro / latent myostatin antibody or antigen-binding fragment thereof can bind to pro / latent myostatin with an affinity of between 5 pM and 500 nM, e.g., between 50 pM and 100 nM, e.g., between 500 pM and 50 nM. The present invention also includes antibodies or antigen-binding fragments that compete with any of the antibodies described herein for binding to pro / latent myostatin and have an affinity of 50 nM or less (e.g., 20 nM or less, 10 nM or less, 500 pM or less, 50 pM or less, or 5 pM or less). The affinity and binding kinetics of anti-pro / latent myostatin antibodies may be tested using any suitable method, including, but not limited to, biosensor technology (e.g., OCTET or BIACORE).

[0234] In some embodiments, disclosed herein are antibodies or antigen-binding fragments thereof that specifically bind to pro / latent myostatin. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein bind to or near the toroid cleavage site or toroid docking site of pro / latent myostatin. In some embodiments, an antibody binds near the toroid cleavage site or toroid docking site if it binds within 15 or fewer amino acid residues of the toroid cleavage site or toroid docking site. In some embodiments, any of the antibodies or antigen-binding fragments provided herein bind within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the toroid cleavage site or toroid docking site. In some embodiments, the antibody binds to or near the toroid cleavage site of GDF8. For example, the antibody binds to the amino acid sequence set forth in SEQ ID NO: 62 PKAPPLRELIDQYDVQRDDSSDGSLEDDDYHAT (SEQ ID NO: 62). In other embodiments, any of the antibodies or antigen-binding fragments provided herein bind to or near the proprotein convertase cleavage site or proprotein convertase docking site of pro / latent myostatin. In some embodiments, the antibody binds near the proprotein convertase cleavage site or proprotein convertase docking site if it binds within 15 or fewer amino acid residues of the proprotein convertase cleavage site or proprotein convertase docking site. In some embodiments, any of the antibodies or antigen-binding fragments provided herein bind within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the proprotein convertase cleavage site or proprotein convertase docking site. In some embodiments, the antibody binds to or near the proprotein convertase cleavage site of GDF8. For example, the antibody can bind to the amino acid sequence set forth in SEQ ID NO: 63 (GLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRC).

[0235] In one example, the anti-pro / latent myostatin antibodies or antigen-binding fragments thereof described herein specifically bind to pro / latent myostatin relative to other forms of myostatin and / or other members of the TGFβ family of growth factors, including, but not limited to, AMH, ARTN, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDNF, INHA, INHBA, INHBB, INHBC, INHBE, LEFTY1, LEFTY2, NODAL, NRTN, PSPN, TGFβ1, TGFβ2, and TGFβ3 proteins. Such antibodies or antigen-binding fragments thereof can bind to pro / latent myostatin with much higher affinity (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1,000-fold higher) than other members of the TGFβ family of growth factors. In some embodiments, such antibodies or antigen-binding fragments thereof can bind to pro / latent myostatin with at least 1,000-fold higher affinity than other members of the TGFβ family of growth factors. In some embodiments, the antibodies or antigen-binding fragments provided herein can bind to pro / latent myostatin with much higher affinity (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1,000-fold higher) than one or more forms of GDF11 or mature myostatin. In some embodiments, the antibodies or antigen-binding fragments provided herein can bind to pro / latent myostatin with at least 1,000-fold greater affinity than one or more forms of GDF11 (e.g., proGDF11, latent GDF11, or mature GDF11) or mature myostatin.Alternatively or additionally, the antibody or antigen-binding fragment thereof may exhibit significantly greater inhibitory activity (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold greater) against proteolytic cleavage of pro / latent myostatin (e.g., by proprotein convertases or thrombin proteases) compared to other members of the TGFβ family, such as pro / latent GDF11. In another embodiment, the antibodies or antigen-binding fragments thereof disclosed herein do not bind to GDF11, thereby avoiding potential toxicity issues associated with antibodies that cross-react with both myostatin and GDF11.

[0236] In some embodiments, antibodies bind to antigens but cannot effectively remove the antigens from plasma. Thus, in some embodiments, the concentration of antigens in plasma can be increased by reducing antigen clearance. However, in some embodiments, the antibodies provided herein (e.g., sweeping antibodies) have pH-sensitive affinity for antigens. Such pH-sensitive antibodies can bind to antigens in plasma at neutral pH and dissociate from the antigen in acidic endosomes, thereby reducing antibody-mediated antigen accumulation and / or antigen clearance from plasma. Promote lance.

[0237] Aspects of the present disclosure relate to sweeping antibodies. As used herein, a "sweeping antibody" or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof that has both pH-sensitive antigen binding and at least a threshold level of binding to cell-surface neonatal Fc receptor (FcRn) at neutral or physiological pH. In some embodiments, the sweeping antibody or antigen-binding portion thereof binds to neonatal Fc receptor FcRn at neutral pH. For example, the sweeping antibody can bind to FcRn at a pH ranging from 7.0 to 7.6. In some embodiments, the sweeping antibody or antigen-binding portion thereof can bind to an antigen at the antigen-binding site and to cellular FcRn via the Fc portion of the antibody. In some embodiments, the sweeping antibody or antigen-binding portion thereof can then be internalized and release the antigen into acidic endosomes, where it may be degraded. In some embodiments, the sweeping antibody or antigen-binding portion thereof no longer binds to the antigen and can then be released back into the serum by cells (e.g., by exocytosis).

[0238] In some embodiments, FcRn in the vascular endothelium (e.g., of a subject) increases the half-life of the sweeping antibody, or antigen-binding portion thereof. In some embodiments, vascular endothelial cells internalize the sweeping antibody, or antigen-binding portion thereof, that binds to an antigen, such as myostatin (e.g., promyostatin, latent myostatin, or primed myostatin). In some embodiments, the sweeping antibody, or antigen-binding portion thereof, is recycled back into the bloodstream. In some embodiments, the sweeping antibody, or antigen-binding portion thereof, has an increased half-life (e.g., in the serum of a subject) compared to its conventional counterpart. In some embodiments, the conventional counterpart of a sweeping antibody refers to the antibody, or antigen-binding portion thereof, from which the sweeping antibody, or antigen-binding portion thereof, is derived (e.g., before engineering the Fc portion of the conventional antibody to bind FcRn with greater affinity at pH 7). In some embodiments, the sweeping antibody or antigen-binding portion thereof has a half-life in the serum of a subject that is at least 1%, 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 100%, 150%, 200%, or 250% longer than its conventional counterpart.

[0239] In some embodiments, the Fc portion of the sweeping antibody binds to FcRn. In some embodiments, the Fc portion of the sweeping antibody binds to FcRn at a pH of 7.4. -3 M to 10 -8 In some embodiments, the sweeping antibody binds to FcRn with a Kd in the range of 10 M at a pH of 7.4. -3 M to 10 -7 M, 10 -3 M to 10 -6 M, 10 -3 M to 10 -5 M, 10 -3 M to 10 -4 M, 10 -4 M to 10 -8 M, 10 -4 M to 10 -7 M, 10 -4 M to 10 -6 M, 10 -4M to 10 -5 M, 10 -5 M to 10 -8 M, 10 -5 M to 10 -7 M, 10 -5 M to 10 -6 M, 10 -6 M to 10 -8 M, 10 -6 M to 10 -7 M or 10 -7 M to 10 -8 In some embodiments, the sweeping antibody Fc region binds with a Kd in the range of M. In some embodiments, the FcRn binds to the CH2-CH3 hinge region of the sweeping antibody. In some embodiments, the FcRn binds to the same region as Protein A or Protein G. In some embodiments, the FcRn binds to a binding site different from that of FcγR. In some embodiments, amino acid residue AA of the sweeping antibody Fc region is required for binding to FcRn. In some embodiments, amino acid residue AA of the sweeping antibody Fc region affects binding to FcRn.

[0240] In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein are engineered to bind to FcRn with higher affinity. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein are engineered to bind to FcRn with higher affinity at pH 7.4. In some embodiments, the affinity of the antibodies or antigen-binding fragments thereof for FcRn is higher than that of their conventional counterparts. The sweeping antibody or antigen-binding portion thereof may be increased to enhance pharmacokinetic (PK) properties. For example, in some embodiments, the sweeping antibody or antigen-binding portion thereof induces fewer adverse reactions due to efficacy at lower doses. In some embodiments, the sweeping antibody or antigen-binding portion thereof is administered less frequently. In some embodiments, the transcytosis of the sweeping antibody or antigen-binding portion thereof to certain tissue types is increased. In some embodiments, the sweeping antibody or antigen-binding portion thereof enhances the efficiency of transplacental delivery. In some embodiments, the sweeping antibody or antigen-binding portion thereof is less expensive to produce.

[0241] In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein are engineered to bind to FcRn with low affinity. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein are engineered to bind to FcRn with low affinity at pH 7.4. In some embodiments, the affinity of the sweeping antibodies or antigen-binding portions thereof to FcRn is reduced to shorten their pharmacokinetic (PK) characteristics compared to their conventional counterparts. For example, in some embodiments, the sweeping antibodies or antigen-binding portions thereof are cleared more rapidly for imaging and / or radioimmunotherapy. In some embodiments, the sweeping antibodies or antigen-binding portions thereof promote the clearance of endogenous pathogenic antibodies as a treatment for autoimmune diseases. In some embodiments, the sweeping antibodies or antigen-binding portions thereof reduce the risk of adverse pregnancy outcomes that may result from transplacental transport of material fetal-specific antibodies.

[0242] In some embodiments, the sweeping antibody or antigen-binding portion thereof has decreased affinity for the antigen at low pH compared to neutral or physiological pH (e.g., pH 7.4). In some embodiments, the sweeping antibody or antigen-binding portion thereof has decreased affinity for the antigen at acidic pH (e.g., a pH in the range of 5.5 to 6.5) compared to physiological pH (e.g., pH 7.4).

[0243] It should be understood that any of the antibodies or antigen-binding fragments thereof provided herein may be engineered to dissociate from an antigen in response to a change in pH (e.g., a pH-sensitive antibody). In some embodiments, the sweeping antibodies or antigen-binding portions thereof provided herein are engineered to bind to an antigen in a pH-dependent manner. In some embodiments, the sweeping antibodies or antigen-binding portions thereof provided herein are engineered to bind to FcRn in a pH-dependent manner. In some embodiments, the sweeping antibodies or antigen-binding portions thereof provided herein are internalized by endocytosis. In some embodiments, the sweeping antibodies or antigen-binding portions thereof provided herein are internalized by FcRn binding. In some embodiments, the endocytosed sweeping antibodies or antigen-binding portions thereof release the antigen into an endosome. In some embodiments, the sweeping antibodies or antigen-binding portions thereof are recycled back to the cell surface. In some embodiments, the sweeping antibodies remain bound to the cells. In some embodiments, the endocytosed sweeping antibodies or antigen-binding portions thereof are recycled back to plasma. It should be understood that the Fc portion of any of the antibodies or antigen-binding fragments thereof provided herein may be engineered to have different FcRn binding activities. In some embodiments, the FcRn binding activity affects the clearance time of an antigen by a sweeping antibody. In some embodiments, the sweeping antibody may be a long-acting or fast-acting sweeping antibody.

[0244] In some embodiments, converting a conventional therapeutic antibody or antigen-binding portion thereof to a sweeping antibody or antigen-binding portion thereof reduces the effective dose. In some embodiments, converting a conventional therapeutic antibody or antigen-binding portion thereof to a sweeping antibody or antigen-binding portion thereof reduces the effective dose by at least 1%, 2%, 5%, 10%, 20%, a 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction. In some embodiments, converting a conventional therapeutic antibody or antigen-binding portion thereof to a sweeping antibody or antigen-binding portion thereof reduces the effective dose by at least 1.5, 2, 3, 4, 5, 6, 8, 10, 15, 20, 50, or 100-fold.

[0245] In some embodiments, selecting an appropriate dose of a sweeping antibody or antigen-binding portion thereof for treatment can be performed empirically. In some embodiments, a high dose of a sweeping antibody or antigen-binding portion thereof may saturate FcRn, resulting in an antibody that stabilizes the antigen in serum without internalizing it. In some embodiments, a low dose of a sweeping antibody or antigen-binding portion thereof may not be therapeutically effective. In some embodiments, the sweeping antibody or antigen-binding portion thereof is administered once daily, once a week, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every eight weeks, once every ten weeks, once every 12 weeks, once every 16 weeks, once every 20 weeks, or once every 24 weeks.

[0246] In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein may be modified or engineered to be sweeping antibodies. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein may be converted into sweeping antibodies using any suitable method. For example, a suitable method for generating sweeping antibodies or antigen-binding portions thereof is described in Igawa et al., (2013) "Engineered Monoclonal Antibody with Novel Antigen-Sweeping Activity In Vivo," PLoS ONE Vol. 8(5):e63236; and Igawa et al., "pH-dependent antigen-binding antibodies as a novel therapeutic modality," Biochimica et Biophysica Acta Vol. 1844 (2014) pp. 1943-1950, the contents of each of which are incorporated herein by reference. However, it should be understood that the methods for producing sweeping antibodies or antigen-binding portions thereof provided herein are not meant to be limiting. Accordingly, additional methods for producing sweeping antibodies or antigen-binding portions thereof are within the scope of this disclosure.

[0247] Some aspects of the present disclosure are based on the recognition that the affinity (e.g., expressed as Kd) of any of the anti-pro / latent myostatin antibodies or antigen-binding fragments thereof provided herein is sensitive to changes in pH. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have an increased Kd for binding to pro / latent myostatin at relatively low pH (e.g., a pH in the range of 4.0 to 6.5) compared to relatively high pH (e.g., a pH in the range of 7.0 to 7.4). In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have an increased Kd for binding to pro / latent myostatin at a pH between 4.0 and 6.5. -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have a Kd for binding to pro / latent myostatin in the range of 10 M when the pH is between 7.0 and 7.4. -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11M. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have a Kd for binding to pro / latent myostatin that is at least 2-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, or at least 10000-fold greater between pH 4.0 and 6.5 compared to between pH 7.0 and 7.4.

[0248] In some embodiments, an epitope within the amino acid sequence set forth in (SEQ ID NO: 64) Provided herein are pro / latent myostatin antibodies or antigen-binding fragments thereof that do not specifically bind to the same epitope as an antibody described in Tables 2a, 11a, 11b, or 13 of International Patent Application Publication No. WO2016 / 098357, published June 23, 2016, based on International Patent Application No. PCT / JP2015 / 006323, filed December 18, 2015. In some embodiments, the pro / latent myostatin antibodies or antigen-binding fragments thereof provided herein do not compete or cross-compete for binding to the same epitope as an antibody described in Tables 2a, 11a, 11b, or 13 of International Patent Application Publication No. WO2016 / 098357, published on June 23, 2016, based on International Patent Application No. PCT / JP2015 / 006323, filed on December 18, 2015. In some embodiments, the pro / latent myostatin antibodies or antigen-binding fragments provided herein do not specifically bind to the same epitope as an antibody comprising a VH and VL pair described in Tables 2a, 11a, 11b, or 13 of International Patent Application Publication No. WO2016 / 098357, published on June 23, 2016, based on International Patent Application No. PCT / JP2015 / 006323, filed on December 18, 2015. In some embodiments, the pro / latent myostatin antibodies or antigen-binding fragments provided herein do not compete or cross-compete for binding to the same epitope as antibodies comprising a VH and VL pair set forth in Tables 2a, 11a, 11b, or 13 of International Patent Application Publication No. WO2016 / 098357, published June 23, 2016, based on International Patent Application No. PCT / JP2015 / 006323, filed December 18, 2015.

[0249] Polypeptides Some aspects of the present disclosure relate to a polypeptide having a sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29. In some embodiments, the polypeptide is a variable heavy chain domain. In some embodiments, the polypeptide is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of the amino acid sequences set forth in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29.

[0250] Some aspects of the present disclosure relate to a polypeptide having a sequence selected from the group consisting of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35. In some embodiments, the polypeptide is a variable light chain domain. In some embodiments, the polypeptide is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of the amino acid sequences set forth in SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35.

[0251] Antibodies or antigen-binding fragments thereof that compete with anti-pro / latent myostatin antibodies or antigen-binding fragments thereof Aspects of the present disclosure relate to antibodies or antigen-binding fragments thereof that compete or cross-compete with any of the antibodies or antigen-binding fragments thereof provided herein. The term "compete," as used herein 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 that the result of binding of the first antibody to that epitope is detectably reduced in the presence of the second antibody compared to binding of the first antibody in the absence of the second antibody. It is possible, but not necessary, that the binding of the second antibody to that epitope is also detectably reduced in the presence of the first antibody. That is, a first antibody can inhibit binding of a second antibody to its epitope without the second antibody inhibiting binding of the first antibody to its respective epitope. However, if each antibody detectably inhibits binding of the other antibody to its epitope or ligand, whether to the same extent, a greater extent, or a lesser extent, the antibodies are said to "cross-compete" with each other for binding their respective epitope(s). Both competing and cross-competing antibodies are within the scope of the present disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), one of skill in the art will understand that such competing and / or cross-competing antibodies are encompassed and may be useful in the methods and / or compositions provided herein.

[0252] Aspects of the present disclosure relate to antibodies or antigen-binding portions thereof that compete or cross-compete with any of the antibodies or antigen-binding fragments thereof provided herein. In some embodiments, the antibody or antigen-binding portion thereof binds at or near the same epitope as any of the antibodies provided herein. In some embodiments, an antibody or antigen-binding portion thereof binds near an epitope if it binds within 15 or fewer amino acid residues of the epitope. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein bind within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the epitope to which any of the antibodies or antigen-binding fragments thereof provided herein bind.

[0253] In another embodiment, the antibody or antigen-binding portion thereof is -6 In other embodiments, the antibody or antigen-binding portion thereof competes or cross-competes for binding to any of the antigens provided herein (e.g., pro / latent myostatin) with an equilibrium dissociation constant, Kd, ​​between the antibody and the protein of less than 10 M. -11 M to 10 -6 Compete or cross-compete for binding to any of the antigens provided herein with a Kd in the range of M.

[0254] Aspects of the present disclosure relate to antibodies or antigen-binding portions thereof that compete with any of the antibodies or antigen-binding fragments thereof provided herein for binding to pro / latent myostatin. In some embodiments, the antibody or antigen-binding portion thereof binds to pro / latent myostatin at the same epitope as any of the antibodies or antigen-binding fragments thereof provided herein. For example, in some embodiments, any of the antibodies provided herein bind to or near the tolloid cleavage site or tolloid docking site of pro / latent myostatin. In other embodiments, any of the antibodies or antigen-binding fragments provided herein bind to or near the proprotein convertase cleavage site or tolloid docking site of pro / latent myostatin. In another embodiment, the antibody or antigen-binding portion thereof binds to or near the proprotein convertase cleavage site or to the proprotein convertase docking site of pro / latent myostatin. -6 In another embodiment, an antibody or antigen-binding portion thereof that competes with any of the antibodies or antigen-binding portions thereof provided herein competes for binding to pro / latent myostatin with an equilibrium dissociation constant Kd between the antibody or antigen-binding portion thereof and pro / latent myostatin of less than 10 M. -11 M to 10 -6 It binds to pro / latent myostatin with a Kd in the range of M.

[0255] Any of the antibodies or antigen-binding fragments thereof provided herein may be characterized using any suitable method. For example, one method is to identify the epitope to which the antigen binds, i.e., "epitope mapping." There are many suitable methods for mapping and characterizing the location of epitopes on proteins, including analyzing the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, "Using Antibodies," a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In a further example, epitope mapping can be used to determine the sequence to which an antibody or its antigen-binding portion binds. The epitope can be a linear epitope, i.e., contained in a single stretch of amino acids, or An epitope may be a conformational epitope formed by three-dimensional interactions of amino acids that may not necessarily be contained in a single stretch (primary linear sequence). Peptides of various lengths (e.g., at least 4-6 amino acids) may be isolated or synthesized (e.g., recombinantly) and used for antibody binding assays. In another example, the epitope to which an antibody or its antigen-binding portion binds can be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody. In a gene fragment expression assay, an open reading frame encoding a target antigen is fragmented, either randomly or by specific genetic construction, and the reactivity of the expressed antigen fragment with the antibody being tested is determined. The gene fragment can be produced, for example, by PCR, and then transcribed and translated into protein in vitro in the presence of radioactive amino acids. Binding of the antibody or its antigen-binding portion to the radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis. Specific epitopes can also be identified using large libraries of random peptide sequences (phage libraries) displayed on the surface of phage particles. Alternatively, defined libraries of overlapping peptide fragments can be tested for binding to a test antibody or its antigen-binding portion in a simple binding assay. In additional examples, mutagenesis of antigen-binding domains, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and / or necessary for epitope binding. For example, domain swapping experiments can be performed using mutants of the target antigen in which various fragments of the pro / latent myostatin polypeptide are replaced (swapped) with sequences from a closely related but antigenically distinct protein, such as another member of the TGFβ protein family (e.g., GDF11). By assessing the binding of an antibody or its antigen-binding portion to mutant pro / latent myostatin, the importance of specific antigen fragments to the binding of the antibody or its antigen-binding portion can be assessed.

[0256] Alternatively, a competition assay may be performed using other antibodies known to bind to the same antigen to determine whether the antibody, or antigen-binding portion thereof, binds to the same epitope as the other antibody, or antigen-binding portion thereof. Competition assays are well known to those of skill in the art.

[0257] Any suitable method, such as the epitope mapping methods described herein, can be applied to determine whether an anti-pro / latent myostatin antibody or antigen-binding portion thereof binds to one or more of the specific residues / segments in pro / latent myostatin described herein. Furthermore, the interaction of an antibody or antigen-binding portion thereof with one or more of these defined residues in pro / latent myostatin can be determined by routine techniques. For example, a crystal structure can be determined, and the distance between a residue in pro / latent myostatin and one or more residues in the antibody or antigen-binding portion thereof can be determined accordingly. Based on such distances, it can be determined whether a particular residue in pro / latent myostatin interacts with one or more residues in the antibody or antigen-binding portion thereof. Furthermore, suitable methods, such as competition assays and targeted mutagenesis assays, can be applied to determine the preferential binding of a candidate anti-pro / latent myostatin antibody or antigen-binding portion thereof to pro / latent myostatin compared to another target, such as mutant pro / latent myostatin.

[0258] Production of anti-pro / latent myostatin antibodies or antigen-binding fragments thereof Numerous methods can be used to obtain the antibodies or antigen-binding fragments thereof of the present disclosure. For example, antibodies and antigen-binding fragments thereof can be produced using recombinant DNA methods. Monoclonal antibodies and antigen-binding fragments thereof can also be produced by the generation of hybridomas by known methods (see, e.g., Kohler and Milstein (1975) Nature, 256:495-499). Hybridomas formed in this manner can be produced by the generation of hybridomas by known methods (see, e.g., Kohler and Milstein (1975) Nature, 256:495-499). The hybridomas are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (e.g., OCTET or BIACORE) analysis, to identify one or more hybridomas that produce an antibody or antigen-binding portion thereof that specifically binds to a particular antigen. Any form of a particular antigen, such as a recombinant antigen, a naturally occurring form, any variant or fragment thereof, and its antigenic peptides (e.g., any of the epitopes described herein within a scaffold, either as a linear epitope or as a conformational epitope) can be used as an immunogen. One exemplary method for producing antibodies and antigen-binding portions thereof involves screening a protein expression library, such as a phage or ribosome display library, that expresses antibodies or fragments thereof (e.g., scFvs). Phage display is described, for example, in Ladner et al., U.S. 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, WO91 / 17271, WO92 / 20791, WO92 / 15679, WO93 / 01288, WO92 / 01047, WO92 / 09690 and WO90 / 02809.

[0259] In addition to using display libraries, a particular antigen (e.g., promyostatin) may be used to immunize a non-human animal, such as a rodent, such as a mouse, hamster, or rat. In one embodiment, the non-human animal is a mouse.

[0260] In another embodiment, monoclonal antibodies are obtained from non-human animals and then modified (e.g., chimeric) using suitable recombinant DNA techniques. Various approaches for producing chimeric antibodies have been described. See, for example, Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 (1985); Takeda et al., Nature 314:452 (1985); Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., European Patent Publication Nos. EP 171496, 0173494, and GB 2177096B.

[0261] For additional antibody production techniques, see Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988. The present disclosure is not necessarily limited to any particular source, method of production or other particular characteristics of the antibodies.

[0262] Some aspects of the present disclosure relate to host cells transformed with a polynucleotide or vector. The host cell may be a prokaryotic or eukaryotic cell. The polynucleotide or vector present in the host cell may be integrated into the genome of the host cell or maintained extrachromosomally. The host cell may be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal, or human cell. In some embodiments, the fungal cell is, for example, of the genus Saccharomyces, specifically the species S. cerevisiae. The term "prokaryote" includes all bacteria that can be transformed or transfected with DNA or RNA molecules for expression of antibodies or corresponding immunoglobulin chains. Prokaryotic hosts may include gram-negative and gram-positive bacteria, such as E. coli, S. typhimurium, Serratia marcescens, and Bacillus subtilis. The term "eukaryote" includes yeast, higher plants, insects, and vertebrate cells, such as mammalian cells, such as NS0 and CHO cells. Depending on the host employed in a recombinant production procedure, the antibody or immunoglobulin chain encoded by the polynucleotide may be glycosylated or may be non-glycosylated. The antibody or the corresponding immunoglobulin chain may also include an initial methionine amino acid residue.

[0263] In some embodiments, once the vector has been incorporated into a suitable host, the host may be maintained under conditions suitable for high-level expression of the nucleotide sequence, followed, if desired, by recovery and purification of the immunoglobulin light chain, heavy chain, light / heavy chain dimer or intact antibody, antigen-binding fragment, or other immunoglobulin form; see Beychok, Cells of Immunoglobulin Synthesis, Academic Press, NY, (1979). The polynucleotide or vector is then introduced into a cell that produces the antibody or antigen-binding fragment. Furthermore, transgenic animals, preferably mammals, containing the aforementioned host cells may be used for large-scale production of antibodies or antibody fragments.

[0264] Transformed host cells may be grown in fermentors and cultured using any suitable technique to achieve optimal cell growth. Once expressed, whole antibodies, their dimers, individual light and heavy chains, other immunoglobulin forms, or antigen-binding fragments may be purified by standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like; see Scopes, "Protein Purification," Springer Verlag, NY (1982). The antibodies or antigen-binding fragments may then be isolated from the growth medium, cell lysates, or cell membrane fractions. Isolation and purification of antibodies or antigen-binding fragments expressed, for example, in microorganisms, may be by any conventional means, including, for example, preparative chromatographic separations and immunological separations, including, for example, those involving the use of monoclonal or polyclonal antibodies directed against the constant regions of the antibodies.

[0265] Aspects of the present disclosure relate to hybridomas that provide an indefinitely sustainable source of monoclonal antibodies. As an alternative to obtaining immunoglobulins directly from hybridoma cultures, immortalized hybridoma cells may be used as a source of rearranged heavy and light chain loci for subsequent expression and / or genetic manipulation. Rearranged antibody genes may be reverse transcribed from the appropriate mRNA to produce cDNA. In some embodiments, the heavy chain constant region may be replaced with one of a different isotype or removed entirely. Variable regions may be linked to encode a single-chain Fv region. Multiple Fv regions may be linked to confer binding ability to more than one target, or chimeric heavy and light chain combinations may be used. Any suitable method may be used for cloning antibody variable regions and generating recombinant antibodies and antigen-binding portions thereof.

[0266] In some embodiments, appropriate nucleic acids encoding the heavy and / or light chain variable regions are obtained and inserted into expression vectors that can be transfected into standard recombinant host cells. A variety of such host cells may be used. In some embodiments, mammalian host cells may be advantageous for efficient processing and production. Exemplary mammalian cell lines useful for this purpose include CHO cells, 293 cells, or NSO cells. Production of antibodies or antigen-binding fragments may be carried out by culturing the modified recombinant host under culture conditions appropriate for host cell growth and expression of the coding sequences. Antibodies or antigen-binding fragments may be recovered by isolating them from the culture. Expression systems may be designed to include a signal peptide so that the resulting antibody is secreted into the medium; however, intracellular production is also possible.

[0267] The present disclosure also includes polynucleotides encoding at least the variable regions of the immunoglobulin chains of the antibodies described herein. In some embodiments, the variable regions encoded by the polynucleotides comprise at least one complementarity-determining region (CDR) of the VH and / or VL variable regions of the antibodies produced by any one of the hybridomas described above.

[0268] The polynucleotide encoding the antibody or antigen-binding fragment may be, for example, DNA, cDNA, RNA, or synthetically produced DNA or RNA, or a recombinantly produced chimeric nucleic acid molecule comprising any of these polynucleotides, alone or in combination. In some embodiments, the polynucleotide is part of a vector. Such vectors may contain additional genes, such as marker genes, that allow for the selection of the vector in a suitable host cell and under suitable conditions.

[0269] In some embodiments, the polynucleotide is operably linked to an expression control sequence that allows expression in prokaryotic or eukaryotic cells. Expression of the polynucleotide includes transcription of the polynucleotide into translatable mRNA. Control elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They may include a control sequence that promotes transcription initiation and, optionally, a polyA signal that promotes transcription termination and transcript stabilization. Additional control elements may include transcriptional and translational enhancers and / or naturally associated or heterologous promoter regions. Possible control elements that allow expression in prokaryotic host cells include, for example, the PL, Lac, Trp, or Tac promoters in E. coli. Examples of control elements that allow expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast, or the CMV promoter, SV40 promoter, RSV promoter (Rous sarcoma virus), CMV enhancer, SV40 enhancer, or globin intron in mammalian and other animal cells.

[0270] Such control elements, in addition to those involved in the initiation of transcription, may also include transcription termination signals downstream of the polynucleotide, such as the SV40 polyA site or the tk polyA site. Furthermore, depending on the expression system used, a leader sequence capable of directing the polypeptide into a cellular compartment or secreting it into the medium may be added to the coding sequence of the polynucleotide, as previously described. The leader sequence(s) is assembled in appropriate phase with the translation, initiation, and termination sequences, and preferably, the leader sequence is capable of directing the secretion of the translated protein or portion thereof, for example, into the extracellular medium. Heterologous polynucleotide sequences encoding fusion proteins containing C- or N-terminal identification peptides that confer desirable characteristics, such as stabilization or simplified purification of the expressed recombinant product, may also optionally be used.

[0271] In some embodiments, the polynucleotides encoding at least the variable domains of the light and / or heavy chains may encode both immunoglobulin chains or only one variable domain. Similarly, the polynucleotides may be under the control of the same promoter or may be separately regulated for expression. Furthermore, some aspects relate to vectors conventionally used in genetic engineering, particularly plasmids, cosmids, viruses, and bacteriophages, that contain polynucleotides encoding the variable domains of an immunoglobulin chain of an antibody or antigen-binding fragment, optionally in combination with polynucleotides encoding the variable domains of other immunoglobulin chains of the antibody.

[0272] In some embodiments, the expression control sequences are provided as eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, although control sequences for prokaryotic hosts may also be used. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses may be used to deliver the polynucleotide or vector to a targeted cell population (e.g., to engineer cells to express an antibody or antigen-binding fragment). A variety of suitable methods may be used to construct recombinant viral vectors. In some embodiments, the polynucleotides and vectors may be reconstituted into liposomes for delivery to target cells. The polynucleotide (e.g., encoding the sequence and expression control sequences) may be expressed in a variety of ways. The vectors containing the heavy and / or light chain variable domain(s) of an immunoglobulin chain may be transferred into a host cell by any suitable method, which varies depending on the type of cellular host.

[0273] qualification The antibodies and antigen-binding fragments of the present disclosure may be modified with a detectable label, including, but not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, non-radioactive paramagnetic metal ions, and affinity labels, for detection and isolation of pro / latent myostatin. The detectable substance may be coupled or conjugated to the polypeptide of the present disclosure either directly or indirectly through an intermediate (e.g., a linker) using suitable techniques. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, or acetylcholinesterase; non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; non-limiting examples of suitable fluorescent materials include biotin, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; non-limiting examples of bioluminescent materials include luciferase, luciferin, and aequorin; an example of a suitable radioactive material is, for example, iodine ( 131 I, 125 I, 123 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115 mIn, 113 mIn, 112 In, 111 In) and technetium ( 99 Tc, 99 mTc), thallium ( 201 Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, Lu, 159 Gd, 149 Pm, 140 La, 175 Yb,166 Ho, 90 Y, 47 Sc, 86 R, 188 Re, 142 Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se and tin ( 113 Sn, 117 Detectable substances include radioactive metal ions such as Sn), e.g., alpha emitters or other radioisotopes. Detectable substances may be coupled or conjugated to the anti-pro / latent myostatin antibodies, or antigen-binding portions thereof, of the present disclosure either directly or indirectly through an intermediate (e.g., a linker) using suitable techniques. Anti-pro / latent myostatin antibodies, or antigen-binding portions thereof, conjugated to a detectable substance can be used for the diagnostic assays described herein.

[0274] Biological effects of myostatin inhibitors, such as anti-pro / latent myostatin antibodies and antigen-binding fragments thereof Myostatin inhibitors encompassed by the present disclosure, such as antibodies and antigen-binding fragments thereof, can be used as pharmaceuticals to achieve a beneficial effect (e.g., a therapeutic effect) in a subject when administered to the subject in an effective amount. Exemplary such biologically beneficial effects are provided herein. A beneficial biological effect in a subject can be achieved by administering a myostatin inhibitor, such as an antibody or antigen-binding fragment thereof described herein that specifically binds to pro / latent myostatin. In some embodiments, the antibody or antigen-binding portion thereof is administered in an amount effective to elicit two or more of the following biological effects. In some embodiments, the myostatin inhibitor, such as an antibody or antigen-binding portion thereof, is administered in an amount effective to elicit three or more of the following biological effects. In some embodiments, the myostatin inhibitor, such as an antibody or antigen-binding portion thereof, is administered in an amount effective to elicit four or more of the following biological effects. In some embodiments, the myostatin inhibitor, such as an antibody or antigen-binding portion thereof, is administered in an amount effective to elicit five or more of the following biological effects. In some embodiments, the myostatin inhibitor, e.g., an antibody or antigen-binding portion thereof, is administered in an amount effective to elicit six or more of the following biological effects: In some embodiments, the myostatin inhibitor, e.g., an antibody or antigen-binding portion thereof, is administered in an amount effective to elicit seven or more of the following biological effects: hi some embodiments, the myostatin inhibitor, e.g., an antibody or antigen-binding portion thereof, is administered in an amount effective to elicit eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen of the following biological effects:

[0275] A. Effects on Muscle Tissue Mass and / or Function in Human Subjects Administration of a myostatin inhibitor, such as an antibody or antigen-binding fragment thereof that specifically binds to pro- or latent myostatin, increases the amount and / or function of muscle tissue in a human subject. In some embodiments, the muscle tissue is selected from the group consisting of smooth muscle tissue, skeletal muscle tissue, and cardiac muscle tissue. Smooth muscle tissue is composed of long, tapered cells, is generally involuntary, and differs from striated muscle by a much higher actin / myosin ratio, the absence of prominent sarcomeres, and the ability to contract to a small fraction of its resting length. Smooth muscle cells are found, inter alia, in the walls of blood vessels, around the intestine, and in the uterus. Cardiac muscle tissue is striated but is an involuntary tissue responsible for the pumping activity of the vertebrate heart. Individual cardiac muscle cells in striated muscle tissue do not fuse into multinucleated structures, as they do in striated muscle tissue. Skeletal muscle tissue is under voluntary control. Muscle fibers are syncytial and contain myofibrils, tandem arrays of sarcomeres. Skeletal muscle fibers are classified into two general types: slow-twitch (type I) and fast-twitch (type II) fibers, according to their expression of specific myosin heavy chain (MHC) isoforms. Slow-twitch fibers are better equipped to work aerobically and are useful for long-term endurance activities such as long-distance running, whereas fast-twitch fibers fatigue quickly but are better equipped to work anaerobically and are used for powerful burst movements such as sprinting. The distinction between slow-twitch and fast-twitch fibers is based on histochemical staining for myosin adenosine triphosphatase (ATPase) and the type of myosin heavy chain. Slow-twitch fibers (type I fibers) are MHC isoform I, and the three fast-twitch isoforms (type II fibers) are MHC isoform IIa, MHC isoform IId, and MHC isoform IIb (S. Schiaffino, J. Muscle Res. Cell. Motil., 10 (1989), pp. 197-205). In some embodiments, the mass and / or function of fast-twitch muscle tissue is increased in a human subject. In other embodiments, the mass and / or function of slow-twitch muscle tissue is increased in a human subject.

[0276] The biological effect of an effective amount of the pharmaceutical compositions provided herein may be related to a change in muscle fiber type phenotype, a process called fiber type switching. In some embodiments, fiber type switching is induced by events such as injury and starvation.

[0277] In one aspect, the disclosure provides a method for promoting fiber type switching in a subject, the method comprising administering to the subject a composition comprising a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin and blocks release of mature myostatin, in an amount effective to promote fiber type switching, thereby promoting fiber type switching in the subject.

[0278] In another aspect, the present disclosure provides a method for preferentially increasing type II or fast twitch muscle fibers over type I or slow twitch muscle fibers in a subject, the method comprising:

[0279] The method includes administering to a subject a myostatin inhibitor, for example, a composition comprising an antibody or its antigen-binding fragment that specifically binds to pro / latent myostatin and blocks the release of mature myostatin, in an amount effective to preferentially increase type II or fast-twitch muscle fibers over type I or slow-twitch muscle fibers, thereby preferentially increasing type II or fast-twitch muscle fibers over type I or slow-twitch muscle fibers in the subject.

[0280] In some embodiments, administering an effective amount of a myostatin inhibitor, such as an antibody or antigen-binding fragment thereof described herein, to a subject can result in an increase in muscle mass. Preferably, such an increase in muscle mass is clinically meaningful to benefit or otherwise improve the subject's health status. For example, clinically meaningful changes in muscle mass can improve a patient's mobility, self-care, metabolism, etc. In some embodiments, the increase in muscle mass is an increase in lean muscle(s). In some embodiments, such an increase in muscle mass is a systemic effect, such that the entire body or substantially the entire body's muscles show a measurable effect. In other embodiments, the effect is localized to a specific group / type of muscle. In some embodiments, the amount of muscle tissue, e.g., lean muscle tissue, is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the amount of muscle tissue, e.g., lean muscle tissue, is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%. Such increases in muscle mass can be estimated or measured by any suitable known method, including measuring cross-sectional area (e.g., forearm cross-section) by MRI, circumference, diaphragm width (e.g., by ultrasound), etc.

[0281] In some embodiments, administration of an effective amount of an antibody or antigen-binding fragment thereof described herein to a subject can result in enhanced muscle function. Muscle function can be assessed by various measures, including, but not limited to, force generation, grip strength (e.g., maximal grip strength), endurance, muscle oxidative capacity, dynamic grip endurance, etc. In some embodiments, serum creatinine levels are used as a validated biomarker of muscle mass, although it has limited sensitivity.

[0282] In some embodiments, muscle tissue function is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, muscle tissue function is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%. In some embodiments, increased muscle function includes an improvement in a rating, e.g., from 1 to 2, from 2 to 3, from 3 to 4, from 4 to 5, from 5 to 6, from 6 to 7, from 7 to 8, from 8 to 9, or from 9 to 10.

[0283] In some embodiments, myostatin inhibitors for use in the methods of the present invention, such as anti-pro / latent myostatin antibodies or antigen-binding fragments thereof, can increase the amount and / or function of muscle tissue in subjects suffering from pathologies resulting from, for example, spinal cord injury. In some embodiments, the subject is in the acute spinal cord injury phase immediately after injury, where it is generally difficult to distinguish between complete and incomplete spinal cord injury. In other embodiments, the subject is in the subacute spinal cord injury phase, where there is a difference between complete and incomplete spinal cord injury and where continued rehabilitation may result in recovery. In yet another embodiment, the subject is in the chronic spinal cord injury phase. The chronic SCI phase occurs approximately four or six months from the date of injury, when the patient shows a substantial decline in the rate of recovery or when rehabilitation attempts reach a plateau despite continued attempts at standard treatment.

[0284] In some embodiments, the amount and / or function of muscle tissue below the lesion is increased in a subject suffering from a lesion, e.g., spinal cord injury. In other embodiments, the amount and / or function of muscle tissue above the lesion is increased in a subject suffering from a lesion, e.g., spinal cord injury. In some embodiments, the muscle is selected from the group consisting of soleus, gastrocnemius, biceps, and triceps. In some embodiments, the amount of muscle tissue is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100 %, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% increase in muscle tissue. In other embodiments, the amount of muscle tissue is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%. In some embodiments, muscle tissue function is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, muscle tissue function is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0285] In some embodiments, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, increases locomotor function in a human subject, e.g., a subject suffering from a pathology. In some embodiments, the locomotor function of the human subject is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the locomotor function of the human subject is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0286] In some embodiments, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, increases motor coordination and balance in a human subject, e.g., a subject suffering from a pathology. In some embodiments, the motor coordination and balance of the human subject is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the motor coordination and balance of the human subject is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0287] In another embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, to a human subject, e.g., a subject suffering from a pathology, increases muscle strength. In some embodiments, the muscle strength of the human subject increases by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the muscle strength of the human subject increases by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0288] In some embodiments, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, can produce clinically meaningful changes in muscle function corresponding to increased functionality in a patient. In some embodiments, increased functionality includes improvements in a patient's mobility, self-care, metabolism, and the like. In some embodiments, administration of an effective amount of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, facilitates or accelerates recovery from conditions such as injury, surgery, and other medical procedures. Suitable such conditions include conditions associated with neuropathy (whether resulting from injury, surgical, or other clinical This may include any adverse events (whether procedurally or otherwise).

[0289] For example, suitable subjects include generally healthy individuals, such as: i) patients with persistent acute injury accompanied by neuropathy affecting muscle function; ii) patients scheduled for surgical procedures (therapeutic or corrective) that may cause unintended nerve damage (e.g., motor neuron injury); iii) patients who have undergone surgical procedures that have caused unintended muscle dysfunction; iv) patients undergoing procedures involving immobilization of a particular muscle or muscle group (e.g., cast immobilization); and v) patients undergoing mechanical ventilation (e.g., as a result of an acute injury). Administration of a myostatin inhibitor as described herein may accelerate recovery in such patients. In some embodiments, such administration may be prophylactic. For example, an antibody may be administered prior to or immediately after a surgical procedure that may cause neuropathy and associated muscle dysfunction to prevent muscle dysfunction. Prevention includes alleviating or reducing the severity of such dysfunction. In these embodiments, administration may be localized, e.g., to or near the site of an area affected by injury, surgery, or the like.

[0290] B. Effects on metabolic rate in human subjects Administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, increases the metabolic rate of a human subject. In some embodiments, administration of an effective amount of such a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, can increase the basal metabolic rate in a subject. Metabolic rate can be calculated by any method known in the art, for example, by measuring oxygen input and carbon dioxide output, or by indirect calorimetry as demonstrated in Example 11 of the present application. In some embodiments, metabolic rate is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the metabolic rate is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0291] C. Effects on insulin sensitivity in human subjects Administration of a myostatin inhibitor, such as an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, increases insulin sensitivity in a human subject. Methods for measuring insulin sensitivity are known in the art, such as the glucose tolerance test and fasting insulin or glucose testing. During a glucose tolerance test, a fasting patient ingests a 75-gram oral dose of glucose, and blood glucose levels are then measured over the next two hours. Blood glucose levels below 7.8 mmol / L (140 mg / dL) are considered normal, blood glucose levels between 7.8 mmol / L and 11.0 mmol / L (140-197 mg / dL) are considered impaired glucose tolerance (IGT), and blood glucose levels greater than or equal to 11.1 mmol / L (200 mg / dL) are considered diabetic. With regard to fasting insulin testing, fasting serum insulin levels greater than 25 mIU / L or 174 pmol / L are considered insulin resistant. In some embodiments, the metabolic rate is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the metabolic rate is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0292] D. Effects on Adipose Tissue Levels in Human Subjects The administration of a myostatin inhibitor, for example, an antibody or its antigen-binding fragment that specifically binds to pro / latent myostatin, affects the level of adipose tissue in human subjects.As used herein, the term "adipose tissue" refers to fat, including connective tissue that stores fat.Adipose tissue is derived from preadipocytes.Its main role is to store energy in the form of lipids, but it also cushions and sequesters the body.The two types of adipose tissue are white adipose tissue (WAT), which stores energy, and brown adipose tissue (BAT), which generates body heat.

[0293] Brown adipose tissue (BAT) is known to function in chemical energy dissipation in response to cold or excessive feeding, and also has the ability to regulate energy balance. Activation of brown adipose tissue has been shown to improve glucose homeostasis and insulin sensitivity in humans, suggesting that BAT activation may be beneficial for anyone with impaired insulin function (Stanford et al., J Clin Invest., 2013, 123(1):215-223).

[0294] Beige adipose tissue results from the browning of WAT, also known as beiging. This occurs when adipocytes within WAT depots develop characteristics of BAT. Beige adipocytes take on a multivesicular appearance (containing several lipid droplets) and increase the expression of uncoupling protein 1 (UCP1). In doing so, these normally energy-storing white adipocytes become energy-releasing adipocytes (Harms et al., Nature Medicine. 2013, 19(10):1252-63).

[0295] Visceral or abdominal fat (also known as organ fat or intra-abdominal fat) is located inside the abdominal cavity and packed between organs (such as the stomach, liver, intestines, and kidneys). Visceral fat differs from subcutaneous fat, which is located under the skin, and intramuscular fat, which is scattered within skeletal muscles. Fat in the lower body, such as the thighs and buttocks, is subcutaneous and not a coherent, spaced tissue, while abdominal fat is mostly visceral and semi-liquid. Excess visceral fat is known as central obesity, or "belly fat," and new developments, such as the Body Volume Index (BVI), are specifically designed to measure abdominal volume and abdominal fat. Excess visceral fat is also associated with type 2 diabetes, insulin resistance, inflammatory diseases, and other obesity-related disorders (Mokdad et al., JAMA: The Journal of the American Medical Association, 2001, 289(1):76-9).

[0296] The amount of adipose tissue can be determined by any method known to those skilled in the art. For example, adipose tissue can be measured by dual-energy X-ray absorptiometry (DXA), as demonstrated in Example 11 of the present application.

[0297] Administration of a myostatin inhibitor, such as an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, increases the level of brown adipose tissue and / or beige adipose tissue in a human subject, while administration of a myostatin inhibitor, such as an anti-pro / latent myostatin antibody or antigen-binding portion thereof, decreases the level of white adipose tissue and visceral adipose tissue in a human subject.

[0298] In some embodiments, levels of brown adipose tissue or beige adipose tissue are increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, levels of brown adipose tissue or beige adipose tissue are increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 80%, 90%, or 100%. increases by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0299] In some embodiments, levels of white adipose tissue or visceral adipose tissue are reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, levels of white adipose tissue or visceral adipose tissue are reduced by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0300] E. Effects on Fat to Muscle Tissue Ratio in Human Subjects Administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, reduces the ratio of adipose tissue to muscle tissue in a human subject. In some embodiments, the ratio of adipose tissue to muscle tissue is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the ratio of adipose tissue to muscle tissue is reduced by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0301] F. Effects on glucose uptake in human subjects Administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, affects glucose uptake by tissue in a human subject. In some embodiments, glucose uptake by muscle tissue is increased. For example, glucose uptake by muscle tissue is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, glucose uptake by muscle tissue is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0302] In other embodiments, glucose uptake by white adipose tissue, liver tissue, and vascular tissue is reduced. In some embodiments, glucose uptake by white adipose tissue, liver tissue, and vascular tissue is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, glucose uptake by white adipose tissue, liver tissue, and vascular tissue is reduced by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0303] G. Effects on muscle protein catabolism and / or amino acid release from muscle in human subjects Administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, reduces muscle protein catabolism and / or muscle amino acid release in a human subject. In some embodiments, muscle protein catabolism and / or muscle amino acid release is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, muscle protein catabolism and / or muscle amino acid release is reduced by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0304] H. Effects on insulin-dependent blood glucose regulation in human subjects Administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, increases insulin-dependent glycemic control in a human subject. In some embodiments, insulin-dependent glycemic control is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, insulin-dependent glycemic control is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0305] I. Effects on intramuscular fat infiltration in human subjects Administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, reduces intramuscular fat infiltration in a human subject. In some embodiments, intramuscular fat infiltration is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, intramuscular fat infiltration is reduced by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0306] J. Effects on Quality of Life of Human Subjects Assessment of quality of life in patients with severe or chronic conditions, such as SCI patients, can involve a comprehensive approach to assessing various aspects of physical, mental, social, and other parameters. Generally, a higher level of quality of life is associated with factors such as availability of assistive technology; community reintegration; lower limb functionality and walking and / or wheeled mobility; mental health; severity of neurological and autonomic impairment; pain management; functional independence and self-care; upper limb strength; and spasticity control. Administration of an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin increases the quality of life of a human subject, achieving clinically meaningful improvements as measured by standardized quality of life tests / systems. A number of suitable tests for assessing quality of life in patients are known in the art, including the Incontinence Quality of Life Questionnaire (I-QOL); Life Satisfaction Questionnaire (LISAT-9, LISAT-11); Quality of Life Index (QLI)-SCI Version; Quality of Life Profile for Adults with Physical Disabilities (QOLP-PD); Quality of Well Being (QWB) and Quality of Well Being-Self-Administered (QWB-SA); Qualiveen; Satisfaction with Life Scale (SWLS, Deiner Scale); Short Form 36 (SF-36); Sickness Impact Profile 68 (SIP 68); and the World Health Organization Quality of Life-BREF (WHOQOL-BREF).

[0307] In some embodiments, quality of life is assessed using the validated scoring system SF- Myostatin scores are assessed according to the SF-36 Quality of Life Scoring System, where a change of 8 points is considered clinically meaningful. Typically, for SCI patients, values ​​are in the low 50s. In some embodiments, administration of an effective amount of an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin results in a clinically meaningful improvement in a standardized quality of life test score. As used herein, the term "clinically meaningful improvement" refers to a significant improvement relative to a standard level. In some embodiments, the SF-36 Quality of Life score of an SCI patient increases by at least 8 points after treatment with an effective amount of an antibody or antigen-binding fragment thereof described herein compared to the patient's score before treatment. In some embodiments, the patient achieves a higher score as assessed by the SF-36 Quality of Life Test, e.g., an increase in the SF-36 Quality of Life Scoring System score of at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 points. In other embodiments, the patient achieves an increase in the SF-36 Quality of Life Scoring System score of at least about 8-10, 10-15, 15-20, 20-30, 30-40, 40-50, 8-20, 8-30, 8-40, or 8-50.

[0308] In some embodiments, the SCI Neurological Quality of Life Test is used to assess a patient's quality of life before and after treatment with the inhibitors of myostatin signaling disclosed herein. Advantages of this test include: i) it is easy to administer; ii) it assesses both physical function and mental health; and iii) it is highly validated for several clinical indications.

[0309] K. Effect on preventing muscle loss or atrophy in human subjects Administration of an effective amount of a myostatin inhibitor, such as an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, prevents muscle loss or atrophy in a human subject at risk of developing muscle loss and / or atrophy. In some embodiments, muscle loss or atrophy is reduced or prevented by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, muscle loss or atrophy is reduced or prevented by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0310] In some embodiments, suitable subjects are those who have not yet developed atrophy but are considered to be at risk of developing atrophy. In some embodiments, the subject has a disease or condition associated with a neurological defect that impairs motor neuron function. In some embodiments, such a condition is caused by muscular dystrophy or atrophy. In some embodiments, the neurological defect is caused by nerve damage. In some embodiments, the nerve damage involves partial denervation of motor neurons, causing partial impairment of function in the affected muscles. In some embodiments, such a condition is caused by SCI. In some embodiments, the subject with SCI is in the acute or subacute phase of SCI (e.g., has not yet reached the chronic phase).

[0311] In some embodiments, when a composition comprising an effective amount of an inhibitor of myostatin signaling described herein is administered to a population of patients at risk of developing muscle atrophy associated with partial denervation of motor neurons, the composition i) prevents the onset or worsening of muscle atrophy in a statistically significant proportion of the patient population, or ii) reduces the severity of muscle atrophy in a statistically significant proportion of the patient population.

[0312] Prevention of muscle loss or muscle atrophy by using a myostatin inhibitor, such as an antibody or antigen-binding fragment thereof described herein, can be easily monitored or evaluated by any suitable method for assessing motor function involving the affected muscles.

[0313] In some embodiments, administration of an effective amount of such an antibody also prevents or reduces early onset axonal polyneuropathy in the affected limb.

[0314] L. Effect on preventing the development of metabolic diseases in subjects Administration of an effective amount of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, prevents the development of metabolic disorders in a subject, e.g., a human subject. In some embodiments, the development of metabolic disorders is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the development of metabolic disorders is reduced by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0315] In some embodiments, suitable subjects are those who have not yet fully developed metabolic disease but are considered to be at risk of developing such a condition. In some embodiments, the subject has a disease or condition associated with muscle dysfunction. In some embodiments, the muscle dysfunction is associated with partial denervation of motor neurons, causing partial impairment of function in the affected muscles. In some embodiments, such a condition is caused by muscular dystrophy or atrophy. In some embodiments, such a condition is caused by SCI. In some embodiments, the subject with SCI is in the acute or subacute phase of SCI (e.g., has not yet reached the chronic phase).

[0316] In some embodiments, when a composition comprising an effective amount of an inhibitor of myostatin signaling described herein is administered to a population of patients at risk of developing a metabolic disorder associated with muscle dysfunction, the composition i) prevents the manifestation or worsening of the metabolic disorder in a statistically significant proportion of the patient population, or ii) reduces the severity of the metabolic disease in a statistically significant proportion of the patient population.

[0317] In some embodiments, effects on metabolism may be monitored or measured by insulin resistance, lipid panels / markers (e.g., leptin), inflammatory and oxidative stress markers including, but not limited to, IL-6, TNF, CRP, plasma total antioxidant status, lipid oxidation, and erythrocyte glutathione peroxidase activity.

[0318] Uses of myostatin inhibitors, such as anti-pro / latent myostatin antibodies and antigen-binding fragments thereof Pharmaceutical Composition Myostatin inhibitors, such as the antibodies or antigen-binding fragments thereof described herein, can be formulated into pharmaceutical compositions suitable for administration to human or non-human subjects. Such pharmaceutical compositions can be intended for therapeutic or prophylactic use. One or more myostatin inhibitors, such as anti-pro / latent myostatin antibodies, can be mixed with pharmaceutically acceptable carriers (excipients), including buffers, to form pharmaceutical compositions for administration to patients who may benefit from reduced myostatin signaling in vivo. "Pharmaceutically acceptable" means that the carrier must be compatible with the active ingredients of the composition (and preferably be able to stabilize the active ingredients) and not harmful to the subject being treated. Examples of pharmaceutically acceptable excipients (carriers), including buffers, will be apparent to those skilled in the art and have been previously described. See, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (2000), Lippincott Williams and Wilkins, edited by K. E. Hoover. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; 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 dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0319] In one example, the pharmaceutical compositions described herein contain more than one myostatin inhibitor, for example, more than one anti-pro / latent myostatin antibody or antigen-binding portion thereof that recognizes different epitopes / residues of a target antigen.

[0320] In some examples, the pharmaceutical compositions described herein include emulsion-based or lipid-based formulations, such as liposomes containing myostatin inhibitors, e.g., anti-pro / latent myostatin antibodies or antigen-binding portions thereof, which can be prepared by any suitable method, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be produced by reverse-phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to obtain liposomes with the desired diameter.

[0321] Myostatin inhibitors, such as anti-pro / latent myostatin antibodies or antigen-binding portions thereof, can also be entrapped in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacylate) microcapsules, respectively, in macroemulsions. Exemplary techniques have been previously described, see, for example, Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing (2000).

[0322] In another example, the pharmaceutical compositions described herein may be formulated in a sustained-release format. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, or antigen-binding portion thereof, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyester hydrogels (e.g., poly(2-hydroxybenzoates)). Examples of suitable poly(lactic acid copolymers) include poly(hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactide lactic acid (U.S. Pat. No. 3,773,919), copolymer of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0323] Pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0324] The pharmaceutical compositions described herein may be in unit dosage form such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories for oral, parenteral, or rectal administration, or for administration by inhalation or insufflation.

[0325] To prepare solid compositions such as tablets, the primary active ingredient may be mixed with pharmaceutical carriers, such as conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gums, and other pharmaceutical diluents, such as water, or non-toxic pharmaceutically acceptable salts thereof, to form solid preformulation compositions containing a homogeneous mixture of the compounds of the present disclosure. When these preformulation compositions are referred to as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition, allowing the composition to be easily divided into equally effective unit dosage forms such as tablets, pills, and capsules. This solid preformulation composition is then divided into unit dosage forms of the type described above containing from 0.1 mg to about 500 mg of the active ingredient of the present disclosure. Tablets or pills of the novel compositions may be coated or otherwise compounded to provide a dosage form offering the advantage of prolonged action. For example, the tablets or pills may comprise an inner dosage and an outer dosage component, the latter in the form of an envelope surrounding the former. The two components may be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0326] Suitable surfactants include, in particular, non-ionic agents such as polyoxyethylene sorbitan (e.g., Tween™ 20, 40, 60, 80, or 85) and other sorbitan (e.g., Span™ 20, 40, 60, 80, or 85). Compositions that include a surfactant conveniently contain 0.05 to 5% surfactant, and may be 0.1 to 2.5%. It will be appreciated that other ingredients, such as mannitol or other pharmaceutically acceptable vehicles, may be added as required.

[0327] Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™.The active ingredient can be dissolved in a premixed emulsion composition, or alternatively, can be dissolved in an emulsion formed by mixing oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and phospholipid (e.g., egg phospholipid, soybean phospholipid or soybean lecithin) with water.It is understood that other ingredients, such as glycerol or glucose, can be added to adjust the tonicity of the emulsion.Suitable Emulsions typically contain up to 20% oil, for example 5 to 20%.

[0328] The emulsion composition may be prepared by mixing an anti-promyostatin antibody with Intralipid™ and its components (soybean oil, egg phospholipids, glycerol, and water).

[0329] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0330] Compositions, preferably in sterile, pharmaceutically acceptable solvents, may be nebulized by the use of gases. Nebulized solutions may be breathed directly from the nebulizing device, or the nebulizing device may be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered from devices that deliver the formulation in an appropriate manner, preferably orally or nasally.

[0331] Subject The pharmaceutical compositions described herein are suitable for administration to human or non-human subjects. Thus, myostatin inhibitors, such as the anti-pro / latent myostatin antibodies and antigen-binding portions thereof described herein, are useful as pharmaceuticals for administration to subjects who may benefit from reduced myostatin signaling. In some embodiments, suitable subjects may include individuals who are healthy but who may benefit from increased muscle mass / function and improved metabolism. In some embodiments, suitable subjects have a pre-existing muscle condition and / or associated metabolic dysfunction. In some embodiments, suitable subjects are at risk for developing such condition(s). In some embodiments, suitable subjects are those undergoing therapy with another therapeutic agent intended to treat a muscle / metabolism condition but with associated adverse effects or toxicity.

[0332] In some embodiments, preferred subjects meet at least two of the following criteria: i) the subject has a condition associated with partial denervation of motor neurons; ii) the condition contains fast-twitch muscle fibers or involves muscles rich in fast-twitch muscle fibers; and iii) the subject retains anabolic capacity (e.g., generally healthy adults with injuries) and / or is in a growing stage (e.g., young children, etc.).

[0333] In some embodiments, such medicaments are suitable for administration in pediatric, adult, and / or geriatric populations.

[0334] Pediatric populations in need of myostatin inhibitors, e.g., anti-pro / latent myostatin antibodies and antigen-binding portions thereof described herein, can range in age from 0 to 6 months, 0 to 12 months, 0 to 18 months, 0 to 24 months, 0 to 36 months, 0 to 72 months, 6 to 36 months, 6 to 36 months, 6 to 72 months, 12 to 36 months, 12 to 72 months. In some embodiments, pediatric populations suitable for receiving myostatin inhibitors, e.g., antibodies or antigen-binding fragments described herein, and who may benefit from such treatment, can range in age from 0 to 6 years, 0 to 12 years, 3 to 12 years, or 0 to 17 years. In some embodiments, the age of the population is at least 5 years old, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 years old. In some embodiments, the pediatric population may be under 18 years old. In some embodiments, the pediatric population is (a) at least 5 years old and (b) 18 years old. It may be less than.

[0335] The adult population in need of myostatin inhibitors, e.g., the anti-pro / latent myostatin antibodies and antigen-binding portions thereof described herein, can be at least 18 years of age, e.g., at least 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65 years of age. In some embodiments, the adult population can be under 65 years of age. In some embodiments, the adult population can be (a) at least 18 years of age and (b) under 65 years of age.

[0336] The elderly population in need of myostatin inhibitors, such as the anti-pro / latent myostatin antibodies and antigen-binding portions thereof described herein, may be 65 years of age or older (i.e., ≥ 65 years of age), for example, at least 70, 75 or 80 years of age.

[0337] Human subjects who may benefit from treatment may be human patients who have, are at risk of developing, or are suspected of having a metabolic disease / disorder associated with defective neural signaling, such as those described below. Subjects with pro / latent myostatin-related diseases or disorders may be identified by routine medical tests, such as laboratory tests, organ function tests, CT scans, or ultrasound. A subject suspected of having any of such diseases / disorders may exhibit one or more symptoms of the disease / disorder. A subject at risk for a disease / disorder may be a subject who has one or more risk factors for the disease / disorder.

[0338] The control subject described herein is a subject that provides a suitable reference for evaluating the effectiveness of a particular treatment or intervention on a test subject or subject.The control subject can be a subject of similar age, race, sex, weight, height, and / or other characteristics to the test subject, or any combination thereof.

[0339] In some embodiments, a myostatin assay (e.g., myostatin ELISA) is used to determine subjects in need of treatment with an anti-pro / latent myostatin antibody. Methods for assaying myostatin are described in Lakshman et al., Molecular and Cell Endocrinology, (2009) 302:26-32 (myostatin ELISA) and Bergen et al., Skeletal Muscle, (2015), both of which are incorporated herein by reference. 5:21 (Liquid Chromatography with Tandem Mass Spectrometry).

[0340] In some embodiments, a method for improving muscle performance in a subject is provided. The subject may or may not have, be at risk of, or not have a condition associated with reduced muscle mass and / or reduced muscle function. As used herein, the term "muscle performance" generally refers to a muscle's ability to contract and / or apply force (e.g., to an external object). In some embodiments, muscle performance may relate to a muscle's ability to consume energy. For example, in some embodiments, muscle performance may relate to a muscle's ability to generate and / or consume adenosine triphosphate (ATP) molecules to facilitate muscle contraction. In some embodiments, muscle performance refers to a muscle's ability to repeatedly contract for a specific duration. In some embodiments, muscle performance refers to a muscle's ability to apply force to an object, e.g., to move an object over a measurable distance. In some embodiments, muscle performance refers to a muscle's ability to apply force to an object over a specific duration (e.g., to move an object over a measurable distance for a specific duration).

[0341] In some embodiments, a myostatin inhibitor, such as an anti-pro / latent myostatin inhibitor described herein, is used. The myostatin antibodies and antigen-binding portions thereof are administered to a subject in need of treatment in an amount sufficient to inhibit proteolytic activation of pro / latent myostatin to active myostatin in vivo by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more). In other embodiments, the myostatin inhibitor, e.g., an antibody or antigen-binding portion thereof, is administered in an amount effective to reduce pro / latent or latent myostatin levels by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more).

[0342] In some embodiments, a myostatin inhibitor, such as an anti-pro / latent myostatin antibody or antigen-binding portion thereof described herein, is administered to a subject who would benefit from increased muscle mass. In some embodiments, a myostatin inhibitor, such as an anti-pro / latent myostatin antibody or antigen-binding portion thereof described herein, is administered to a subject who would benefit from an increased muscle-to-fat ratio. In some embodiments, a myostatin inhibitor, such as an anti-pro / latent myostatin antibody or antigen-binding portion thereof described herein, is administered to a subject who would benefit from increased muscle function. In some embodiments, the subject may have, not have, be at risk for, or not be at risk for a condition associated with decreased muscle mass and / or decreased muscle function. In some embodiments, the subject has or is at risk for a condition associated with decreased muscle mass and / or decreased muscle function.

[0343] The methods of the present invention further include selecting a subject. In some embodiments, the subject has or is at risk of developing a muscle condition or disorder. In some embodiments, the subject has or is at risk of developing a metabolic disorder. In some embodiments, the subject has or is at risk of developing a disease or disorder associated with defective neural signaling.

[0344] Route of administration To practice the methods described herein, an effective amount of the pharmaceutical composition described above can be administered to a subject (e.g., a human) in need of treatment via a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation, or topical. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be directly nebulized, or lyophilized powders can be nebulized after reconstitution. Alternatively, anti-pro / latent myostatin antibodies can be aerosolized using a fluorocarbon formulation and a metered-dose inhaler, or inhaled as a lyophilized, milled powder.

[0345] Conventional methods known to those skilled in the art of medicine may be used to administer pharmaceutical compositions to subjects depending on the type or location of the disease being treated. The compositions may also be administered via other conventional routes, such as orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Additionally, the compositions may be administered to subjects via injectable depot routes of administration, such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.

[0346] The injectable composition may contain vegetable oil, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propanediol, propyl paraben ... The antibody may contain a variety of carriers, such as propylene glycol, liquid polyethylene glycol, etc. For intravenous injection, water-soluble antibodies may be administered by infusion, in which a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. For intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the antibody may be dissolved in a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% glucose solution and administered.

[0347] In one embodiment, the myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody or antigen-binding portion thereof, is administered via site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of the myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody or antigen-binding portion thereof, or local delivery catheters such as infusion catheters, indwelling catheters, or needle catheters, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site-specific carriers, direct injection, or direct application. See, e.g., PCT Publication No. WO00 / 53211 and U.S. Patent No. 5,981,568.

[0348] The particular administration regimen used in the methods described herein, eg, dosage, timing, and repetition, will depend on the particular subject and that subject's medical history.

[0349] The efficacy of a treatment for a disease / disorder associated with myopathy can be evaluated using any suitable method. For example, the efficacy of a treatment for a disease / disorder associated with myopathy can be evaluated by assessing muscle weakness (e.g., assessing the pattern and severity of weakness), electromyography, assessing blood chemistry (e.g., assessing electrolytes, assessing endocrine causes, measuring creatine kinase levels, determining erythrocyte sedimentation rate, and performing antinuclear antibody assays), and evaluating biopsies (e.g., by histological, histochemical, electron microscopic, biochemical, and genetic analysis).

[0350] "Effective amount," as used herein, refers to the amount of each active agent, either alone or in combination with one or more other active agents, required to confer a therapeutic effect on a subject. For example, an effective amount may be a biological effect, such as an increase in muscle mass or muscle fiber diameter, a switch in muscle fiber type, an increase in the amount of force produced by a muscle, an increase in the mass and / or function of muscle tissue in a subject; an increase in the metabolic rate of a subject; an increase in insulin sensitivity in a subject; an increase in the level of brown adipose tissue in a subject; an increase in the level of beige adipose tissue in a subject; a decrease in the level of white adipose tissue in a subject; a decrease in the level of visceral adipose tissue in a subject; a decrease in the ratio of adipose tissue to muscle tissue in a subject; an increase in glucose uptake by brown adipose tissue, beige adipose tissue, or muscle tissue in a subject; a decrease in glucose uptake by white adipose tissue or hepatic tissue; a decrease in muscle catabolism of protein and / or release of amino acids from muscle in a subject; an increase in insulin-dependent blood glucose regulation in a subject; or a decrease in intramuscular fat infiltration in a subject; or a clinically significant outcome, such as partial or complete recovery of the ability to perform a physical task after injury; "Myostatin inhibitory effect" refers to the amount of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, of the present disclosure, sufficient to achieve a clinically meaningful improvement in quality of life as assessed by a standardized system, such as the NIH System; prevention of muscle loss or muscle atrophy in a subject; and / or prevention of the development of metabolic disease in a subject.

[0351] As will be appreciated by those skilled in the art, an effective amount will depend on the particular condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex and weight, the duration of treatment, the nature of concurrent therapy (if any), the particular route of administration and the knowledge of the medical practitioner. The dosage may vary depending on factors such as the dosage and similar factors within professional opinion. These factors are well known to those skilled in the art and can be addressed with no more than routine experimentation. It is generally preferred that the maximum dosage of each component or combination thereof, i.e., the highest safe dosage according to sound medical judgment, be used. However, it will be understood by those skilled in the art that a patient may insist on a lower or tolerable dosage for medical reasons, psychological reasons, or virtually any other reason.

[0352] In some embodiments, with respect to increasing the level of promyostatin in the target muscle, the increase is at least 1-fold, 1.2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold (or any range bracketed by any of these values) compared to the control level of promyostatin. In one embodiment, the increase in the level of promyostatin in the target muscle is in the range of 1-fold to 3-fold, 1.2-fold to 10-fold, 2-fold to 9-fold, 3-fold to 8-fold, 4-fold to 7-fold, 2-fold to 7-fold, etc., compared to the control level of promyostatin.

[0353] In some embodiments, with respect to the increase in latent myostatin in the target muscle after the administering step, the increase is detectable within 4 hours, 24 hours, 48 ​​hours, 7 days, 14 days, 21 days, 28 days, or 30 days (or any range of time bracketed by any of the recited durations) after the administering step. In one embodiment, the increase in latent myostatin in the target muscle after the administering step is detectable for at least 5 days, 7 days, 14 days, 21 days, 28 days, or 30 days (or any range of time bracketed by any of the recited durations) after the administering step. In one embodiment, the increase in the level of latent myostatin in the target muscle after the administering step is at least 1-fold, 1.2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold (or any range bracketed by any of these values) compared to the level of latent myostatin in the target muscle before the administering step. In one embodiment, the increase in the level of latent myostatin in the target muscle after the administering step is in the range of 1-fold to 3-fold, 1.2-fold to 10-fold, 2-fold to 9-fold, 3-fold to 8-fold, 4-fold to 7-fold, 2-fold to 7-fold, etc., compared to the level of latent myostatin in the target muscle before the administering step.

[0354] In some embodiments, with respect to the increase in circulating latent myostatin after the administering step, the increase is detectable within 4 hours, 24 hours, 48 ​​hours, 7 days, 14 days, 21 days, 28 days, or 30 days (or any range of times lumped around any of the durations recited) after the administering step. In one embodiment, the increase in circulating latent myostatin after the administering step is detectable for at least 5 days, 7 days, 14 days, 21 days, 28 days, or 30 days (or any range of times lumped around any of the durations recited) after the administering step. In one embodiment, the increase in circulating latent myostatin levels after the administering step is at least 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold or more (or any range of times lumped around any of these values) compared to the level of circulating latent myostatin before the administering step. In one embodiment, the increase in the level of latent myostatin in the target muscle after the administering step is in the range of 1-fold to 3-fold, 1.2-fold to 10-fold, 2-fold to 9-fold, 3-fold to 8-fold, 4-fold to 7-fold, 2-fold to 7-fold, etc. compared to the level of latent myostatin in the target muscle before the administering step.

[0355] In some embodiments, with respect to a reduction in levels of circulating latent myostatin, the reduction is at most 1-fold, 1.2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or less (or less) compared to control levels of latent myostatin. In one embodiment, the reduction in circulating latent myostatin levels is in the range of 1 / 3, 1.2-10, 2 / 2-9, 3 / 8, 4 / 7, 2 / 2-7, etc., relative to control levels of latent myostatin.

[0356] As noted above, in some embodiments, with respect to administration of a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof, to a subject, an effective amount is an amount effective to increase the mass of a target muscle in the subject compared to control muscle mass. In some embodiments, muscle treated with an effective amount of the antibody increases by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, etc., compared to control muscle mass not treated with an effective amount of the antibody. In some embodiments, such increases in muscle mass are achieved in selected muscle groups or types in the subject.

[0357] In some embodiments, with respect to administration of a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof, to a subject, an effective amount is an amount effective to switch fiber type in the subject. In some embodiments, an effective amount of the antibody can promote fiber type switching from type I to type II. In some embodiments, an effective amount of the myostatin inhibitor, e.g., an antibody or antigen-binding portion thereof, can promote fiber type switching from type I to type IIB. In some embodiments, an effective amount of the myostatin inhibitor, e.g., an antibody or antigen-binding portion thereof, can promote type II fibers over other fiber types. In some embodiments, an effective amount of the myostatin inhibitor, e.g., an antibody or antigen-binding portion thereof, can promote type IIB fibers over other fiber types. In some embodiments, such fiber phenotype switching can occur without a significant change in overall muscle mass. In other embodiments, such fiber phenotype switching can occur concomitantly with an increase in overall muscle mass.

[0358] In some embodiments, with respect to administration of a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof, to a subject, an effective amount is an amount effective to increase muscle fiber diameter in the subject compared to control muscle fibers. In some embodiments, the increase in muscle fiber diameter is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 4-fold, at least 5-fold, or more, compared to control muscle fibers. In some embodiments, the increase in muscle fiber diameter is in the range of 1-fold to 5-fold, 2-fold to 10-fold, 1-fold to 1.5-fold, 1-fold to 2-fold, etc., compared to control muscle fibers.

[0359] In some embodiments, with respect to administration of a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof, to a subject, an effective amount is an amount effective to increase muscle-to-fat ratio in the subject compared to control muscle mass. In some embodiments, the increase in muscle-to-fat ratio is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 4-fold, at least 5-fold, or more, compared to a control subject. In some embodiments, the increase in muscle-to-fat ratio is in the range of 1-fold to 5-fold, 2-fold to 10-fold, 1-fold to 1.5-fold, 1-fold to 2-fold, etc., compared to a control subject.

[0360] In some embodiments, myostatin inhibitors, such as pro / latent myostatin inhibitors, With respect to administration of the antibody or antigen-binding fragment thereof to a subject, an effective amount is an amount effective to reduce intramuscular fat infiltration in the subject relative to control muscle mass. In some embodiments, the reduction in intramuscular fat infiltration is at most 1.1-fold, at most 1.2-fold, at most 1.3-fold, at most 1.4-fold, at most 1.5-fold, at most 1.6-fold, at most 1.7-fold, at most 1.8-fold, at most 1.9-fold, at most 2-fold, at most 4-fold, at most 5-fold, or less, relative to control subjects. In some embodiments, the reduction in intramuscular fat infiltration is in the range of 1- to 5-fold, 2- to 10-fold, 1- to 1.5-fold, 1- to 2-fold, etc., relative to control subjects.

[0361] In some embodiments, a method for preventing muscle mass loss and / or increasing muscle mass in a human subject comprises administering to the subject a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof, that inhibits the formation of mature myostatin via proteolysis by tolloid proteases. In one embodiment, inhibition of proteolytic cleavage of promyostatin or latent myostatin by tolloid proteases results in a progressive increase in muscle mass. In one embodiment, the subject exhibits a progressive increase in muscle mass over at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, or 20 weeks (or any range bracketed by any of these values). In some embodiments, a method for preventing muscle mass loss and / or increasing muscle mass in a human subject comprises administering to the subject a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof, comprising more than two doses. In one embodiment, the step of administering a myostatin inhibitor, e.g., a pro / latent myostatin antibody or its antigen-binding fragment, includes at least a first dose and a second dose, wherein the first dose and the second dose are administered to the subject at least about 2 weeks apart, 4 weeks apart, 6 weeks apart, 8 weeks apart, or 12 weeks apart.

[0362] In some embodiments, with respect to administration of a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof, to a subject, an effective amount is an amount effective to increase target muscle function in the subject compared to control muscle function. In some embodiments, the increase in muscle function is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 4-fold, at least 5-fold, or more compared to control muscle function. In some embodiments, the increase in muscle function is in the range of 1-fold to 5-fold, 2-fold to 10-fold, 1-fold to 1.5-fold, 1-fold to 2-fold, etc., increase compared to control muscle function.

[0363] As used herein, the term "control muscle mass" refers to a reference standard useful for evaluating the effect of a condition (e.g., treatment with a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof) on a target muscle mass in a subject. In some embodiments, the control muscle mass is a predetermined value. In some embodiments, the control muscle mass is experimentally determined. In some embodiments, the control muscle mass is a target muscle mass in a subject who has not been administered a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof. In some embodiments, the control muscle mass is a target muscle mass (e.g., an average mass) in a population of subjects who have not been administered a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof. In some embodiments, the control muscle mass is a target muscle mass in a subject before (e.g., immediately before) being administered a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof. In some embodiments, the control muscle mass is measured using a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof, instead of a pro / latent myostatin antibody or antigen-binding fragment thereof. The control muscle mass is the target muscle mass in a subject administered a conventional antibody (e.g., of the same isotype as the pro / latent myostatin antibody) obtained from an animal that was not exposed to the antigen to which the original binding fragment is directed. In some embodiments, the control muscle mass is the target muscle mass in a subject administered a vehicle, e.g., saline, instead of a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof.

[0364] In some embodiments, in terms of administering a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof, to a subject, an effective amount is an amount effective to increase the force-generating capacity of a target muscle in the subject (e.g., maximal force generation as determined in vitro using a muscle lever system adapted for horizontal perfusion bathing) compared to a control force-generating capacity. In some embodiments, the increase in force-generating capacity is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 4-fold, at least 5-fold, or more, compared to the control force-generating capacity. In some embodiments, the increase in force-generating capacity is in the range of 1-fold to 5-fold, 2-fold to 10-fold, 1-fold to 1.5-fold, 1-fold to 2-fold, etc., compared to the control force-generating capacity.

[0365] As used herein, the term "control force-generating capacity" refers to a reference standard useful for comparing the effect of a condition (e.g., treatment with a pro / latent myostatin antibody or antigen-binding fragment thereof) on the force-generating capacity of a muscle in a subject. In some embodiments, the control force-generating capacity is a predetermined value. The control force-generating capacity is experimentally determined. In some embodiments, the control force-generating capacity is the force-generating capacity of a target muscle in a subject who has not been administered a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof. In some embodiments, the control force-generating capacity is the force-generating capacity (e.g., average force-generating capacity) of a target muscle in a population of subjects who have not been administered a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof. In some embodiments, the control force-generating capacity is the force-generating capacity of a target muscle in a subject before (e.g., immediately before) being administered a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof. In some embodiments, the control muscle force-generating capacity is the force-generating capacity of the target muscle in a subject that has been administered a myostatin inhibitor, e.g., a pro / latent myostatin antibody, with a normal antibody (e.g., of the same isotype as the pro / latent myostatin antibody) obtained from an animal that has not been exposed to the antigen to which the pro / latent myostatin antibody is directed. In some embodiments, the control force-generating capacity is the force-generating capacity of the target muscle in a subject that has been administered a vehicle, e.g., saline, with a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof.

[0366] In some embodiments, the target muscle is a plantar flexor. In some embodiments, the target muscle is a muscle containing type 2 fibers. In some embodiments, the target muscle is a muscle containing fast oxidative fibers or fast glycolytic fibers. In some embodiments, the target muscle is a muscle containing type IIB fibers. In some embodiments, administration of a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof, results in an increase in the cross-sectional area of ​​type IIB fibers of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% (or any range bracketed by any of these values) compared to the cross-sectional area before the administering step.

[0367] Dosage Empirical considerations such as half-life generally contribute to determining dosage. Antibodies and antigen-binding portions thereof that are compatible with the human immune system, such as humanized or fully human antibodies, may be used to extend the half-life of the antibody and prevent it from being attacked by the host's immune system. The frequency of administration may be determined and adjusted over the course of treatment, and generally Therapeutic effects of myostatin include, but are not necessarily based on the treatment and / or suppression and / or amelioration and / or delay of a disease / disorder associated with myopathy. Alternatively, sustained continuous release formulations of myostatin inhibitors, such as anti-pro / latent myostatin antibodies or antigen-binding portions thereof, may also be appropriate. Various formulations and devices for achieving sustained release will be apparent to those skilled in the art and are within the scope of this disclosure.

[0368] In one example, the dosage for a myostatin inhibitor described herein, e.g., an anti-pro / latent myostatin antibody or antigen-binding fragment thereof, can be empirically determined in an individual given one or more administrations of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof. The individual is given increasing dosages of an antagonist. Disease / disorder indicators can be tracked to assess the efficacy of the antagonist.

[0369] Generally, for administration of any of the antibodies or antigen-binding fragments thereof described herein, the initial candidate dose may be about 2 mg / kg. For purposes of this disclosure, a typical daily dose may range anywhere from about 0.1 μg / kg to 3 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg to 30 mg / kg to 100 mg / kg or more, depending on the factors discussed above. For repeated administration over several days or longer, treatment is continued until desired symptomatic suppression occurs or until a sufficient therapeutic level is achieved to alleviate a disease or disorder associated with pro- or latent myostatin or its symptoms, depending on the condition. An exemplary dosing regimen includes an initial dose of about 2 mg / kg, followed by weekly maintenance doses of about 1 mg / kg of the antibody or antigen-binding fragment thereof, or by maintenance doses of about 1 mg / kg every other week. However, other dosing regimens are useful depending on the pattern of pharmacodynamic decay the clinician desires to achieve. For example, dosing one to four times per week is contemplated. In some embodiments, about 3 μg / mg to about 2 mg / kg (e.g., about 3 μg / mg, about 10 μg / mg, about 30 μg / mg, about 100 μg / mg, about 300 μg / mg, about 1 mg / kg, and about 2 mg / kg) are used. In some embodiments, the administration frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks, or once every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 8 months, every 10 months, every year, or longer. The progress of this treatment can be easily monitored by conventional techniques and assays. The administration regimen (including the antibody used) may be modified over time.

[0370] In some embodiments, administration of a myostatin inhibitor, e.g., any of the antibodies or antigen-binding fragments thereof described herein, comprises a single dose. In some embodiments, administration of a myostatin inhibitor, e.g., any of the antibodies or antigen-binding fragments thereof described herein, comprises multiple doses (e.g., at least two, three, four, five, six, seven, eight, nine, or ten doses). The administering step may comprise more than two doses. In some embodiments, administration comprises at least a first dose and a second dose of a therapeutically effective amount of a myostatin inhibitor, e.g., an antibody or antigen-binding portion thereof. In one embodiment, the first and second doses are administered to a subject at least about 4 weeks apart, 6 weeks apart, 8 weeks apart, or 12 weeks apart.

[0371] In some embodiments, for a normal weight adult patient, a dose ranging from about 0.3 to 5.00 mg / kg may be administered. The particular dosing regimen, e.g., dose, timing, and repetition, will depend on the particular individual and their medical history and the characteristics of the individual drug (such as the drug's half-life and other relevant considerations).

[0372] For purposes of this disclosure, an appropriate dosage of a myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody or antigen-binding fragment thereof, will depend on the specific antibody (or composition thereof) used. ), the type and severity of the disease / disorder, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to the antagonist, and the judgment of the attending physician. In some embodiments, a clinician will administer a myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody or antigen-binding portion thereof, until a dosage is reached that achieves the desired result. Administration of a myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody or antigen-binding portion thereof, whether the purpose of administration is therapeutic or prophylactic, can be continuous or intermittent, depending, for example, on the physiological condition of the recipient and other factors known to a skilled physician. Administration of a myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody or antigen-binding fragment thereof, whether for therapeutic or prophylactic purposes, can be essentially continuous or intermittent, depending, for example, on the physiological condition of the recipient and other factors known to a skilled physician. Administration of a myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody or antigen-binding fragment thereof, can be essentially continuous over a preselected period of time, or can be in a series of spaced doses, e.g., either before, during, or after the onset of a disease or disorder associated with pro / latent myostatin.

[0373] As used herein, the term "treating" refers to the application or administration of a composition containing one or more active agents to a subject having a disease / disorder, symptom of a disease / disorder, or predisposition to a disease / disorder involving myopathy to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, symptom of the disease, or predisposition to the disease / disorder.

[0374] Alleviating a disease / disorder associated with pro / latent myostatin includes delaying the development or progression of the disease or reducing the severity of the disease. Alleviating a disease does not necessarily require a curative outcome. As used herein, "delaying" the onset of a disease / disorder associated with pro / latent myostatin means deferring, hindering, slowing, retarding, stabilizing, and / or postponing the progression of the disease. This delay may be for various lengths of time depending on the history of the disease and / or the individual being treated. A method of "delaying" or alleviating the onset of a disease or delaying the onset of a disease is a method that reduces the likelihood of the onset of one or more symptoms of the disease in a given time frame and / or reduces the severity of the symptoms in a given time frame compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to produce statistically significant results.

[0375] Combination therapy The present invention encompasses pharmaceutical compositions and related methods used as combination therapies to treat subjects who may benefit from in vivo myostatin inhibition. In any of these embodiments, such subjects can receive combination therapy comprising a first composition containing at least one myostatin inhibitor, such as an antibody or antigen-binding portion thereof described herein, in combination with a second composition containing at least one additional therapeutic agent intended to treat the same or an overlapping disease or clinical condition. The first and second compositions can act on the same or distinct cellular targets. In some embodiments, the first and second compositions can treat or alleviate the same or overlapping set of symptoms or aspects of a disease or clinical condition. In some embodiments, the first and second compositions can treat or alleviate separate sets of symptoms or aspects of a disease or clinical condition. For example, the first composition can treat myopathy associated with a disease, while the second composition can treat inflammation or fibrosis associated with the same disease. Such combination therapies can be administered in conjunction with each other. The phrase "in conjunction with," in reference to combination therapy, means that the therapeutic effect of a first therapy overlaps temporally and / or spatially with the therapeutic effect of a second therapy in a subject receiving the combination therapy. Thus, the combination therapy may be formulated as a single formulation for simultaneous administration or as separate formulations for sequential administration of the therapies. .

[0376] In a preferred embodiment, the combination therapy produces a synergistic effect in treating the disease. The term "synergistic" refers to an effect (e.g., greater efficacy) that is greater than the additive effect of each monotherapy taken together.

[0377] In some embodiments, a combination therapy comprising a pharmaceutical composition described herein produces overall efficacy comparable to that produced by another therapy (e.g., monotherapy with a second agent), but produces fewer undesirable adverse effects or less severe toxicity associated with the second agent compared to monotherapy with the second agent. In some embodiments, such a combination therapy allows for lower dosages of the second agent while maintaining overall efficacy. Such a combination therapy may be particularly suitable for patient populations where long-term treatment is warranted and / or involving pediatric patients.

[0378] Thus, the present invention provides pharmaceutical compositions and methods for use in combination therapy to enhance muscle mass / function and treat or prevent metabolic diseases or diseases associated with defective neural signaling, including diabetes, obesity, and spinal cord injury. Thus, the methods or pharmaceutical compositions further include a second treatment. In some embodiments, the second treatment may be useful in treating or preventing metabolic diseases or diseases associated with defective neural signaling. The second treatment can attenuate or treat at least one symptom(s) associated with the targeted disease. The first and second treatments may exert their biological effects through similar or unrelated mechanisms of action, or one or both of the first and second treatments may exert their biological effects through multiple mechanisms of action.

[0379] It should be understood that the pharmaceutical compositions described herein may have the first and second therapies for each of the described embodiments in the same pharmaceutically acceptable carrier or in different pharmaceutically acceptable carriers. It should further be understood that the first therapeutic agent and the second therapy may be administered simultaneously or sequentially within the described embodiments.

[0380] One or more anti-myostatin antibodies or other myostatin inhibitors of the present invention may be combined with one or more additional therapeutic agents. Examples of additional therapeutic agents that may be used with the anti-myostatin antibodies of the present invention include, but are not limited to, drugs for treating diabetes, drugs for treating complications of diabetes, drugs for treating cardiovascular disease, antihyperlipidemic agents, hypotensive or antihypertensive agents, anti-obesity agents, drugs for treating non-alcoholic steatohepatitis (NASH), chemotherapeutic agents, immunotherapeutic agents, immunosuppressants, etc. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding potential toxicities or complications associated with various monotherapies. Examples of agents for treating diabetes include insulin preparations (e.g., animal insulin preparations extracted from bovine or porcine pancreas; human insulin preparations synthesized by genetic engineering techniques using microorganisms or methods), insulin sensitizers, pharmaceutically acceptable salts, hydrates, or solvates thereof (e.g., pioglitazone, troglitazone, rosiglitazone, netoglitazone, balaglitazone, rivoglitazone, tesaglitazar, farglitazar, C LX-0921, R-483, NIP-221, NIP-223, DRF-2189, GW-7282, TAK-559, T-131, RG-12525, LY-510929, LY-519818, BMS-298585, DRF-2725, GW-1536, GI-262570, KRP-297, TZD18 (Merck), DRF-2655, etc.), alpha-glycosidase inhibitors (e.g., voglibose, acarbose, miglitol, emiglitate, etc.), biguanides ( For example, phenformin, metformin, buformin, etc.) or sulfonylureas (for example, tolbutamide, glibenclamide, gliclazide, chlorpropamide, tolazamide, acetohexamide, glyclopyramide, glimepiride, etc.), as well as other insulin secretagogues (for example, repaglinide, senaglinide, nateglinide, mitiglinide, GLP-1, etc.), amylin agonists (for example, pramlintide, etc.), phosphotyrosine phosphatase inhibitors (for example, vanadate, etc.), etc. Examples of drugs for treating diabetic complications include, but are not limited to, aldose reductase inhibitors (e.g., tolrestat, epalrestat, zenarestat, zopolrestat, minalrestat, fidarestat, SK-860, CT-112, etc.), neurotrophic factors (e.g., NGF, NT-3, BDNF, etc.), PKC inhibitors (e.g., LY-333531, etc.), advanced glycation end products (AGE) inhibitors (e.g., ALT946, pimagedine, etc.), and the like. , pyridoxamine, phenacylthiazolium bromide (ALT766), etc.), active oxygen quenchers (e.g., thioctic acid or its derivatives, bioflavonoids such as flavones, isoflavones, flavanones, procyanidins, anthocyanidins, pycnogenol, lutein, lycopene, vitamin E, coenzyme Q, etc.), cerebral vasodilators (e.g., tiapride, mexiletine, etc.). Antihyperlipidemic agents include, for example, statin-based compounds (e.g., pravastatin, simvastatin, lovastatin, atorvastatin, fluvastatin, rosuvastatin, etc.) that are cholesterol synthesis inhibitors, squalene synthetase inhibitors or fibrate compounds (e.g., fenofibrate, gemfibrozil, bezafibrate, clofibrate, simfibrate, clinofibrate, etc.) that have triglyceride-lowering effects, niacin, PCSK9 inhibitors, triglyceride-lowering agents, or cholesterol sequestrants. Antihypertensive agents include, for example, angiotensin-converting enzyme inhibitors (e.g., captopril, enalapril, delapril, benazepril, cilazapril, enalapril, enalaprilat, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, trandolapril, etc.), or angiotensin II antagonists (e.g., losartan, candesartan cilexetil, olmesartan medoxomil, eprosartan, valsartan, telmisartan, irbesartan, tasosartan, pomisartan, ripisartan forasartan, etc.), or calcium channel blockers (e.g., amlodipine), or aspirin.

[0381] Non-alcoholic steatohepatitis (NASH) treatment agents include, for example, ursodiol, pioglitazone, orlistat, betaine, and rosiglitazone. Anti-obesity agents include, for example, central anti-obesity agents (e.g., dexfenfluramine, fenfluramine, phentermine, sibutramine, amfepramone, dexamphetamine, mazindol, phenylpropanolamine, clobenzorex, etc.), gastrointestinal lipase inhibitors (e.g., orlistat, etc.), beta3-adrenergic receptor agonists (e.g., CL-316243, SR-58611-A, UL-TG-307, SB-226552, AJ-9677, BMS-196085, etc.), peptide-based appetite suppressants (e.g., leptin, CNTF, etc.), cholecystokinin agonists (e.g., lintitript, FPL-15849, etc.), and the like. Chemotherapeutic agents include, for example, alkylating agents (e.g., cyclophosphamide, ifosfamide, etc.), metabolic antagonists (e.g., methotrexate, 5-fluorouracil, etc.), anticancer antibiotics (e.g., mitomycin, adriamycin, etc.), plant-derived anticancer agents (e.g., vincristine, vindesine, taxol, etc.), cisplatin, carboplatin, etoposide, etc. Among these substances, 5-fluorouracil derivatives such as furtulon and neofurtulon are preferred. Immunotherapeutic agents include, for example, microbial or bacterial components (e.g., muramyl dipeptide derivatives, picibanil, etc.), polysaccharides with immunopotentiating activity (e.g., lentinan, sizofiran, krestin, etc.), cytokines obtained by genetic engineering techniques (e.g., interferon, interleukin (IL), etc.), colony-stimulating factors (e.g., granulocyte colony-stimulating factor, erythropoietin, etc.), etc., and preferred substances among these are IL-1, IL-2, IL-12, etc. Immunosuppressants include, for example, calcineurin inhibitors / immunophilin modulators, such as cyclosporine (Sandimmune, Gengraf, Neoral), tacrolimus (Prograf, FK506), ASM981, sirolimus (RAPA, rapamycin, Rapamune), or its derivative SDZ-RAD, glucocorticoids (prednisone, prednisolone, methylprednisolone, dexamethasone, etc.), purine synthesis inhibitors (mycophenolate mofetil, MMF, Cellcept®, azathioprine, cyclophosphamide), interleukin antagonists (basiliximab, daclizumab, deoxyspergualin), lymphodepleting agents, such as antithymocyte globulin (thymoglobulin, lymphoglobulin), anti-CD3 antibodies (OKT3), etc. Additionally, agents whose cachexia-ameliorating effects have been established in animal models or clinical settings, such as cyclooxygenase inhibitors (e.g., indomethacin, etc.), progesterone derivatives (e.g., megestrol acetate), glucosteroids (e.g., dexamethasone, etc.), metoclopramide-based agents, tetrahydrocannabinol-based agents, lipid metabolism-improving agents (e.g., eicosapentaenoic acid, etc.), growth hormone, IGF-1, TNF-α, LIF, IL-6, and antibodies against oncostatin M, may also be used in conjunction with the anti-myostatin antibodies of the present invention. Additional therapeutic agents for use in treating diseases or conditions associated with metabolic disorders and / or defective neural signaling will be apparent to those of skill in the art and are within the scope of the present disclosure.

[0382] In some embodiments, a second agent suitable for administration as a combination therapy with an antibody described herein is an anti-fibrotic agent, such as a TGFβ1 inhibitor.

[0383] In some embodiments, second agents suitable for administration as combination therapy with an antibody described herein are modulators (e.g., agonists and antagonists) of certain members of the TGFβ superfamily of growth factors, such as BMP6, BMP7, GDF11, TGFβ2, TGFβ3, RGMc, etc.

[0384] Any of the above agents may be administered in combination with the myostatin antibodies of the present invention to treat metabolic diseases or diseases associated with defective neural signaling between neurons and target tissues, such as spinal cord injury, muscular atrophy, and muscular dystrophies.

[0385] Use of myostatin inhibitors, such as anti-pro / latent myostatin antibodies and antigen-binding fragments thereof, to treat diseases / disorders The pharmaceutical compositions described herein are suitable for administration to human patients to treat or prevent diseases and conditions in which reducing myostatin signaling is desirable. Such diseases and conditions include, but are not limited to, muscle conditions or disorders, metabolic disorders, and diseases associated with defective neural signaling, such as spinal cord injury. Exemplary conditions for which the compositions and methods of the present invention may be useful are further described below.

[0386] A. Muscle Conditions and Disorders In some embodiments, the methods of the present invention are suitable for treating or preventing muscle conditions and disorders. As used herein, the term "muscle condition" or "muscle disorder" refers to a disease, condition, or disorder in which muscles do not function normally, or a disease in which muscles function normally but produce less force due to a reduced amount of available muscle. "Muscle condition" refers to a condition or disorder. Muscle conditions or disorders may include, but are not limited to, myopathy, muscle atrophy, muscular dystrophy, etc. Such conditions may be caused by motor neuron loss(es), genetic mutations, or damage such as nerve injury.

[0387] In one embodiment, the muscle condition is myopathy.As used herein, the term "myopathies" refers to muscle conditions characterized by defects in muscle structure or function, which generally result in muscle weakness."Myopathies" may also include muscle conditions characterized by normal muscle structure but defective or abnormal neuronal input, which in turn affects muscle function."Myopathies" may also include inflammatory myopathies and / or autoimmune myopathies, such as myasthenia gravis.

[0388] Myopathies include muscle conditions that are neuromuscular or musculoskeletal in nature. In some embodiments, the myopathy is a hereditary myopathy. Hereditary myopathies include, but are not limited to, dystrophies, myotonias, congenital myopathies (e.g., nemaline myopathy, multi / minicore myopathy, and centronuclear myopathy), mitochondrial myopathies, familial periodic myopathies, inflammatory myopathies, and metabolic myopathies (e.g., glycogen storage diseases and lipid storage disorders). In some embodiments, the myopathy is an acquired myopathy. Acquired myopathies include, but are not limited to, exogenous substance-induced myopathies (e.g., drug-induced myopathies and glucocorticoid myopathies, alcoholic myopathies, and myopathies due to other toxic agents), myositis (e.g., dermatomyositis, polymositis, and inclusion body myositis), myositis ossificans, rhabdomyolysis, and myoglobinuria, and disuse atrophy. In some embodiments, the myopathy is disuse atrophy, which can be caused by prolonged muscle inactivity leading to a deterioration of normal muscle function. Disuse atrophy can be the result of hospitalization, a fracture (e.g., a hip fracture), or nerve injury. In some embodiments, the myopathy is associated with a disease or disorder such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), cachexia syndrome due to renal failure, AIDS, a cardiac condition, and / or cancer. In some embodiments, the myopathy is associated with aging. In some embodiments, the myopathy is associated with sarcopenia. In some embodiments, the myopathy is associated with paraspinal muscle atrophy (PMA).

[0389] In some embodiments, the myopathy is a primary myopathy. In one embodiment, the primary myopathy comprises disuse atrophy. In some embodiments, the disuse atrophy is associated with hip fracture, elective joint replacement, critical care myopathy, spinal cord injury, or stroke. In some embodiments, the myopathy is genetic muscle weakness, e.g., associated with muscular dystrophy.

[0390] In some embodiments, the myopathy is a secondary myopathy in which muscle loss or dysfunction occurs secondary to a disease condition. In some embodiments, the secondary myopathy includes denervation or cachexia. In some embodiments, the secondary myopathy is caused by denervation associated with monitor neuron dysfunction. In some embodiments, the motor neuron dysfunction is due to a mutation(s) in a gene affecting motor neurons. Diseases known to involve motor neuron mutations include, but are not limited to, amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). In some embodiments, the secondary myopathy is cachexia associated with renal failure, AIDS, a cardiac condition, cancer, or aging. In some embodiments, the secondary myopathy is caused by nerve damage, including unwanted nerve damage sustained during a medical procedure such as surgery. The adverse effects of such damage on the function of target tissues (e.g., target muscles) are ameliorated by administration of a myostatin inhibitor as described herein. For example, such administration can prevent and / or alleviate myopathy and / or facilitate recovery.

[0391] In some embodiments, the methods of the present invention are suitable for treating or preventing muscular conditions and disorders, including vocal cord paresis / paralysis. As used herein, the term "vocal cord paresis / paralysis" refers to a condition resulting from abnormal nerve input to the voice box muscles (laryngeal muscles). Paralysis can involve complete interruption of nerve impulses, resulting in immobility; paresis can involve partial interruption of nerve impulses, resulting in weak or abnormal laryngeal muscle movement. In some embodiments, the anti-myostatin antibody or antigen-binding fragment thereof is administered locally, for example, by direct local injection into the affected vocal cord muscle(s). If only one side of the vocal cord is affected, for example, due to unilateral nerve damage, unilateral injection is necessary. In other embodiments, if both nerves are damaged, both sides of the vocal cord are affected, and therefore bilateral injection is preferred.

[0392] The anti-myostatin antibody or antigen-binding fragment thereof for use in the methods of the present invention can locally increase vocal cord muscle mass to close the gap between the two vocal fold...

Claims

1. 1. A pharmaceutical composition for use in a method for treating a muscle condition in a subject, comprising: The pharmaceutical composition comprises an antibody or fragment thereof that specifically binds to pro / latent myostatin and inhibits release of mature myostatin; and The pharmaceutical composition is administered to a subject in the following manner: (a) increasing muscle tissue mass and / or function in said subject; (b) increasing the metabolic rate of said subject; (c) increasing insulin sensitivity in said subject; (d) increasing the level of brown adipose tissue in the subject; (e) increasing the level of beige adipose tissue in the subject; (f) reducing the level of white adipose tissue in said subject; (g) reducing the level of visceral adipose tissue in said subject; (h) reducing the ratio of adipose tissue to muscle tissue in said subject; (i) increasing glucose uptake by brown adipose tissue, beige adipose tissue, or muscle tissue in said subject; (j) a decrease in glucose uptake by white adipose tissue or hepatic tissue; (k) reducing muscle catabolism of protein and / or muscle release of amino acids in said subject; (l) increasing insulin-dependent blood glucose regulation in the subject; (m) a reduction in intramuscular fat infiltration in said subject; (n) improvement in quality of life as assessed by a standardized quality of life test; (o) preventing muscle loss or muscle atrophy in said subject; and / or (p) preventing the development of metabolic dysregulation associated with muscle dysfunction in the subject. A pharmaceutical composition that causes two or more of the following:

2. 2. The pharmaceutical composition for use according to claim 1, wherein the muscle condition is associated with a defect in neural signaling between neurons and target muscles.

3. 3. The pharmaceutical composition for use according to claim 2, wherein the muscle condition comprises motor neuron dysfunction.

4. 3. The pharmaceutical composition for use according to claim 2, wherein the muscle condition is caused by an injury.

5. 3. The pharmaceutical composition for use according to claim 2, wherein the injury comprises an incomplete SCI.

6. 3. The pharmaceutical composition for use according to claim 2, wherein the muscle condition is associated with a genetic defect.

7. 3. The pharmaceutical composition for use according to claim 2, wherein the muscle condition is amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), or myasthenia gravis.

8. 10. The pharmaceutical composition for use according to any one of the preceding claims, wherein the subject has or is at risk of developing a metabolic disorder.

9. A pharmaceutical composition for use according to any one of the preceding claims, wherein the antibody or fragment does not bind to mature myostatin or GDF11.

10. 10. The method of claim 9, wherein the antibody is a fully human or humanized antibody. Pharmaceutical compositions for use.

11. The antibody is IgG ４ 10. The pharmaceutical composition for use according to any one of the preceding claims, which is a subtype.

12. 10. The pharmaceutical composition for use according to any one of the preceding claims, wherein the antibody is a pH-sensitive antibody.

13. 10. The pharmaceutical composition for use according to any one of the preceding claims, wherein said method comprises administering said pharmaceutical composition at a dosage of 0.1 to 30 mg / kg of said antibody.

14. 10. The pharmaceutical composition for use according to any one of the preceding claims, wherein said method comprises weekly, biweekly or monthly administration.

15. 10. The pharmaceutical composition for use according to any one of the preceding claims, wherein said method comprises IV injection or subcutaneous administration.

Images