Methods for treating metabolic diseases by inhibiting myostatin activation

Abstract

To provide a composition for treating or preventing metabolic diseases.SOLUTION: The present invention provides a composition comprising an antibody, or antigen-binding fragment thereof that specifically binds pro / latent-myostatin and blocks release of mature myostatin, for use as a medicament in treatment or prevention of a metabolic disease in a human subject, comprising steps of: selecting a human subject suffering from or at risk of developing a metabolic disease; and administering to the human subject the composition comprising an effective amount of the antibody, or antigen-binding fragment thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims priority to the following patent applications: U.S. Provisional Patent Application No. 62 / 443,455, filed January 6, 2017, European Priority Application No. 1750586.0, U.S. Provisional Patent Application No. 62 / 530,311, filed July 10, 2017, and U.S. Provisional Patent Application No. 62 / 608,069, filed December 20, 2017. The entire contents of these priority applications, including electronic sequence listings submitted electronically in ASCII format, 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. Said ASCII copy, created on January 5, 2018, is titled "SR16-WO-PCT_Sequence_Listing_127036-00220.txt" and is 108,914 bytes in size. [Background technology]

[0003] Millions of people worldwide suffer from metabolic disease, and patients with metabolic disease typically experience loss of lean or lean muscle mass, excessive gain in body fat mass, low metabolic rate, insulin resistance, inability to control blood sugar, weight gain, and increased body mass index.Therefore, these patients are at risk of developing major complications such as diabetes, obesity, coronary artery disease, hypertension, stroke, atherosclerosis, heart failure (including congestive heart failure) such as chronic heart failure (CHF), metabolic bone disorders, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as polycystic ovarian syndrome, breast cancer, prostate cancer, and colon cancer, as well as increased incidence of complications of general anesthesia.

[0004] In addition to the serious health consequences of these metabolic diseases, serious economic costs are associated with these diseases. For example, the total cost of treating diabetes and its complications in the United States is estimated at $245 billion per year. The estimated annual medical costs of obesity-related diseases are a staggering $190.2 billion, or nearly 21% of annual medical costs. Not only the direct medical costs of these metabolic diseases, but also the indirect costs, including lost productivity due to metabolic disease-related morbidity and premature death, impose substantial costs on both society and its citizens.

[0005] Myostatin, also known as growth differentiation factor 8 or GDF-8, is a member of the transforming growth factor-β (TGF-β) superfamily. Myostatin is produced and released by muscle cells and is an important autocrine / paracrine inhibitor of skeletal muscle growth (Non-Patent Document 1). Myostatin has been primarily evaluated for its use in treating diseases related to muscle function.

[0006] To date, metabolic diseases remain largely undertreated. Current treatments do not adequately meet the needs of patients, and no effective treatments are available for the majority of the affected patient population. Thus, there is an unmet need for treatments for patients suffering from metabolic diseases. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Mouisel et al., Am J Physiol Regul Integr Comp Physiol, 2014, Volume 307(4): R444-54 Summary of the Invention

[0008] The present disclosure encompasses the recognition that myostatin can act as a key regulator to directly mediate the function and regulation of muscle as an endocrine organ that controls metabolism, including body composition.

[0009] According to the present invention, the control of myostatin signaling can affect many metabolic parameters central to the control of energy production and consumption by selectively mobilizing the body's three main energy pools: glucose, lipids, and proteins.Without wishing to be bound by theory, it is contemplated that myostatin can play a role in the process both as a molecular sensor of energy consumption and as an effector to affect metabolic normalization.The present invention is concerned with the broader role of myostatin in metabolic control, including nitrogen mobilization, osmoregulation, calcium metabolism, and acid-base and electrolyte balance.

[0010] Thus, the present invention provides methods and compositions for treating or preventing metabolic disease in a human subject using an anti-pro / latent myostatin inhibitor, e.g., an antibody. The present invention is based, at least in part, on the discovery that administering a myostatin inhibitor, e.g., an antibody, or an antigen-binding fragment thereof, that specifically binds to pro / latent myostatin to a subject with a metabolic disease, e.g., spinal cord injury (SCI), significantly improves both the physiological and functional characteristics of the injured subject. In particular, the inventors have surprisingly discovered that administration of a myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody, significantly increases metabolic rate or energy expenditure in a subject with a metabolic disease, e.g., spinal cord injury (SCI). Administration of a myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody, also significantly attenuated SCI-induced reductions in sublesional muscle mass and total body weight, while simultaneously reducing the amount of undesirable adipose tissue, such as white adipose tissue and visceral adipose tissue. Additionally, subjects treated with myostatin inhibitors, such as anti-pro / latent myostatin antibodies, showed significant improvements in locomotor function, muscle strength, and motor coordination and balance skills.

[0011] The present invention is further based, at least in part, on the surprising discovery that administration of a myostatin inhibitor, such as an anti-pro / latent myostatin antibody or antigen-binding fragment thereof, not only increases bone volume in weight-bearing bones, but also increases bone volume in non-weight-bearing bones, such as rodent vertebrae. It is well known in the art that weight-bearing activity is an important stimulus for bone mass gain, which could potentially explain the increase in bone volume in weight-bearing bones after administration of a myostatin inhibitor. However, the surprising increase observed in non-weight-bearing bone volume demonstrated upon administration of the myostatin inhibitors disclosed herein further confirms that myostatin inhibitors have a broader metabolic effect, i.e., they not only act to increase bone, for example via increased muscle stimulation, but also act as an important regulator for increasing general metabolic effects, including bone health.

[0012] Finally, the present invention provides a method for promoting the improvement of body composition, for example, muscle-fat ratio. Such a method can be effective in achieving a solid weight loss in both healthy subjects, for example, bodybuilders, or subjects with obesity, for example, diet-induced obesity, metabolic syndrome, NASH, NAFLD, and / or diabetes. Compared to diet alone, which causes weight loss in both fat and muscle, administration of the myostatin inhibitors disclosed herein in combination with diet leads to weight loss or a greater muscle-to-fat ratio due to preferential loss of fat stores compared to muscle loss. Specifically, administration of the myostatin inhibitors in combination with diet, for example, calorie-restricted diet, leads to a more solid weight loss compared to diet alone; the benefit of improved cardiac metabolism (lipid profile, glucose metabolism, cardiovascular risk, etc.); and a greater reduction in visceral fat levels and other harmful fat levels, due in part to the maintenance of a higher metabolic rate. Such beneficial effects can be further enhanced when combined with moderate exercise.

[0013] Thus, in one aspect, disclosed herein is 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, for use as a medicament in treating or preventing metabolic disease in a human subject, comprising the steps of: selecting a human subject suffering from or at risk of developing a metabolic disease; and administering to the human subject an effective amount of 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. In one embodiment, the subject is a pediatric subject.

[0014] In some embodiments, the subject does not have a myopathy, and optionally the myopathy is a primary or secondary myopathy. In some embodiments, the subject is an adult human subject suffering from growth hormone (GH) deficiency, and optionally the subject is concurrently receiving recombinant GH therapy or GH gene therapy.

[0015] In some embodiments, 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 some embodiments, the obesity is sarcopenic obesity.

[0016] In some embodiments, 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.

[0017] In some embodiments, the Cushing's disease is selected from the group consisting of corticosteroid-induced Cushing's disease and tumor-induced Cushing's disease.

[0018] In some embodiments, 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.

[0019] In some embodiments, administration of the composition comprises the following: a) increasing muscle tissue mass and / or function in a human subject; b) increasing fast muscle tissue mass and / or function in a human subject; c) increasing the mass and / or function of slow muscle tissue in a human subject; d) increasing the metabolic rate of a human subject; e) increasing insulin sensitivity in a human subject; f) increasing the level of brown adipose tissue in a human subject; g) increasing the levels of beige adipose tissue in human subjects; h) reducing the level of white adipose tissue in a human subject; i) reducing the level of visceral adipose tissue in a human subject; j) reducing the ratio of fat to muscle tissue in a human subject; k) increasing glucose uptake by a target tissue in a human subject, wherein the target tissue is selected from the group consisting of brown adipose tissue, beige adipose tissue, and muscle tissue; 1) reducing glucose uptake by a target tissue in a human subject, wherein the target tissue is selected from the group consisting of white adipose tissue and liver tissue; m) reducing muscle catabolism of protein and / or muscle release of amino acids in a human subject; n) increasing insulin-dependent glycemic control in a human subject; o) reducing intramuscular fat infiltration in human subjects; p) improving standardized quality of life test scores; q) preventing muscle loss or atrophy in human subjects; r) reducing bone loss; s) increasing bone cross-sectional area and / or cortical bone thickness; t) reducing the frequency or severity of fractures; and / or u) Reducing fluid overload or edema in chronic heart failure (CHF); at least one of, for example, 2, 3, 4, 5, 6, 7, 2 to 4, 2 to 5, 2 to 6, 3 to 5, or 3 to 6.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof does not bind to GDF11 or activin. In some embodiments, the antibody or antigen-binding fragment thereof does not bind to mature (fully processed, free and active) myostatin. In some embodiments, the antibody or antigen-binding fragment thereof comprises a) 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, or b) a heavy chain comprising the amino acid sequence of SEQ ID NO:50 and a light chain comprising the amino acid sequence of SEQ ID NO:51.

[0021] In another aspect, the 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 or at risk of developing a metabolic disease; and administering to the human subject an effective amount of a myostatin inhibitor, e.g., a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin and blocks the release of mature myostatin, thereby treating or preventing the metabolic disease in the human subject.

[0022] In one embodiment, the subject does not have a myopathy, hi one embodiment, the myopathy is a primary or secondary myopathy.

[0023] In one embodiment, the subject is an adult human subject suffering from growth hormone (GH) deficiency, hi one embodiment, the subject is concurrently receiving recombinant GH therapy or GH gene therapy.

[0024] 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 conditions, double diabetes, Cushing's disease, and obesity syndrome. In one embodiment, the obesity is sarcopenic obesity. In one embodiment, the hypometabolic conditions are selected from the group consisting of conditions associated with prolonged immobilization, conditions associated with bed rest, conditions associated with cast immobilization, conditions associated with stroke, conditions associated with amputation, and post-surgery 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 genetic disorders, and obesity syndrome associated with hypothalamic disorders.

[0025] In some embodiments, the hypometabolic condition is a post-surgical condition, e.g., paraspinal muscle atrophy following lumbar surgery. In one embodiment, the paraspinal muscle atrophy is nerve damage-dependent muscle atrophy. In one embodiment, the surgery is spinal surgery. In one embodiment, the spinal surgery is a lumbar surgery or lumbar procedure, e.g., a lumbar fusion procedure, a lumbar non-fusion procedure, a posterior lumbar fusion procedure, an anterior lumbar fusion procedure, a minimally invasive (MIS) posterior lumbar decompression procedure, a minimally invasive (MIS) posterior lumbar fusion procedure, a non-MIS equivalent procedure, and the like.

[0026] In one embodiment, administration of the composition increases muscle tissue mass and / or function in the human subject. In one embodiment, administration of the composition increases fast muscle tissue mass and / or function in the human subject. In one embodiment, administration of the composition increases slow muscle tissue mass and / or function in the human subject. In one embodiment, administration of the composition increases metabolic rate in the human subject. In one embodiment, administration of the composition increases insulin sensitivity in the human subject. In one embodiment, administration of the composition increases brown adipose tissue levels in the human subject. In one embodiment, administration of the composition increases beige adipose tissue levels in the human subject. In one embodiment, administration of the composition decreases white adipose tissue levels in the human subject. In one embodiment, administration of the composition decreases visceral adipose tissue levels in the human subject. In one embodiment, administration of the composition decreases fat to muscle tissue ratio in the human subject. In one embodiment, the human subject is a pediatric human subject.

[0027] In one embodiment, administration of the composition increases glucose uptake by a target tissue in a human subject, wherein the target tissue is selected from the group consisting of brown adipose tissue, beige adipose tissue, and muscle tissue. In one embodiment, administration of the composition decreases glucose uptake by a target tissue in a human subject, wherein the target tissue is selected from the group consisting of white adipose tissue and liver tissue. In one embodiment, administration of the composition decreases muscle catabolism of protein and / or release of amino acids from muscle in a human subject. In one embodiment, the human subject is a pediatric human subject.

[0028] In one embodiment, administration of the composition increases insulin-dependent glycemic control in a human subject. In one embodiment, administration of the composition reduces intramuscular fat infiltration in a human subject. In one embodiment, administration of the composition achieves a clinically meaningful improvement in quality of life scores as assessed by a standardized quality of life test. In some embodiments, the clinically meaningful improvement is an increase of at least 8 points in the SF-36 Quality of Life Scoring System. In one embodiment, administration of the composition prevents muscle loss or muscle atrophy in a human subject. In one embodiment, the human subject is a pediatric human subject.

[0029] In one aspect, a method for inhibiting myostatin activation in a subject includes 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, and obtaining in the subject: (a) an increase in muscle tissue mass and / or function; (b) an increase in the metabolic rate of the subject; (c) an increase in insulin sensitivity of 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) a decrease in the level of white adipose tissue in the subject; (g) a decrease in the level of visceral tissue in the subject. Disclosed herein are methods comprising administering to a subject a compound effective to cause two or more of: (a) a decrease in the level of adipose tissue; (h) a decrease in the ratio of fat 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 glycemic control 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 as assessed by a standardized quality of life test (e.g., an increase of at least 8 points in the SF-36 Quality of Life Scoring System); (o) prevention of muscle loss or 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 who would benefit from reduced myostatin signaling. In one embodiment, the human subject is a pediatric human subject.

[0030] In one embodiment, the method further comprises selecting a subject suffering from a muscle condition or disorder. In another embodiment, the method further comprises selecting a subject suffering from or at risk of developing a metabolic disorder. In one embodiment, the method further comprises selecting a pediatric human subject.

[0031] In one embodiment, the subject exhibits i) elevated levels of promyostatin in target muscle compared to a control level of promyostatin, or ii) decreased levels of latent myostatin in the circulation compared to a control level of latent myostatin. In one embodiment, the subject exhibits both i) and ii). In one embodiment, the human subject is a pediatric human subject.

[0032] 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 the muscle, or defective signaling between the motor neurons and the target muscle. In one embodiment, the nerve damage is a SCI. In another embodiment, the SCI is a partial / incomplete SCI. In one embodiment, the SCI in the human subject comprises a lesion between i) T1-T6; ii) T7-L5; iii) C6-C7; iv) C5-C6; or v) C3-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 comprises insulin resistance, inflammation, abnormal lipid metabolism, or increased intramuscular fat infiltration. In one embodiment, the muscle atrophy comprises glucocorticoid-induced muscle atrophy. In one embodiment, the human subject is a pediatric human subject.

[0033] In one embodiment, the subject has a metabolic disease selected from the group consisting of type I diabetes, type II diabetes, obesity, metabolic syndrome / pre-diabetes, cardiovascular disease, non-alcoholic steatohepatitis (NASH), spinal cord injury (SCI), hypometabolic states, double diabetes, Cushing's disease, and obesity syndrome. In one embodiment, the human subject is a pediatric human subject.

[0034] 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.

[0035] In another aspect, disclosed herein is a method of treating or preventing a disease associated with defective neural signaling between a neuron and a target tissue in a human subject, comprising the steps of selecting a human subject suffering from a disease associated with defective neural signaling between a neuron and a target tissue; and administering to the human subject a myostatin inhibitor, e.g., 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 treat or prevent the disease, thereby treating or preventing the disease associated with defective neural signaling in the human subject. In one embodiment, the human subject is a pediatric human subject.

[0036] 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.

[0037] In another aspect, disclosed herein is a method for treating a lesion that causes a defect but not a complete loss of signaling between neurons and target muscles in a subject. Such a method comprises administering to the subject a composition comprising a myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody, in an amount effective to treat the muscle located under the lesion in the subject. In some embodiments, the amount is an amount effective to prevent muscle loss or muscle atrophy under the lesion in the subject. In some embodiments, the amount is an amount effective to increase the amount and / or function of the muscle under the lesion in the subject.

[0038] In some embodiments, the pathology is associated with an incomplete spinal cord injury.

[0039] In one embodiment, the muscle contains fast twitch fibers. In another embodiment, the muscle located under the lesion is selected from the group 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 relative to mature GDF11. In some embodiments, the antibody specifically binds to mature myostatin but not mature GDF11. In some embodiments, the antibody binds to and / or blocks the myostatin receptor.

[0040] In one embodiment, the subject has an incomplete spinal cord injury (SCI). In one embodiment, the incomplete SCI in a human subject includes a lesion between i) T1-T6; ii) T7-L5; iii) C6-C7; iv) C5-C6; or v) C3-C8.

[0041] 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 muscle tissue mass and / or function in the subject; (b) increase metabolic rate in 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) increase glucose uptake by brown adipose tissue, beige adipose tissue, or muscle tissue in the subject; (j) decrease glucose uptake by white adipose tissue or hepatic tissue; (k) decrease muscle catabolism of protein and / or release of amino acids from muscle in the subject; (l) increase insulin-dependent glycemic control in the subject; (m) decrease intramuscular fat infiltration in the subject; (n) decrease SF-36 Quality of Life Scoring in the subject; (n) decrease insulin sensitivity ... (o) preventing muscle loss or atrophy in the subject; and / or (p) preventing the development of metabolic dysregulation associated with muscle dysfunction in the subject. In one embodiment, the human subject is a pediatric human subject.

[0042] In another aspect, the disclosure provides a method of treating or preventing a metabolic disease in a human subject, the method comprising the steps of selecting a human subject suffering from a metabolic disease; and administering to the human subject an effective amount of 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. In one embodiment, the human subject is a pediatric human subject.

[0043] 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 conditions, double diabetes, Cushing's disease, and obesity syndrome. In one embodiment, the obesity is sarcopenic obesity. In one embodiment, the hypometabolic conditions are selected from the group consisting of conditions associated with prolonged immobilization, conditions associated with bed rest, conditions associated with cast immobilization, conditions associated with stroke, conditions associated with amputation, and post-surgery 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 genetic disorders, and obesity syndrome associated with hypothalamic disorders.

[0044] In another aspect, the disclosure provides a method of treating or preventing a disease associated with defective neural signaling between neurons and a target tissue in a human subject, 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). In one embodiment, the human subject is a pediatric human subject.

[0045] 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.

[0046] In one embodiment, the target tissue is selected from the group consisting of muscle tissue, adipose tissue, brain tissue, liver tissue, and vascular tissue.

[0047] In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases the amount 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 amount and / or function of fast 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 amount and / or function of slow muscle tissue in a human subject. In one embodiment, the human subject is a pediatric human subject.

[0048] In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases the metabolic rate of the human subject. In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases insulin sensitivity in the human subject. In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases the level of brown adipose tissue in the human subject. In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases the level of beige adipose tissue in the human subject. In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, decreases the level of white adipose tissue in the human subject. In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, decreases the level of visceral adipose tissue in the human subject. In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, decreases the ratio of adipose tissue to muscle tissue in the human subject. In one embodiment, the human subject is a pediatric human subject.

[0049] 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. In one embodiment, the human subject is a pediatric human subject.

[0050] 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. In one embodiment, the human subject is a pediatric human subject.

[0051] 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 would 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.

[0052] 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, such as 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.

[0053] 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, such as 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.

[0054] In another aspect, disclosed herein is a method for increasing insulin-dependent glycemic control in a human subject, comprising the steps of selecting a human subject who would benefit from increased insulin-dependent glycemic control; 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 insulin-dependent glycemic control in the human subject.

[0055] In another aspect, disclosed herein is a method for reducing muscle catabolism of protein and / or muscle release of amino acids in a human subject, comprising the steps of selecting a human subject who would benefit from reduced muscle catabolism of protein and / or muscle release of amino acids; 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 reducing muscle catabolism of protein and / or muscle release of amino acids in the human subject.

[0056] 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, such as 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.

[0057] In one embodiment, the target tissue comprises macrophages, smooth muscle cells and foam cells.

[0058] 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, and obtaining in the human subject: (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; (f) an increase in the level of white adipose tissue in the human subject. Disclosed herein is a method comprising administering to a human subject a human subject in an amount effective to cause at least two or more of: (a) a decrease in the level of visceral adipose tissue in the human subject; (b) a decrease in the level of visceral adipose tissue in the human subject; (c) a decrease in the ratio of adipose tissue to muscle tissue in the human subject; (d) an increase in glucose uptake by white adipose tissue, hepatic tissue or vascular tissue in the human subject; (e) a decrease in muscle catabolism of protein and / or release of amino acids from muscle in the human subject; and / or (f) an increase in insulin-dependent blood glucose regulation in the human subject, thereby treating or preventing a metabolic disease in the human subject. In one embodiment, the human subject is a pediatric human subject.

[0059] In another aspect, disclosed herein is a method for increasing the mass and / or function of muscle located under a lesion in a subject suffering from a lesion, the method comprising the steps of selecting a subject suffering from a lesion; 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 mass and / or function of muscle located under the lesion in the human subject.

[0060] In one embodiment, the pathology is caused by 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.

[0061] 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 an antibody or antigen-binding fragment thereof increases the mass and / or function of slow switch muscles.

[0062] 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%.

[0063] 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%.

[0064] 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 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 locomotor function 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%. In one embodiment, the human subject is a pediatric human subject.

[0065] 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, motor coordination and balance in 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, motor coordination and balance in 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%. In one embodiment, the human subject is a pediatric human subject.

[0066] 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 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%. In one embodiment, the human subject is a pediatric human subject.

[0067] 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 levels of white adipose tissue in a human subject. In some embodiments, levels of white 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, 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%. In one embodiment, the human subject is a pediatric human subject.

[0068] In one embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, increases the total body weight of 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%. In one embodiment, the human subject is a pediatric human subject.

[0069] 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%. In one embodiment, the human subject is a pediatric human subject.

[0070] In one embodiment, the muscle is selected from the group of soleus, gastrocnemius, biceps and triceps.

[0071] In one embodiment, the myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, is administered to the human subject less than 5, 10, 20, 30, 40, 50, or 60 minutes after the human subject is afflicted with 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 is afflicted with 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, 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 after the human subject develops 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 1-30 days, about 1-50 days, about 1-100 days, about 1-200 days, or about 1-300 days.

[0072] 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.

[0073] In one embodiment, the methods disclosed herein further comprise administering a second treatment to the human subject. In one embodiment, the second treatment is selected from the group consisting of insulin, insulin sensitizers, 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 reducing agents, cholesterol sequestrants, angiotensin converting enzyme inhibitors, angiotensin II antagonists, calcium channel blockers, ursodiol, pioglitazone, orlistat, betaine, rosiglitazone, central anti-obesity agents, and the like. agents), gastrointestinal lipase inhibitors, beta3-adrenergic receptor agonists, peptide-based appetite suppressants, cholecystokinin agonists, dopamine agonists, DPP-4 inhibitors, glucagon-like peptides, meglitinides, sulfonylureas, sodium glucose transporter (SGLT) 2 inhibitors, cyclooxygenase inhibitors, progesterone derivatives, metoclopramide-based agents, tetrahydrocannabinol-based agents, and lipid metabolism improvers.

[0074] In one embodiment, the myostatin inhibitor, e.g., an 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., an antibody or antigen-binding fragment thereof, is administered intraperitoneally, intravenously, intramuscularly, or subcutaneously.

[0075] In one embodiment, the antibody or antigen-binding fragment thereof does not bind to GDF11 or activin, hi one embodiment, the antibody or antigen-binding fragment thereof does not bind to mature myostatin.

[0076] 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 the toroid protease. In one embodiment, the antibody or antigen-binding fragment thereof inhibits the formation of mature myostatin via proteolysis by the toroid protease with an IC50 of less than 1 μM.

[0077] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising a complementarity determining region 3 (CDRH3) comprising a 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 a 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 a sequence set forth in any one of SEQ ID NOs: 1 to 3, CDRH2 comprises a sequence set forth in any one of SEQ ID NOs: 4 to 9, CDRH3 comprises a sequence set forth in any one of SEQ ID NOs: 10 to 11 and 66, CDRL1 comprises a sequence set forth in any one of SEQ ID NOs: 12 to 17, CDRL2 comprises a sequence set forth in any one of SEQ ID NOs: 18 to 21, and CDRL3 comprises a sequence set forth in any one of SEQ ID NOs: 22 to 23 and 67.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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: 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] In one embodiment, the antibody or antigen-binding fragment thereof is -6 The 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 The range is M.

[0086] 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.

[0087] 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 that creates an IgG1-like hinge and allows for interchain disulfide bond formation. In one embodiment, the antibody or antigen-binding portion thereof does not bind to GDF11. In one embodiment, the antibody or antigen-binding portion thereof does not bind to mature (fully processed, free / soluble, active) myostatin. In one embodiment, the antibody or antigen-binding portion thereof selectively or preferentially binds to tissue-bound myostatin (e.g., the proform of myostatin, i.e., proMyostatin or pro-myostatin). In one embodiment, the antibody or antigen binding portion thereof binds to both the pro and latent forms of myostatin (promyostatin and latent myostatin), but does not bind to mature myostatin. [Brief description of the drawings]

[0088] [Figure 1] Figures 1A-1B show the domain structure and promyostatin assembly of myostatin (also known as GDF8). 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 precursor protein assembled in an inactive conformation in which the prodomain (dark grey) encapsulates the growth factor (light grey) by a "straight jacket" assembly. This figure is an adaptation of the structure of latent TGFβ1 (Shi et al., Nature, 2011). [Diagram 2]Figure 2 shows that activation of myostatin involves two distinct protease events to generate three major myostatin species. The biosynthetic precursor protein, promyostatin, is processed by two separate proteases. Cleavage of promyostatin (and proGDF11) is performed by proprotein convertases such as furin / PACE3 (Paired Basic Amino acid Cleaving Enzyme3) or PCSK5 (Proprotein Convertase Subtilisin / Kexin type5), which cleave at a conserved RXXR site between the prodomain and the mature growth factor. This cleavage produces a latent complex in which the mature growth factor is shielded from binding to its receptor by the prodomain. Activation and release of the active growth factor is achieved after cleavage by further proteases of the BMP / Tolloid family, such as TLL-2 (Tolloid-like protein 2) or BMP1 (Bone morphogenetic protein 1). These cleavage events result in a mature form of myostatin, which may be referred to as active or mature myostatin. [Diagram 3] Figure 3 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 reflects a significant difference from sham, and an asterisk * at the bottom of the bar reflects a significant difference from SCI-Ab1. [Figure 4] Figure 4 shows muscle wet weights (mass) of sham, SCI-veh, SCI-IgG, SCI-Ab1 treated groups 2 weeks after SCI. Muscles excised include the sublesional soleus and gastrocnemius and the supralesional biceps and triceps. [Diagram 5] FIG. 5 shows the analysis of total fat-free (lean) mass and fat mass in the sham, SCI-veh, SCI-IgG, and SCI-Ab1 treated groups 2 weeks after SCI. [Figure 6]FIG. 6 shows lean mass as a percentage of body weight in sham, SCI-veh, SCI-IgG, SCI-Ab1 treated groups 2 weeks after SCI. [Figure 7] Figure 7 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 a combination of the SCI / veh+SCI / IgG groups from the top graph. [Figure 8] Figure 8 shows BMS locomotor 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. [Figure 9] Figure 9 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. [Figure 10] FIG. 10 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. [Figure 11]11A-11D 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 a week for 8 weeks with a 4-week recovery period. Control animals were administered 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). FIG. 11A 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. FIG. 11B 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 11C is a graph showing the mean % change in lean mass in leg muscles of Ab2-treated and vehicle control animals measured at week 8. Figure 11D is a graph showing the mean % change in lean mass in leg muscles of Ab2-treated and vehicle control animals measured at week 12. [Figure 12] 12A-12B are graphs showing the effect of treatment with Ab2 on muscle weight in biceps and gastrocnemius muscles taken 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 a week 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. [Figure 13] FIG. 13 shows the mean % change in lean mass from baseline (day 0) and the percentage difference in muscle weight compared to vehicle control in healthy cynomolgus monkeys treated with Ab2. [Figure 14]Figures 14A and 14B show latent myostatin levels in serum samples from healthy cynomolgus monkeys treated with Ab2 and control animals, 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 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. [Figure 15] FIG. 15 shows changes in lean mass due to Ab2-mediated myostatin inhibition. [Figure 16] FIG. 16 shows differentially expressed genes (DEGs) in the Ab2-treated group. [Figure 17] FIG. 17 shows suppression of muscle atrophy-causing genes (atrogenes) following Ab2-mediated myostatin inhibition. [Figure 18] FIG. 18 shows the expression of muscle-specific markers following Ab2-mediated myostatin inhibition. [Figure 19] FIG. 19 shows expression of markers of respiratory capacity following Ab2-mediated myostatin inhibition. [Figure 20] FIG. 20 shows the expression of adipocytes and adipogenic markers following Ab2-mediated myostatin inhibition. [Figure 21] FIG. 21 shows the regulation of pyruvate dehydrogenase. [Figure 22] FIG. 22 shows the expression levels of pyruvate dehydrogenase and regulators of fatty acid oxidation. [Diagram 23] FIG. 23 shows an immunofluorescence assay performed on frozen sections of tibialis anterior muscles from healthy mice, co-stained with laminin using Ab2. [Figure 24]Figures 24A-24B show cross sections of tibialis anterior muscle probed with anti-pro / latent GDF8 antibodies, Ab10 or a non-specific targeting antibody (Figure 24A) and HuNeg (Figure 24B), counterstained with DAPI. Scale bar is 0.01 cm. [Diagram 25] Figures 25A-25C show cross sections of tibialis anterior muscle probed with anti-pro / latent GDF8 antibody Ab10 incubated in blocking buffer alone (Figure 25A), anti-pro / latent GDF8 antibody Ab10 incubated in blocking buffer with a 10-fold molar excess of recombinant mouse GDF8 (Figure 25B), or anti-pro / latent GDF8 antibody Ab10 incubated in blocking buffer with a 10-fold molar excess of recombinant mouse GDF11 (Figure 25C). Figures 25A-25C are counterstained with DAPI. [Figure 26] Figures 26A-26C 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). [Figure 27] Figures 27A-27C demonstrate the reduction of SCI-induced intramuscular fat infiltration by a monoclonal antibody that inhibits myostatin activation. [Figure 28] 28A-28B show the effect of a monoclonal antibody that inhibits myostatin activation in a cardiotoxin-induced injury model. [Figure 29] FIG. 29 demonstrates that antibody-treated animals showed a statistically significant increase in mean total cross-sectional bone area and cortical bone thickness compared to controls (PBS). [Diagram 30] Figure 30 demonstrates that antibody-treated animals exhibited increased trabecular bone volume, trabecular thickness, and trabecular number compared to controls. Additionally, antibody-treated animals exhibited decreased trabecular spacing compared to controls. [Diagram 31]FIG. 31 demonstrates that animals treated with myostatin inhibitors exhibited increased bone volume in non-weight-bearing bones, such as vertebrae. [Diagram 32] FIG. 32 demonstrates that mice treated with Abl exhibited a 14.4% weight gain at day 50 compared to control mice (PBS treated). [Diagram 33] FIG. 33 shows the increase in weight of several muscles after treatment with Abl: gastrocnemius, TA, EDL, soleus, and masseter. [Diagram 34] Figure 34A shows a 23% increase in plantar flexion force (maximum torque) after treatment with Abl versus PBS control, and a 20% increase in plantar flexion force maximum torque / limb length after treatment with Abl versus PBS control. Figure 34B shows masseter muscle force after treatment with Abl versus control. [Diagram 35] Figure 35 shows the histological data of the high-dose SMN-C1 cohort and shows histograms of total fiber cross-sectional area (CSA) and CSA distribution in control (vehicle) versus Abl-treated animals, demonstrating a trend toward increased fiber CSA that was entirely attributable to type IIb fibers (data not shown).

[0089] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090] The present invention is based, at least in part, on the discovery that administering a myostatin inhibitor, such as an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, to a subject with a metabolic disease, such as spinal cord injury (SCI), significantly improves both the physiological and functional characteristics of the affected subject. In particular, the inventors have surprisingly discovered that administration of a myostatin inhibitor, such as an anti-pro / latent myostatin antibody or an antigen-binding portion thereof, significantly enhances metabolic rate or energy expenditure in subjects with metabolic disease or dysfunction. Administration of a myostatin inhibitor, such as an anti-pro / latent myostatin antibody, significantly attenuates SCI-induced reductions in sublesional muscle mass and total body weight, while also reducing the amount of undesirable adipose tissue, such as white adipose tissue and visceral adipose tissue. Furthermore, subjects treated with a myostatin inhibitor, such as an anti-pro / latent myostatin antibody, showed significant improvements in motor function, muscle strength, and motor coordination and balance skills.

[0091] Thus, the present invention provides methods of using myostatin inhibitors, such as anti-pro / latent myostatin antibodies or antigen-binding portions thereof, to treat or prevent metabolic disease in a human subject. The present invention also provides methods of using myostatin inhibitors, such as anti-pro / latent myostatin antibodies or antigen-binding portions thereof, to treat or prevent diseases associated with defective neural signaling, increased metabolic rate, increased levels of brown adipose tissue, increased levels of beige adipose tissue, increased insulin-dependent blood glucose regulation, decreased muscle catabolism of protein and / or release of amino acids from muscle, and decreased glucose uptake by target tissues in a human subject. The present invention further provides methods of using myostatin inhibitors, such as anti-pro / latent myostatin antibodies and antigen-binding portions thereof, to increase the mass and / or function of muscle underlying a lesion in a subject afflicted with the lesion.

[0092] Thus, the invention includes the use of antibodies and antigen-binding portions thereof that specifically bind to promyostatin and / or latent myostatin in vivo and block activation of mature myostatin in subjects, e.g., human subjects, who would benefit from reduced myostatin signaling. The invention includes methods of treating or preventing conditions associated with myostatin dysregulation using myostatin inhibitors, e.g., antibodies or antigen-binding portions 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.

[0093] 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.

[0094] Except in the operating examples or where otherwise indicated, the numbers expressing the quantities of ingredients or reaction conditions used herein should be understood to be modified in all cases by the term "about". The term "about" when used in relation to percentages can mean ±1% of the mean. Additionally, the term "about" can mean within ±1% of the value.

[0095] 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 an agent to a subject's system or to a specific area within or on a subject (systemic administration and local administration, respectively).

[0096] 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, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to 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 herein by reference in their entirety. Antibody variants known in the art are also encompassed by the present invention.

[0097] 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, VH, CL and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment consisting of the VH domain (Ward et al., (1989), Nature, 341:544-546); and (vi) an isolated complementarity determining region (CDR). Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but can be joined by a synthetic linker using recombinant methods to make them into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv)); see, for example, 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).

[0098] 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 non-specified elements, whether essential or not.

[0099] 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 embodiments.

[0100] 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, and the like.

[0101] 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, or 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.

[0102] The term "decrease" as used herein refers to a statistically significant reduction in such levels with respect to symptoms of a disease. The 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 the detection method. The 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 the detection method. In certain embodiments, the reduction is to a level that is accepted as being within the normal range for individuals without such a disorder, which can also be referred to as normalization of the level.

[0103] 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 denervation (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 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.

[0104] "Determining," as used herein, is understood as performing an assay or using a method to ascertain the status of someone or something, e.g., the presence, absence, level, or degree of a particular condition, biomarker, disease state, or physiological condition.

[0105] "Onset" or "progression" of a disease refers to the initial manifestation of the disease and / or subsequent progression. Onset of a disease may be detectable and can be assessed using standard clinical techniques. However, onset also refers to progression that may be undetectable. For purposes of this disclosure, onset or progression refers to the biological course of a symptom. "Onset" includes emergence, recurrence, and onset. As used herein, "onset" or "onset" of a myopathy-related disease / disorder includes initial onset and / or recurrence.

[0106] Although methods and materials similar or equivalent to those described herein may 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."

[0107] 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, phosphoryls, or sulfonyls, 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 when 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.

[0108] 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, with 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.

[0109] The relevant measure of the intended purpose can be objective (i.e., measurable by some assay or marker) or subjective (i.e., the subject shows an indication of or feels an effect). In some embodiments, a therapeutically effective amount is an amount that, when administered to a patient population that meets certain clinical criteria for a disease, disorder, or condition (e.g., as determined by manifested symptoms, disease progression / stage, genetic profile, etc.), results in a statistically significant therapeutic response in the population.

[0110] In some embodiments, an effective amount is an amount that, when administered according to a particular regimen, produces a positive clinical outcome, with adverse effects (e.g., toxicity) at a reasonably tolerable level, such that, if adverse effects are present, the treatment regimen is well tolerated by the patient and the benefits of treatment outweigh the risks of toxicity. 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.

[0111] The therapeutically effective amount is generally administered in a dosage regimen that may include a number of unit doses.For any particular pharmaceutical product, the therapeutically effective amount (and / or the appropriate unit dose within an effective dosage regimen) may vary, for example, depending on the route of administration, depending on the combination with other pharmaceutical products.In some embodiments, the specific therapeutically effective amount (and / or unit dose) for any particular patient may depend on various factors, including the disorder and the severity of the disorder to be treated; the activity of the specific pharmaceutical product used; the specific composition used; the age, weight, overall health, sex and diet of the patient; the time of administration, the route of administration, and / or the rate at which the specific pharmaceutical product used is excreted or metabolized; the duration of treatment; and similar factors well known in the medical field.

[0112] 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 antibody 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 mouse, are grafted onto human framework sequences.

[0113] For example, the term "elevation / increase" in relation to a symptom of a disease, such as 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 the 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 the detection method. In certain embodiments, an increase is to a level that is accepted as being within the normal range for individuals without such a disorder, which can also be referred to as normalization of the level. 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 for the disease. In certain embodiments, the method comprises increasing the amount and / or function of muscle tissue after treating a subject with an antibody that specifically binds pro / latent myostatin. In certain embodiments, the method comprises increasing the level of promyostatin in a target muscle compared to a control level of promyostatin.

[0114] The term "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to pro / latent myostatin is substantially free of antibodies that specifically bind to antigens other than pro / latent myostatin). However, an isolated antibody that specifically binds to pro / latent myostatin may have cross-reactivity to other antigens, such as pro / latent myostatin molecules from other species. Additionally, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0115] The term "mature myostatin" refers to the fully processed biologically active form of myostatin, unless otherwise specified. Biologically active forms of myostatin can bind to and / or activate myostatin receptors. The wild-type sequence of mature myostatin is provided as SEQ ID NO: 52. In some cases, mature myostatin can contain one or more mutations that may indicate altered structure / function or stability.

[0116] 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 (including, for example, fragments thereof, such as domain antibodies (dAbs) described in U.S. Pat. Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; and 6,696,245), a small molecule inhibitor, an adnectin, an affibody, a DARPin, an anticalin, an avimer, a versabody, or a 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 can 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 can be used to treat diseases involving fast muscle fibers as described herein. In another embodiment, myostatin inhibitors can be used to provide a therapeutic effect beneath a lesion as described herein.

[0117] As used herein, the phrase "circulating latent myostatin" or "circulating latent myostatin" refers to latent myostatin in the blood, plasma, or serum.

[0118] As used herein, the term "pro / latent myostatin" refers to promyostatin, latent myostatin, or both (i.e., the pro-form or precursor of myostatin).

[0119] "Specific" and "specificity" refer to the selective reactivity of an interaction with respect to interactions between members of a specific binding pair (e.g., ligand and binding site, antibody and 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 a binding preference of at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1,000-fold over a control nonspecific antigen, epitope, receptor ligand, or binding partner. on , K off , and K D For example, a ligand may have a Koff of 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 than, or 10 -6 sec -1 or less; and / or K D 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, or 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 bind more than one ligand, but still be considered to have specificity based on binding preference compared to non-specific antigens and / or by having certain binding kinetic parameters. How to select an appropriate non-specific control is within the capabilities of a person skilled in the art. Binding assays are generally performed under physiological conditions.

[0120] 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 used frequently, are more postural, and help enable long 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 burst 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-fast muscle fibers (type IIA) and fast-twitch muscle fibers (type IIB or IIx). Intermediate-fast 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 least 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 alteration of muscle physiology.

[0121] As used herein, the terms "subject" and "patient" may 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, a subject is a human who would benefit from or is in need of treatment. In one embodiment, a subject is a human subject. In one embodiment, a subject is a pediatric subject.

[0122] 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 time period following administration of a therapeutically effective amount of a myostatin inhibitor, such as an anti-pro / latent myostatin antibody described herein. A sustained increase can be continuous or discontinuous, but overall results in an increase in muscle mass over a specified time period.

[0123] "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, aggravation or worsening, 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%.

[0124] Myostatin Myostatin, also known as GDF8, is a member of the TGFβ superfamily and belongs to a subfamily that contains 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). The domain structure and nomenclature are shown in FIG. 1A. FIG. 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."

[0125] Myostatin is a well-characterized negative regulator of skeletal muscle mass that is released by two separate protease cleavage steps from an autoinhibitory N-terminal prodomain. These cleavage events in the myofiber microenvironment can be referred to as, for example, 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. There is interest in myostatin as a target for the treatment of muscle wasting. Several therapeutics 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 predominantly complexed with its inhibitory prodomain.

[0126] 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 to specifically block 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, it has been found that activation-blocking antibodies can protect mice from dexamethasone-induced muscle atrophy. Evaluation of serum and muscle samples from healthy animals and from animals undergoing dexamethasone-induced atrophy demonstrated altered biodistribution of precursor forms during atrophy, a unique finding with important implications in 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 a significant increase 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 way to prevent myostatin signaling, a technique that provides a novel therapeutic strategy that may also be applied to other members of the TGFβ superfamily.

[0127] Activation and release of mature myostatin is achieved by several distinct protease cleavage events. The first cleavage step of promyostatin and proGDF11 is performed by a proprotein convertase that 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 is 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 as well as fragments of full-length mature myostatin that retain biological activity.

[0128] The term "promyostatin," also known as "proGDF8," refers to an inactive precursor of mature myostatin comprising a disulfide-linked homodimer, each molecule of which contains an amino-terminal prodomain covalently linked to a carboxyl-terminal mature myostatin domain. In one embodiment, "promyostatin" has not been cleaved by either proprotein convertases or BMP / thoroid family proteases. Exemplary promyostatin sequences, variants thereof, and methods for producing promyostatin are well known in the art and are described in more detail herein.

[0129] As used herein, the term "latent myostatin" refers to an inactive precursor of mature myostatin that comprises a disulfide-linked homodimer, each molecule of the homodimer containing an amino-terminal prodomain non-covalently 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 / Tolloid 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.

[0130] Exemplary pro-GDF8 sequences in 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 240-243 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 that additional pro-GDF8 sequences from other species, including any isoforms thereof, are within the scope of the present disclosure.

[0131] Pro-GDF8 (human):

[0132] [ka]

[0133] Pro-GDF8 (rat):

[0134] [ka]

[0135] Pro-GDF8 (mouse):

[0136] [ka]

[0137] Pro-GDF8 (Cynomolgus monkey):

[0138] [ka]

[0139] As previously described (WO2014 / 182676), the prodomain of a myostatin polypeptide consists of several structural domains. These include, for example, the Straight Jacket region, the Fastner region, the Arm region, Fingers region 1, Fingers region 2, the Latency Loop, the Alpha-1 helical region, and the Bowtie region. In some embodiments, a preferred antibody or fragment thereof binds to an epitope within the arm region of the myostatin prodomain. In some embodiments, the epitope is located within the arm region of the prodomain. JPEG2025020230000005.jpg1251 (SEQ ID NO: 118) polypeptide stretch. In some embodiments, the amino acid residues in the arm regions of the prodomain that contact the antibody when bound to the antigen are residues that are not conserved between myostatin and GDF11. In some embodiments, such residue(s) are K, E, and / or N of the polypeptide stretch (shown in bold above).

[0140] Myostatin and GDF11 share a relatively high degree of conservation between their mature growth factor domains, with 90 percent identity, but are less well conserved in their prodomain regions, with less than 50 percent 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). Engagement of myostatin with type I and type II receptors initiates a signaling cascade that results in SMAD phosphorylation and transcriptional activation of muscle 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.

[0141] In some embodiments, provided herein is a pro / latent myostatin antibody that specifically binds to a chimeric construct containing a growth factor domain and an N-terminal propeptide portion of GDF11 and a C-terminal portion of the propeptide of GDF8. This chimeric construct, described below, is referred to as GDF11Arm8.

[0142] >GDF11Arm8 (SEQ ID NO: 65)

[0143] [ka]

[0144] The role of myostatin in muscle homeostasis and metabolic control 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 believed to play a central role in maintaining muscle homeostasis in both healthy and disease states. Myostatin can induce muscle atrophy by inhibiting myoblast proliferation, increase ubiquitin-proteasome activity, and downregulate the activity of the IGF-Akt pathway. These well-recognized effects are seen in numerous situations where atrophy is caused, including injury, diseases such as cachexia, disuse, and space flight, thereby demonstrating the importance of the myostatin signaling mechanism. Based on this central role, significant research has been pursued to inhibit the action of myostatin in vivo. Indeed, antagonism of myostatin signaling has been shown to favor muscle growth / augmentation.

[0145] Moreover, muscle is known to be the main protein reservoir of the body and thus contributes to amino acid homeostasis / metabolism. Together with glucose (mainly made and stored as glycogen in 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). Deficiencies or imbalances in the utilization or mobilization of these energy sinks in the body may underlie, at least in part, various types of metabolic dysregulation. Thus, it is contemplated that myostatin may play a direct role in controlling metabolism by coordinating the balance between the breakdown and synthesis / storage of glucose, fat and / or muscle in the body. Indeed, myostatin has been primarily considered a key regulator of muscle growth / loss since its discovery in 1997, but the findings presented in more detail herein suggest a more extensive role of myostatin as a metabolic regulator.

[0146] Since muscle homeostasis correlates with amino acid / protein metabolism, it is further contemplated that myostatin inhibition can then control nitrogen metabolism and nitrogen mobilization in the body. In muscle catabolism, muscle tissue is broken down into its building blocks, amino acids, which can be considered the main reservoir (and therefore source) of nitrogen. Nitrogen is a component of ammonia, which is highly toxic to the body and is excreted in the form of urea in humans. When nitrogen metabolism is dysregulated, possible consequences include an imbalance in fluid retention, which can appear as generalized or local edema (e.g., congestion; fluid overload). For example, pulmonary edema and renal congestion are frequently observed in patients with heart failure associated with reduced cardiac output. Pulmonary congestion is actually the most common cause of hospitalization in this clinical setting and correlates with poor prognosis.

[0147] Similarly, in pathological conditions involving defects in osmoregulation, affected individuals may be particularly sensitive to salt intake that can cause or exacerbate fluid overload.

[0148] Thus, for subjects with fluid retention or volume-overload, such as subjects with defective osmoregulation and cardiac failure, e.g., chronic cardiac failure, current guidelines suggest that decongestion should be attempted with diuretic therapy (e.g., Regolisti et al., Nephrology@Point of Cre 2016, 2(1):e73-e87). However, in many cases, diuretic treatment is ineffective or the subject is refractory to diuretic treatment. Myostatin inhibition according to the present disclosure may provide clinical benefit to such patients. Specifically, the methods of the present invention are suitable for enhancing the response of subjects who are refractory to diuretic treatment or who respond poorly to diuretic treatment.

[0149] For example, administration of a myostatin inhibitor reduces the required diuretic dosage and / or results in improved control of symptoms, such as CHF symptoms, improves cardiac function, and / or prevents pathological cardiac remodeling or other chronic deterioration of cardiac function. Myostatin inhibition using the inhibitors described herein also reduces the risk of CHF exacerbations, such as the development of acute pulmonary edema.

[0150] For other volume overload conditions requiring high doses of diuretics, such as renal failure or liver disease, the myostatin inhibitors disclosed herein reduce the required diuretic dose, provide improved control of conditions such as peripheral edema or congestion internally (including ocular volume overload within the eye which can cause pleural effusion, ascites, hepatic congestion, or retinal detachment), and / or reduce the risk of pulmonary edema.

[0151] For subjects at higher risk of developing acute pulmonary edema, such as those receiving intravenous fluids, blood transfusions, or fluid transfers, a myostatin inhibitor disclosed herein may be administered prophylactically. For example, a subject with congestive heart failure who needs to receive a blood transfusion may be administered a myostatin inhibitor prophylactically during the transfusion to prevent the development of acute pulmonary edema during the transfusion.

[0152] For subjects with CHF and / or other volume overload conditions who develop hyponatremia, either due to volume overload itself or from the diuretics used to treat volume overload, the myostatin inhibitors disclosed herein can be administered to treat the hyponatremia and / or allow for the use of higher doses of diuretics when administration of diuretics is limited by hyponatremia as a side effect. Generally speaking, however, the myostatin inhibitors disclosed herein can be used to treat hyponatremia regardless of the underlying etiology.

[0153] 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 the lack of specificity observed in these myostatin antagonists described elsewhere may pose a greater risk to certain patient populations because they block additional biological pathways, such as those listed above, in addition to myostatin. This therefore potentially limits the patient population that can be safely treated due to unacceptable adverse effects such as abnormal bleeding, wound healing, or reproductive issues 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 thus binding to activin A limits 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) patient populations requiring 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.

[0154] 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-present myostatin precursors, such as promyostatin and latent myostatin, rather than the mature growth factor, given the predominance of latent complexes in the circulation. Without wishing to be bound by any particular theory, the myostatin inhibitors provided herein, such as antibodies or antigen-binding fragments thereof, can prevent the activation of promyostatin and / or latent myostatin via proteolysis to mature myostatin, which is considered the "active" form of myostatin that can activate the myostatin pathway, for example by binding to type I (ALK4 / 5) and type II (ACTRIIA / B) receptors.

[0155] 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.

[0156] 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, can provide an advantageous way of selectively inhibiting 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 specifically, the present invention includes the surprising finding that by specifically inhibiting myostatin activation, it is possible to achieve not only an increase in muscle mass, but also an enhancement of muscle function and prevention of metabolic dysregulation.Unexpectedly, advantageous therapeutic effects can also be achieved even under pathology in subjects with defective, but not complete loss of signaling between neurons and target tissues, such as target muscles.

[0157] An antibody (used interchangeably with the plural) is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., 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, and an antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chain, an immunoglobulin can be assigned to various classes. There are five main 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.

[0158] The antibodies or antigen-binding fragments thereof described herein can bind to pro / latent myostatin, thereby inhibiting 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.

[0159] 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, the subject exhibits a progressive increase in muscle mass for 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 FIG. 3 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 the inhibitory activity of anti-pro / latent myostatin antibodies against proteolytic cleavage of pro / latent myostatin. In some embodiments, inhibition of pro / latent myostatin cleavage (e.g., by proprotein convertase and / or throid protease) provides a measure of inhibitor potency and can be reflected as an inhibition constant (Ki), which is the concentration of inhibitor (e.g., anti-pro / latent myostatin antibody) required to reduce the activity of a protease (e.g., of a proprotein convertase or throid protease) by half, and is independent of either the enzyme or substrate concentration.

[0160] 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 that 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). The proprotein convertase, in some embodiments, is obtained, e.g., purified, from any mammal, including, but not limited to, human, monkey, or rodent (e.g., mouse, rat, hamster). In another embodiment, the proprotein convertase is recombinantly produced.

[0161] 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).

[0162] 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).

[0163] In some embodiments, the toroid protease for use according to the present disclosure includes, but is not limited to, BMP-1, mTLL-1 and mTLL-2. The toroid protease can be obtained from any mammal, including, but not limited to, human, monkey or rodent (e.g., mouse, rat, 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.

[0164] A toroid protease cleavage site, in some embodiments, is an amino acid sequence that can be cleaved by a toroid (e.g., BMP-1, mTLL-1, and mTLL-2). Exemplary toroid protease cleavage sites for human, rat, mouse, and cynomolgus monkey myostatin are underlined in SEQ ID NOs: 52-55. In some embodiments, a toroid cleavage site comprises the amino acid sequence QR, where Q is glutamine and R is arginine.

[0165] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can bind to pro / latent 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%, such as 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.

[0166] It should be understood that the extent of proteolytic cleavage of myostatin, for example by proprotein convertases and / or thrombolytic proteases, can be measured and / or quantified using any suitable method. In some embodiments, the extent of proteolytic cleavage of myostatin is measured and / or quantified using an enzyme-linked immunosorbent assay (ELISA). For example, an ELISA can be used to measure the level of released growth factor (e.g., mature myostatin). As another example, 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 quantify the extent of proteolytic cleavage of myostatin. In some embodiments, the extent of proteolytic cleavage of myostatin is measured and / or quantified using immunoprecipitation followed by SDS-PAGE or mass spectrometry of tryptic peptides, fluorescence anisotropy-based techniques, FRET assays, hydrogen-deuterium exchange mass spectrometry, and / or NMR spectroscopy.

[0167] 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 main classes: A, D, E, G and M, some of which are subclasses (isotypes), e.g., IgG. 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 and IgA 2 Each light chain can be further divided into 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 LA domain consists of four regions of relatively conserved sequences called framework regions (FR1, FR2, FR3 and FR4) that form a scaffold for three regions of hypervariable sequences (complementarity determining regions, CDRs). CDRs contain most of the residues involved in the specific interaction of the antibody with the antigen. CDRs are designated CDR1, CDR2 and CDR3. Thus, the CDR components on the heavy chain are designated CDRH1, CDRH2 and CDRH3, while the CDR components on the light chain are designated CDRL1, CDRL2 and CDRL3. CDRs typically refer to Kabat CDRs as described in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (1991), edited by Kabat et al. Another criterion for characterizing antigen-binding sites refers to the hypervariable loops described by Chothia. See, e.g., Chothia, D. et al. (1992) J. Mol. Biol. 227:799-817 and Tomlinson et al. (1995) EMBO J. 14:4628-4638. Yet another standard is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains in the Antibody Engineering Lab Manual (Duebel, S. and Kontermann, R., eds., Springer-Verlag, Heidelberg). The embodiments described with respect to Kabat CDRs can alternatively be implemented using the relationships similarly described with respect to Chothia hypervariable loops or with respect to AbM-defined loops, or with respect to any combination of these methods.

[0168] Anti-pro / latent myostatin antibodies or antigen-binding fragments thereof suitable for use in the methods of the invention include those described in International Patent Application Nos. PCT / US15 / 59468 and PCT / US16 / 52014, the entire contents of each of the foregoing applications being incorporated herein by reference in their entirety.

[0169] In some embodiments, the anti-pro / latent myostatin antibodies or antigen-binding fragments thereof of the present disclosure, and nucleic acid molecules of the present disclosure encoding the antibodies or antigen-binding fragments thereof, comprise the CDR amino acid sequences shown in Tables 1-3.

[0170] [Table 1]

[0171] [Table 2-1]

[0172] [Table 2-2]

[0173] [Table 2-3]

[0174] [Table 2-4]

[0175] [Table 3-1]

[0176] [Table 3-2]

[0177] [Table 3-3]

[0178] [Table 3-4]

[0179] In some embodiments, the anti-pro / latent myostatin antibodies, or antigen-binding portions thereof, of the present disclosure include any antibody or antigen-binding fragment thereof that includes 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, the anti-pro / latent myostatin antibodies, or antigen-binding portions thereof, include 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 that encodes a molecule that includes 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 for an antigen. Thus, the anti-pro / latent myostatin antibodies or antigen-binding portions thereof of the present disclosure, or nucleic acid molecules thereof, may comprise at least the heavy chain and / or light chain CDR3 of the antibodies shown in Tables 1-3.

[0180] An embodiment of the present disclosure relates 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.

[0181] In some embodiments, CDRH1 comprises a sequence set forth in any one of SEQ ID NOs: 1-3. In some embodiments, CDRH2 comprises a sequence set forth in any one of SEQ ID NOs: 4-9. In some embodiments, CDRH3 comprises a 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 a sequence set forth in any one of SEQ ID NOs: 12-17. In some embodiments, CDRL2 comprises a sequence set forth in any one of SEQ ID NOs: 18-21. In some embodiments, CDRL3 comprises a sequence set forth in any one of SEQ ID NOs: 22-23, 67, 72, 77, 82, 87, 92, 97, 102, 107, and 112.

[0182] In some embodiments (e.g., for 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.

[0183] 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 antigen-binding portion thereof binds to pro / latent myostatin.

[0184] In some embodiments (e.g., for anti-pro / latent myostatin antibody Ab3 or antigen-binding portion thereof 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 antigen-binding portion thereof binds to pro / latent myostatin.

[0185] In some embodiments (e.g., for anti-pro / latent myostatin antibody Ab5 or antigen-binding portion thereof shown in Table 1), CDRH1 comprises the sequence set forth in SEQ ID NO: 1 or 3, CDRH2 comprises the sequence set forth in SEQ ID NO: 8 or 9, CDRH3 comprises the sequence set forth in SEQ ID NO: 11, CDRL1 comprises the sequence set forth in SEQ ID NO: 16 or 17, CDRL2 comprises the sequence set forth in SEQ ID NO: 20 or 21, and CDRL3 comprises the sequence set forth in SEQ ID NO: 23, and the antibody or antigen-binding portion thereof binds to pro / latent myostatin.

[0186] 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 variations compared to the corresponding CDR regions 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.

[0187] In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding portions thereof of the present disclosure include any antibody that includes 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 that includes 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).

[0188] Aspects of the 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 any of the heavy chain variable sequences 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, the 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.

[0189] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993, modified as described in Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the protein molecule of interest. When gaps exist between the two sequences, gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0190] In some embodiments, conservative mutations may be introduced into CDR or framework sequences at positions where the residue is unlikely to be involved in interactions with pro / latent myostatin as determined based on the crystal structure. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change 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 modifying polypeptide sequences known to those skilled in the art, such as those found in references that summarize 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 FMAusubel 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.

[0191] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein contain mutations that confer desirable properties to the antibodies or antigen-binding fragments thereof. For example, to avoid possible difficulties with Fab arm exchange that is known to occur in natural IgG4 mAbs, the antibodies or antigen-binding portions thereof provided herein may contain a stabilizing "Adair" mutation in which serine 228 (EU numbering, Kabat numbering residue 241) is converted to proline to produce 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).

[0192] 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 may be attached to all or part of a heavy chain, such as IgA, IgD, IgE, IgG and IgM, and any isotype subclass. The antibody may include a suitable constant region (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within this scope may include any V domain combined with any suitable constant region. H and V L It may comprise a domain, or an antigen-binding portion thereof.

[0193] In certain embodiments, V H and / or VL The domains may be reverted to their germline sequences, e.g., the FRs of these domains are mutated using conventional molecular biology techniques to match those produced by germline cells. H and / or V L The domains may be reverted to the germline sequences of IgHV3-30 (SEQ ID NO:36) and / or IgLV1-44 (SEQ ID NO:37), respectively. H and / or V L It should be understood that any of the domains may be reverted to any suitable germline sequence. In other embodiments, the FR sequences remain deviated from the consensus germline sequence.

[0194] IgHV3-30

[0195] [ka]

[0196] IgLV1-44

[0197] [ka]

[0198] 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: 24-35. In some embodiments, the anti-pro / latent myostatin antibody is a murine antibody and includes murine framework region sequences.

[0199] 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 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). When such binding profiles are measured using OCTET or BIACORE, the assay is typically performed according to the manufacturer's instructions unless otherwise specified.

[0200] 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, the antibodies bind near the toroid cleavage site or near the toroid docking site when they bind 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 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 toroid cleavage site or toroid docking site. In some embodiments, the antibodies bind to or near the toroid cleavage site of GDF8. For example, the antibodies can bind to the amino 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 to or near the proprotein convertase docking site of pro / latent myostatin. In some embodiments, the antibody binds near the proprotein convertase cleavage site or near the 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).

[0201] 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 000-fold higher affinity than other members of the TGFβ family of growth factors. In some embodiments, the antibodies or antigen-binding fragments thereof 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 much higher inhibitory activity (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold higher) against proteolytic cleavage of pro / latent myostatin (e.g., by proprotein convertase or toroidal protease) compared to other members of the TGFβ family, such as pro / latent GDF11. In another embodiment, the antibody or antigen-binding fragment thereof disclosed herein does not bind to GDF11. This avoids potential toxicity issues associated with antibodies that cross-react with both myostatin and GDF11.

[0202] In some embodiments, the antibody binds to the antigen, but cannot effectively remove the antigen from the plasma. Thus, in some embodiments, the concentration of the antigen in the plasma can be increased by reducing the clearance of the antigen. However, in some embodiments, the antibody provided herein (e.g., sweeping antibody) has an affinity to the antigen that is sensitive to pH. Such pH-sensitive antibody can bind to the antigen in the plasma at neutral pH and dissociate from the antigen in acidic endosome, thereby reducing antibody-mediated antigen accumulation and / or promoting antigen clearance from the plasma.

[0203] Aspects of the present disclosure relate to sweeping antibodies. As used herein, "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 antigen at the antigen-binding site and can bind to cellular FcRn via the Fc portion of the antibody. In some embodiments, the sweeping antibody or antigen-binding portion thereof can then release the antigen into acidic endosomes, where it may be internalized and degraded. In some embodiments, the sweeping antibody or antigen-binding portion thereof no longer binds to antigen and can then be released back into serum by cells (e.g., by exocytosis).

[0204] 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, the vascular endothelial cells internalize the sweeping antibody or antigen-binding portion thereof, which in some embodiments 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.

[0205] 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 with a 10 -3 M to 10 -8 In some embodiments, the sweeping antibody binds to FcRn with a Kd in the range of 10 M at pH 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 different binding site than FcγR. In some embodiments, the amino acid residue AA of the sweeping antibody Fc region is required for binding to FcRn. In some embodiments, the amino acid residue AA of the sweeping antibody Fc region affects binding to FcRn.

[0206] 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 to FcRn is increased to extend their pharmacokinetic (PK) properties compared to their conventional counterparts. For example, in some embodiments, the sweeping antibodies or antigen-binding portions thereof induce fewer adverse reactions due to efficacy at lower doses. In some embodiments, the sweeping antibodies or antigen-binding portions thereof are administered less frequently. In some embodiments, the transcytosis of the sweeping antibodies or antigen-binding portions thereof to certain tissue types is increased. In some embodiments, the sweeping antibodies or antigen-binding portions thereof enhance the efficiency of transplacental delivery. In some embodiments, the sweeping antibodies or antigen-binding portions thereof are less costly to produce.

[0207] 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) properties 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.

[0208] In some embodiments, the sweeping antibody or antigen-binding portion thereof has a 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 a 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).

[0209] It should be understood that any of the antibodies or antigen-binding fragments thereof provided herein may be engineered to dissociate from the antigen in response to a pH change (e.g., a pH-sensitive antibody). In some embodiments, the sweeping antibodies or antigen-binding portions thereof provided herein are engineered to bind to the 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 the 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 activity. In some embodiments, the FcRn binding activity affects the clearance time of the antigen by the sweeping antibody. In some embodiments, the sweeping antibody may be a long-acting or fast-acting sweeping antibody.

[0210] 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%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%. 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.

[0211] In some embodiments, selecting the appropriate dose of the sweeping antibody or antigen-binding portion thereof for treatment may be performed empirically. In some embodiments, a high dose of the 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 the 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 a day, once a week, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every eight weeks, once every ten weeks, once every twelve weeks, once every sixteen weeks, once every twenty weeks, or once every twenty-four weeks.

[0212] In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein may be modified or engineered to become a sweeping antibody. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein may be converted into a sweeping antibody using any suitable method. For example, suitable methods for making sweeping antibodies or antigen-binding portions thereof have been previously described in Igawa et al., (2013) "Engineered Monoclonal Antibody with Novel Antigen-Sweeping Activity In Vivo", PLoS ONE vol. 8(5):e63236; and Igawa et al., "pH-dependent antigen-binding antibodies as a novel therapeutic modality", Biochimica et Biophysica Acta vol. 1844 (2014) pp. 1943-1950, the contents of each of which are incorporated herein by reference. However, it should be understood that the methods for making sweeping antibodies or antigen-binding portions thereof provided herein are not meant to be limiting. Thus, additional methods for making sweeping antibodies or antigen-binding portions thereof are within the scope of this disclosure.

[0213] 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 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). -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 -11 M. 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.

[0214] In some embodiments, provided herein is a pro / latent myostatin antibody or antigen-binding fragment thereof that does not specifically bind to an epitope within the amino acid sequence set forth in (SEQ ID NO: 64). In some embodiments, provided herein is a pro / latent myostatin antibody or antigen-binding fragment thereof that does not specifically bind to the same epitope as an antibody set forth in Tables 2a, 11a, 11b, or 13 of International Patent Application Publication No. WO2016 / 098357, published June 23, 2016, based on International Patent Application No. PCT / JP2015 / 006323, filed December 18, 2015. In some embodiments, the pro / latent myostatin antibodies or antigen-binding fragments 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 an antibody comprising a VH and VL pair set forth 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.

[0215] Polypeptides Some aspects of the 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 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.

[0216] Some aspects of the 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.

[0217] Antibody or antigen-binding fragment thereof that competes with anti-pro / latent myostatin antibody or antigen-binding fragment thereof An embodiment of the present disclosure relates to an antibody or antigen-binding fragment thereof that competes or cross-competes with any of the antibodies or antigen-binding fragments thereof provided herein. The term "competes" as used herein with respect to an antibody means that a first antibody binds to an epitope of a protein (e.g., latent myostatin) in a manner sufficiently similar to the binding of a second antibody that the result of the binding of the first antibody to the epitope is detectably reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. There can be other cases, but this is not necessary, where the binding of the second antibody to the epitope is also detectably reduced in the presence of the first antibody. That is, the first antibody can inhibit the binding of the second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, if each antibody detectably inhibits the binding of the other antibody to its epitope or ligand, whether to the same extent, a greater extent, or a lesser extent, the antibodies are said to "cross-compete" with each other for the binding of their respective epitope(s). Both competing and cross-competing antibodies are within the scope of the present disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), those skilled in the art will understand that such competing and / or cross-competing antibodies are encompassed and may be useful in the methods and / or compositions provided herein.

[0218] 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 antibodies or antigen-binding portions thereof bind 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.

[0219] In another embodiment, the antibody or antigen-binding portion thereof is -6 In another embodiment, 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 -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.

[0220] Aspects of the disclosure relate to antibodies or antigen-binding portions thereof that compete for binding to pro / latent myostatin with any of the antibodies or antigen-binding fragments thereof provided herein. In some embodiments, the antibodies or antigen-binding portions thereof bind 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 toroid cleavage site or toroid 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 to proprotein convertase docking site of pro / latent myostatin. In another embodiment, the antibodies or antigen-binding portions thereof bind to or near the proprotein convertase cleavage site or to 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 M range.

[0221] 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 the antibody-antigen complex, competitive assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In an additional example, epitope mapping can be used to determine the sequence to which an antibody or antigen-binding portion thereof binds. The epitope may be a linear epitope, i.e., contained in a single stretch of amino acids, or may be a conformational epitope formed by three-dimensional interactions of amino acids that may not necessarily be contained in a single stretch (primary structure linear sequence). Peptides of various lengths (e.g., at least 4-6 amino acids long) may be isolated or synthesized (e.g., recombinantly) and used for binding assays with antibodies. In another example, the epitope to which an antibody or antigen-binding portion thereof binds may be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody. With gene fragment expression assays, an open reading frame encoding a target antigen is fragmented, either randomly or by specific genetic construction, and the reactivity of the expressed antigen fragments with the antibody being tested is determined. Gene fragments may be produced, for example, by PCR, and then transcribed and translated in vitro into proteins in the presence of radioactive amino acids. Binding of the antibody or antigen-binding portion thereof to the radiolabeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes may also be identified using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries).Alternatively, a defined library of overlapping peptide fragments can be tested for binding to a test antibody or antigen-binding portion thereof in a simple binding assay. In additional examples, mutagenesis of the antigen-binding domain, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient and / or necessary for epitope binding. For example, domain swapping experiments can be performed using mutants of the target antigen in which various fragments of the pro / latent myostatin polypeptide are replaced (swapped) with sequences from a closely related but antigenically distinct protein, such as another member of the TGFβ protein family (e.g., GDF11). By assessing the binding of the antibody or antigen-binding portion thereof to mutant pro / latent myostatin, the importance of a particular antigen fragment to the binding of the antibody or antigen-binding portion thereof can be evaluated.

[0222] Alternatively, competitive assays 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. Competitive assays are well known to those of skill in the art.

[0223] Any suitable method, such as the epitope mapping method 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 distance, 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.

[0224] 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, the 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, for example, Kohler and Milstein (1975) Nature, 256:495-499). Hybridomas formed in this manner are then screened using standard methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (e.g., OCTET or BIACORE) 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 recombinant antigen, naturally occurring form, any variant or fragment thereof, and antigenic peptides thereof (e.g., any of the epitopes described herein within a scaffold as a linear epitope or as a conformational epitope) can be used as an immunogen. One exemplary method of making antibodies and antigen-binding portions thereof involves screening a protein expression library, such as a phage or ribosome display library, which expresses antibodies or fragments thereof (eg, scFv). 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.

[0225] In addition to the use of 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.

[0226] In another embodiment, monoclonal antibodies are obtained from non-human animals and then modified using suitable recombinant DNA techniques (e.g., chimeric).Various approaches to making 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 No. EP 171496; European Patent Publication No. 0173494; and British Patent No. GB ​​2177096B.

[0227] For additional antibody production techniques, see Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988. This disclosure is not necessarily limited to any particular source, method of production or other special characteristics of the antibodies.

[0228] Some aspects of the disclosure relate to a host cell 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 may be maintained extrachromosomally. The host cell may be any prokaryotic or eukaryotic cell, such as bacterial, insect, fungal, plant, animal or human cells. In some embodiments, the fungal cell is, for example, of the genus Saccharomyces, specifically of the species S. cerevisiae. The term "prokaryote" includes all bacteria that can be transformed or transfected with DNA or RNA molecules for the expression of antibodies or corresponding immunoglobulin chains. Prokaryotic hosts may include gram-negative and gram-positive bacteria, such as, for example, 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 NSO 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.

[0229] 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). Thus, 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.

[0230] The transformed host cells may be grown in fermenters 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, such as 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.

[0231] Aspects of the present disclosure relate to hybridomas that provide an indefinitely extended 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 engineering. Rearranged antibody genes may be reverse transcribed from appropriate mRNA to produce cDNA. In some embodiments, the heavy chain constant region may be replaced with that 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 capability 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.

[0232] In some embodiments, suitable nucleic acids encoding the variable regions of the heavy and / or light chains are obtained and inserted into expression vectors that can be transfected into standard recombinant host cells. A variety of such host cells 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, 293 or NSO cells. Production of the antibody or antigen-binding fragment may be carried out by culturing the modified recombinant host under culture conditions suitable for host cell growth and expression of the coding sequence. The antibody or antigen-binding fragment may be recovered by isolating it from the culture. The expression system may be designed to include a signal peptide so that the resulting antibody is secreted into the medium; however, intracellular production is also possible.

[0233] 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 include at least one complementarity determining region (CDR) of the VH and / or VL of the variable regions of the antibodies produced by any one of the hybridomas described above.

[0234] 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 a vector may contain additional genes, such as marker genes, that allow the selection of the vector in a suitable host cell and under suitable conditions.

[0235] 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 a translatable mRNA. Control elements that ensure expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They may include a control sequence that promotes initiation of transcription and optionally a polyA signal that promotes termination of transcription and stabilization of the transcript. 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, PL, Lac, Trp or Tac promoters in E. coli, and examples of control elements that allow expression in eukaryotic host cells are the AOX1 or GAL1 promoters in yeast or the CMV promoter, SV40 promoter, RSV promoter (Rous sarcoma virus), CMV enhancer, SV40 enhancer or globin intron in mammalian and other animal cells.

[0236] Such control elements, other than 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, leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the polynucleotide and have been previously described. The leader sequence(s) are 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 a portion thereof, for example, into the extracellular medium. Heterologous polynucleotide sequences may optionally be used that encode fusion proteins containing C- or N-terminal identification peptides that confer desirable characteristics, for example stabilization or simplified purification of the expressed recombinant product.

[0237] 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, in particular plasmids, cosmids, viruses and bacteriophages, that contain polynucleotides encoding the variable domains of the immunoglobulin chains of an antibody or antigen-binding fragment, optionally in combination with polynucleotides encoding the variable domains of the other immunoglobulin chains of the antibody.

[0238] In some embodiments, the expression control sequences are provided as eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, although control sequences for prokaryotic hosts may also be used. Expression vectors derived from viruses, such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses, may be used to deliver polynucleotides or vectors to targeted cell populations (e.g., to engineer cells to express antibodies or antigen-binding fragments). 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 vectors containing the polynucleotides (e.g., sequences and expression control sequences encoding heavy and / or light chain variable domain(s) of immunoglobulin chains) may be transferred into host cells by suitable methods that vary depending on the type of cellular host.

[0239] qualification The antibodies and antigen-binding fragments of the 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 either directly to the polypeptides of the disclosure or indirectly through an intermediate (e.g., a linker, etc.) 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 includes, for example, iodine ( 131 I, 125 I, 123 I, 121 I), Carbon ( 14 C), Sulfur ( 35 S), tritium ( 3 H), Indium ( 115m In, 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, 47S c, 86 R, 188 Re, 142 Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se and tin ( 113 Sn, 117 Detectable substances include radioactive metal ions such as radioactive ions (e.g., Sn), e.g., alpha emitters or other radioisotopes. The detectable substance may be coupled or conjugated to the anti-pro / latent myostatin antibody, or antigen-binding portion thereof, of the present disclosure, either directly or indirectly through an intermediate (e.g., a linker, etc.) using suitable techniques. The anti-pro / latent myostatin antibody, or antigen-binding portion thereof, conjugated to a detectable substance can be used for the diagnostic assays described herein.

[0240] 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, may 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 administration of 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 cause two or more of the biological effects described below. In some embodiments, the myostatin inhibitor, such as an antibody or antigen-binding portion thereof, is administered in an amount effective to cause three or more of the biological effects described below. In some embodiments, the myostatin inhibitor, such as an antibody or antigen-binding portion thereof, is administered in an amount effective to cause four or more of the biological effects described below. In some embodiments, the myostatin inhibitor, such as an antibody or antigen-binding portion thereof, is administered in an amount effective to cause five or more of the biological effects described below. In some embodiments, the myostatin inhibitor, e.g., an antibody or antigen-binding portion thereof, is administered in an amount effective to cause six or more of the biological effects described below. In some embodiments, the myostatin inhibitor, e.g., an antibody or antigen-binding portion thereof, is administered in an amount effective to cause seven or more of the biological effects described below. In some embodiments, the myostatin inhibitor, e.g., an antibody or antigen-binding portion thereof, is administered in an amount effective to cause eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen of the biological effects described below.

[0241] 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 / 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, among others, 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 do not fuse into multinucleated structures as they are in striated muscle tissue. Skeletal muscle tissue is under voluntary control. Muscle fibers are syncytial and contain myofibrils, tandem arrays of sarcomeres. There are two general types of skeletal muscle fibers, slow (type I) and fast (type II), according to their expression of specific myosin heavy chain (MHC) isoforms. Slow muscles are better equipped to work aerobically and are useful for enabling long endurance movements such as long-distance running, whereas fast muscles, which fatigue quickly, are better equipped to work anaerobically and are used for powerful burst movements such as sprinting. The distinction between slow and fast muscles is based on histochemical staining for myosin adenosine triphosphatase (ATPase) and the type of myosin heavy chain. Slow muscles (type I) are MHC isoform I, and the three fast muscles (type II) are MHC isoform IIa, MHC isoform IId, and MHC isoform IIb (S. Schiaffino, J. Muscle Res. Cell. Motil., vol. 10 (1989), pp. 197-205). In some embodiments, the mass and / or function of fast muscle tissue is increased in a human subject. In other embodiments, the mass and / or function of slow muscle tissue is increased in a human subject.

[0242] The biological effect of an effective amount of a pharmaceutical composition 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.

[0243] In one aspect, the disclosure provides a method for promoting fiber type switching in a subject, the method comprising administering to the subject a myostatin inhibitor, e.g., 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 promote fiber type switching, thereby promoting fiber type switching in the subject.

[0244] In another aspect, the disclosure provides a method for preferentially increasing type II or fast twitch fibers over type I or slow twitch fibers in a subject, the method comprising administering to the subject a myostatin inhibitor, e.g., 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 fibers over type I or slow twitch fibers, thereby preferentially increasing type II or fast twitch fibers over type I or slow twitch fibers in the subject.

[0245] In some embodiments, administering an effective amount of a myostatin inhibitor, such as an antibody or antigen-binding fragment thereof, 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 health status of the subject. For example, clinically meaningful changes in muscle mass can improve a patient's mobility, self-care, metabolism, and the like. 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 of muscle shows a measurable effect. In other embodiments, the effect is localized to a particular 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 by MRI (e.g., forearm cross-section), circumference, diaphragm width (e.g., by ultrasound), and the like.

[0246] 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 a variety of measures, including, but not limited to, force generation, grip strength (e.g., maximal grip strength), endurance, muscle oxidative capacity, dynamic grip endurance, and the like. In some embodiments, serum creatinine levels are used as a validated biomarker of muscle mass, although with limited sensitivity.

[0247] 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 score, 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.

[0248] In some embodiments, the 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 pathology resulting from spinal cord injury, for example. In some embodiments, the subject is in the acute spinal cord injury stage immediately after injury, where it is generally difficult to diagnose between complete and incomplete injury. In other embodiments, the subject is in the subacute spinal cord injury stage, 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 stage. The chronic SCI stage occurs approximately 4 or 6 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.

[0249] In some embodiments, the amount and / or function of muscle tissue below the lesion is increased in a subject suffering from a pathology, 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 pathology, 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%, 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%. In some embodiments, the function 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, 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%.

[0250] 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, locomotor function in 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 locomotor function 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%.

[0251] 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, motor coordination and balance in 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%.

[0252] In another embodiment, administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, increases muscle strength in a human subject, e.g., a subject suffering from a pathology. In some embodiments, the muscle strength 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 muscle strength 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%.

[0253] 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 result in clinically meaningful changes in muscle function that correspond to increased functionality in the patient. In some embodiments, increased functionality includes improved patient 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 can include conditions associated with neuropathy (whether due to injury or due to surgical or other clinical procedures).

[0254] 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 to undergo 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 that involve immobilization of a particular muscle or muscle group (e.g., cast immobilization, etc.); and v) patients on mechanical ventilation (e.g., as a result of acute injury). Administration of the myostatin inhibitors described herein may accelerate recovery in such patients. In some embodiments, such administration may be prophylactic. For example, the antibody may be administered prior to or immediately after undergoing 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, for example, to the site or near the area affected by injury, surgery, etc.

[0255] B. Effects on Metabolic Rate 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 the metabolic rate of a human subject. In some embodiments, administration of an effective amount of such a myostatin inhibitor, such as 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, such as by examining oxygen input and carbon dioxide output, or by indirect calorimetry as demonstrated by 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%.

[0256] 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 takes an oral dose of 75 grams of glucose, and then blood glucose levels are measured over the next two hours. A blood glucose level below 7.8 mmol / L (140 mg / dl) is considered normal, a blood glucose level between 7.8 mmol / L and 11.0 mmol / L (140-197 mg / dl) is considered impaired glucose tolerance (IGT), and a blood glucose level greater than or equal to 11.1 mmol / L (200 mg / dl) is considered diabetes. With regard to fasting insulin testing, a fasting serum insulin level greater than 25 mIU / L or 174 pmol / L is 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%.

[0257] D. Effects on Adipose Tissue Levels in Human Subjects Administration of a myostatin inhibitor, such as an antibody or an antigen-binding fragment thereof 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 the 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.

[0258] 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 is beneficial for anyone with impaired insulin function (Stanford et al., J Clin Invest., 2013, vol. 123(1): pp. 215-223).

[0259] 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).

[0260] Visceral or abdominal fat (also known as organ fat or intraperitoneal fat) is located inside the abdominal cavity and packed between the organs (stomach, liver, intestines, kidneys, etc.). Visceral fat is different from subcutaneous fat under the skin and intramuscular fat scattered within skeletal muscles. Fat in the lower body, such as the thighs and buttocks, is subcutaneous and not a coherently spaced tissue, whereas fat in the abdomen is mostly visceral and semi-liquid. Excess visceral fat is known as central obesity, or "belly fat", where the abdomen is overly protruding, 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 diseases (Mokdad et al., JAMA: The Journal of the American Medical Association., 2001, vol. 289(1):76-9).

[0261] 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.

[0262] Administration of a myostatin inhibitor, such as an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, increases the levels of brown adipose tissue and / or beige adipose tissue in a human subject, whereas administration of a myostatin inhibitor, such as an anti-pro / latent myostatin antibody or antigen-binding portion thereof, decreases the levels of white adipose tissue and visceral adipose tissue in a human subject.

[0263] In some embodiments, levels of brown 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 or beige adipose tissue are 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%.

[0264] 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%.

[0265] 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 fat to muscle tissue in a human subject. In some embodiments, the ratio of fat 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 fat 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%.

[0266] Administration of a myostatin inhibitor, such as an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, also increases the ratio of muscle tissue to fat in a human subject. In some embodiments, the ratio of muscle tissue to fat 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 ratio of muscle tissue to fat 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%.

[0267] 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%.

[0268] 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%.

[0269] 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 catabolism of protein and / or release of amino acids from muscle 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%.

[0270] H. Effects on insulin-dependent glycemic control 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-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%.

[0271] 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%.

[0272] 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 assess various aspects of physical, mental, social and other parameters. In general, a higher degree of quality of life is associated with factors such as availability of assistive technology; return to the community; functionality of the lower limbs and walking and / or wheeled mobility; mental health; severity of neurological and autonomic disorders; pain management; functional independence and self-care; upper limb strength; and control of spasticity. 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 such that a clinically meaningful improvement is achieved as measured by a standardized quality of life test / system. A number of suitable tests are known in the art for assessing quality of life in patients, including: 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 (SIP68); and World Health Organization Quality of Life-BREF (WHOQOL-BREF).

[0273] In some embodiments, quality of life is assessed according to the SF-36 Quality of Life Scoring System, a validated 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.

[0274] In some embodiments, the SCI Neurological Quality of Life Test is used to assess the quality of life of patients 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 a number of clinical indications.

[0275] K. Effects 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%.

[0276] In some embodiments, suitable subjects are subjects who have not developed atrophy but are considered to be at risk of developing atrophy. In some embodiments, the subject has a disease or condition associated with neurological deficits that impair motor neuron function. In some embodiments, such conditions are caused by muscular dystrophy or atrophy. In some embodiments, such neurological deficits are caused by nerve damage. In some embodiments, nerve damage involves partial denervation of motor neurons, which causes partial impairment of function in affected muscles. In some embodiments, such conditions are 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).

[0277] 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 manifestation 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.

[0278] 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 readily monitored or evaluated by any suitable method for assessing motor function involving the affected muscles.

[0279] In some embodiments, administration of an effective amount of such an antibody also prevents or reduces early onset axonal polyneuropathy in the affected limb.

[0280] L. Effect on preventing the onset of metabolic disease in a subject 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 disease in a subject, e.g., a human subject. In some embodiments, the development of metabolic disease 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 disease 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%.

[0281] In some embodiments, suitable subjects are subjects 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 related to muscle dysfunction. In some embodiments, the muscle dysfunction is related to partial denervation of motor neurons, which causes partial defects of function in 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).

[0282] 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 impaired muscle function, 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.

[0283] 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, total plasma antioxidant status, lipid oxidation and erythrocyte glutathione peroxidase activity.

[0284] Pharmaceutical Compositions Myostatin inhibitors, such as antibodies or antigen-binding fragments thereof described herein, can be formulated into pharmaceutical compositions suitable for administration in human or non-human subjects. Such pharmaceutical compositions can be for therapeutic or prophylactic use. One or more of the 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. By "pharmaceutically acceptable" it is meant 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 described previously. See, for example, Remington: The Science and Practice of Pharmacy 20th Edition (2000), Lippincott Williams and Wilkins, ed. KE Hoover.Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, or other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin. , proteins such as 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.

[0285] 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.

[0286] 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 may 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 may be produced by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to obtain liposomes with the desired diameter.

[0287] 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 in 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).

[0288] In other examples, the pharmaceutical compositions described herein may be formulated in sustained release form. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or an antigen-binding portion thereof, the matrices being in the form of shaped articles, e.g., films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactide lactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0289] Pharmaceutical compositions used for in vivo administration must be sterile. This is easily 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 that can be pierced by a hypodermic injection needle.

[0290] 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.

[0291] To prepare solid compositions such as tablets, the main 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 pharma- ceutically 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 evenly dispersed throughout the composition, so that the compositions can 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 0.1 mg to about 500 mg of the active ingredient of the present disclosure. The tablets or pills of the novel compositions may be coated or otherwise compounded to provide a dosage form that provides the advantage of prolonged action. For example, the tablets or pills may include an inner dosage and an outer dosage component, the latter being in the form of an envelope over 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.

[0292] Suitable surfactants include in particular non-ionic agents such as polyoxyethylene sorbitan (e.g. Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g. Span™ 20, 40, 60, 80 or 85). Compositions containing 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 pharma- ceutically acceptable vehicles, may be added as required.

[0293] Suitable emulsions may be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient may be dissolved in a premixed emulsion composition, or alternatively, in an emulsion formed by mixing oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and phospholipids (e.g., egg phospholipids, soybean phospholipids or soybean lecithin) with water. It is understood that other ingredients, such as glycerol or glucose, may be added to adjust the tonicity of the emulsion. Suitable emulsions typically contain up to 20% oil, for example 5 to 20% oil.

[0294] The emulsion composition may be prepared by mixing anti-promyostatin antibody with Intralipid™ and its components (soybean oil, egg phospholipids, glycerol and water).

[0295] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable aqueous or organic solvents or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0296] Compositions, preferably in sterile pharma- ceutically acceptable solvents, may be nebulized by the use of gases. Nebulized solutions may be directly inhaled 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.

[0297] Subject The pharmaceutical compositions described herein are suitable for administration in human or non-human subjects. Thus, myostatin inhibitors, such as 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 may benefit from increased muscle mass / function, as well as improved metabolism. In some embodiments, suitable subjects have pre-existing muscle conditions and / or associated metabolic dysfunction. In some embodiments, suitable subjects are at risk for developing such condition(s). In some embodiments, suitable subjects are subjects undergoing therapy that includes another therapeutic agent intended to treat a muscle / metabolism condition but with associated adverse effects or toxicity. In some embodiments, the subject is a pediatric subject, such as a human patient from birth to less than 18 years of age.

[0298] 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 fibers or involves muscles rich in fast-twitch fibers; and iii) the subject retains anabolic capacity (e.g., a generally healthy adult with an injury) and / or is in a period of growth (e.g., a young child, etc.).

[0299] In some embodiments, such medicaments are suitable for administration in pediatric, adult, and / or geriatric populations.

[0300] Pediatric populations in need of a myostatin inhibitor, such as an anti-pro / latent myostatin antibody and antigen binding portion 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 a myostatin inhibitor, such as an antibody or antigen binding fragment 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, 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 may be (a) at least 5 years old and (b) under 18 years old.

[0301] The age of an adult population in need of a myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody and antigen-binding portion 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.

[0302] The age of the elderly population in need of myostatin inhibitors, such as the anti-pro / latent myostatin antibodies and antigen-binding portions thereof described herein, can be 65 years of age or older (i.e., ≧65 years of age), for example, at least 70, 75 or 80 years of age.

[0303] A human subject who may benefit from treatment may be a human patient who has, is at risk of developing, or is suspected of having a metabolic disease / disorder associated with defective neuronal signaling, such as those described below. A subject who has a pro / latent myostatin-related disease or disorder may be identified by routine medical tests, such as laboratory tests, organ function tests, CT scans, or ultrasound. A subject who is suspected of having any of such diseases / disorders may exhibit one or more symptoms of the disease / disorder. A subject who is at risk for a disease / disorder may be a subject who has one or more risk factors for the disease / disorder.

[0304] The control subject described herein is a subject that provides a suitable reference for evaluating the effect of a particular treatment or intervention on a test subject or subject.The control subject may be a subject of similar age, race, sex, weight, height, and / or other characteristics, or any combination thereof, to the test subject.

[0305] In some embodiments, a myostatin assay (e.g., myostatin ELISA) is used to determine subjects in need of treatment with anti-pro / latent myostatin antibodies. Methods for assaying myostatin can be found in Lakshman et al., Molecular and Cell Endocrinology, (2009) 302:26-32 (myostatin ELISA) and Bergen et al., Skeletal Muscle, (2015) 5:21 (liquid chromatography with tandem mass spectrometry), both of which are incorporated herein by reference.

[0306] In some embodiments, a method for improving muscle performance in a subject is provided. The subject may or may not have, may be at risk of, or may 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).

[0307] In some embodiments, a myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody and antigen-binding portion thereof described herein, is 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, a myostatin inhibitor, e.g., an antibody or antigen-binding portion thereof, is administered in an amount effective to reduce pro / latent myostatin or latent myostatin levels by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more).

[0308] 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 of having, or not be at risk of having a condition associated with reduced muscle mass and / or reduced muscle function. In some embodiments, the subject has or is at risk of having a condition associated with reduced muscle mass and / or reduced muscle function.

[0309] The method of the present invention further comprises the step of selecting a subject. In some embodiments, the subject suffers from or is at risk of developing a muscle condition or disorder. In some embodiments, the subject suffers from or is at risk of developing a metabolic disorder. In some embodiments, the subject suffers from or is at risk of developing a disease or disorder associated with defective neural signaling.

[0310] Route of Administration To carry out 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, and 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 and milled powder.

[0311] Conventional methods known to those skilled in the art of medicine may be used to administer the pharmaceutical composition to a subject depending on the type of disease or site of disease to be treated. The composition may be administered via other conventional routes, such as orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. It may also be administered to a subject via an injectable depot route of administration, such as using 1, 3 or 6 month depot injectable or biodegradable materials and methods.

[0312] Injectable compositions may contain a variety of carriers, such as vegetable oils, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies may be administered by drip infusion, in which a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is injected. The physiologically acceptable excipient may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipient. For intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the antibody may be dissolved and administered in a pharmaceutical excipient, such as water for injection, 0.9% saline, or 5% glucose solution.

[0313] In one embodiment, the myostatin inhibitor, for example, anti-pro / latent myostatin antibody or its antigen-binding portion, 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 myostatin inhibitor, for example, anti-pro / latent myostatin antibody or its antigen-binding portion, 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, for example, PCT Publication No. WO00 / 53211 and U.S. Patent No. 5,981,568.

[0314] The particular dosing regimen used in the methods described herein, including dosage, timing and repetition, will depend on the particular subject and that subject's medical history.

[0315] The efficacy of treatment for myopathy-associated disease / disorder can be evaluated using any suitable method.For example, the efficacy of treatment for myopathy-associated disease / disorder 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 assay), and evaluating biopsy (e.g., by histological, histochemical, electron microscopic, biochemical and genetic analysis).

[0316] "An effective amount," as used herein, refers to the amount of each active agent needed to confer a therapeutic effect on a subject, either alone or in combination with one or more other active agents. 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 metabolic rate in 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 following injury; Myostatin inhibitor refers to an amount of a myostatin inhibitor, such as an antibody or antigen-binding fragment thereof, sufficient to achieve a clinically meaningful improvement in quality of life as assessed by a standardized system such as the IQM System; prevention of muscle loss or muscle atrophy in a subject; and / or prevention of the onset of metabolic disease in a subject.

[0317] As recognized by those skilled in the art, effective amounts vary depending on the specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex and weight, duration of treatment, nature of concurrent treatment (if any), specific route of administration and similar factors within the knowledge and professional opinion of medical personnel. These factors are well known to those skilled in the art and can be addressed with no more than routine experimentation. It is generally preferred that the maximum dose of each component or combination thereof, i.e., the highest safe dose according to sound medical judgment, is used. However, it is understood by those skilled in the art that a patient may insist on a lower dose or a tolerable dose for medical reasons, psychological reasons or virtually any other reason.

[0318] 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.

[0319] In some embodiments, with respect to an increase in latent myostatin in the target muscle following 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 into any of the durations recited) following the administering step. In one embodiment, the increase in latent myostatin in the target muscle following 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 into any of the durations recited) following 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 more (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.

[0320] In some embodiments, with respect to an 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 together into 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 together into 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 lumped together into 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 administration 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 administration step.

[0321] In some embodiments, with respect to a reduction in circulating levels of 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 any range bracketed by any of these values) compared to control levels of latent myostatin. In one embodiment, the reduction in circulating levels of latent myostatin is in the range of 1- to 3-fold, 1.2- to 10-fold, 2- to 9-fold, 3- to 8-fold, 4- to 7-fold, 2- to 7-fold, etc., reduction compared to control levels of latent myostatin.

[0322] 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 as compared to a control muscle mass. In some embodiments, a muscle treated with an effective amount of the antibody is increased 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. as compared to a control muscle mass not treated with an effective amount of the antibody. In some embodiments, such an increase in muscle mass is achieved in selected muscle groups or types in the subject.

[0323] 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 an antibody can promote fiber type switching from type I to type II. In some embodiments, an effective amount of a 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 a myostatin inhibitor, e.g., an antibody or antigen-binding portion thereof, can promote type II fibers relative to other types of fibers. In some embodiments, an effective amount of a myostatin inhibitor, e.g., an antibody or antigen-binding portion thereof, can promote type IIB fibers relative to other types of fibers. In some embodiments, such fiber phenotype switching can occur without significant changes in overall muscle mass. In other embodiments, such fiber phenotype switching can occur concomitantly with an increase in overall muscle mass.

[0324] 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 diameter of muscle fibers in the subject as 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 increase as 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. increase as compared to control muscle fibers.

[0325] 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 as compared to a 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 increase as 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. increase as compared to a control subject.

[0326] 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 reduce intramuscular fat infiltration in the subject as compared 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, as compared to a control subject. In some embodiments, the reduction in intramuscular fat infiltration is in the range of 1-5-fold, 2-10-fold, 1-1.5-fold, 1-2-fold, etc., reduction as compared to a control subject.

[0327] In some embodiments, a method of 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 toroid proteases. In one embodiment, inhibition of proteolytic cleavage of promyostatin or latent myostatin by toroid proteases results in a progressive increase in muscle mass. In one embodiment, the subject exhibits a progressive increase in muscle mass for 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 of 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 comprises 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 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.

[0328] 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 as 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 increase as 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 as compared to control muscle function.

[0329] 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., average amount) 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 prior to (e.g., immediately before) administration of a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof. In some embodiments, the control muscle mass is the target muscle mass in a subject administered a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof, instead of a normal antibody (e.g., of the same isotype as the pro / latent myostatin antibody) obtained from an animal that was not exposed to the antigen against which the pro / latent myostatin antibody or antigen-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.

[0330] 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 determined in vitro using a muscle lever system adapted for a horizontal perfusion bath) 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 increase compared to the control force-generating capacity. In some embodiments, the increase in force-generating capacity is in the range of 1-5-fold, 2-fold-10-fold, 1-fold-1.5-fold, 1-fold-2-fold, etc. increase compared to the control force-generating capacity.

[0331] As used herein, the term "control force generation 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 generation capacity of a muscle in a subject. In some embodiments, the control force generation capacity is a predetermined value. The control force generation capacity is experimentally determined. In some embodiments, the control force generation capacity is the force generation 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 generation capacity is the force generation capacity (e.g., average force generation 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 generation capacity is the force generation capacity of a target muscle in a subject prior to (e.g., immediately prior to) being administered a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof. In some embodiments, the control muscle force generation capacity is the force generation capacity of the target muscle in a subject administered a myostatin inhibitor, e.g., a pro / latent myostatin antibody, instead of a normal 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 pro / latent myostatin antibody is directed. In some embodiments, the control force generation capacity is the force generation capacity of the target muscle 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.

[0332] 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 prior to the administering step.

[0333] Dosage Empirical considerations such as half-life generally contribute to determining the 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 is generally, but not necessarily, based on the treatment and / or suppression and / or reversal and / or delay of the disease / disorder involving 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.

[0334] In one example, the dosage for the myostatin inhibitors described herein, e.g., anti-pro / latent myostatin antibodies or antigen-binding fragments thereof, can be empirically determined in individuals given one or more administrations of the myostatin inhibitor, e.g., antibodies or antigen-binding fragments thereof. Individuals are given increasing dosages of the antagonist. Indicators of disease / disorder can be followed to assess efficacy of the antagonist.

[0335] In general, 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 mentioned above. For repeated administration over several days or longer, depending on the condition, treatment is sustained until desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate the disease or disorder associated with pro / latent myostatin or its symptoms. 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 followed by alternate weekly maintenance doses of about 1 mg / kg. However, other dosing regimens are useful depending on the pattern of pharmacodynamic decay the clinician wishes to achieve. For example, dosing 1-4 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 frequency of administration 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 changed over time.

[0336] In some embodiments, the administration of the myostatin inhibitor, e.g., any of the antibodies or antigen-binding fragments thereof described herein, comprises a single dose. In some embodiments, the administration of the myostatin inhibitor, e.g., any of the antibodies or antigen-binding fragments thereof described herein, comprises multiple doses (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses). The administering step may comprise more than two doses. In some embodiments, the administration comprises at least a first dose and a second dose of a therapeutically effective amount of the myostatin inhibitor, e.g., an antibody or antigen-binding portion thereof. In one embodiment, the first dose and the second dose are administered to the subject at least about 4 weeks apart, 6 weeks apart, 8 weeks apart, or 12 weeks apart.

[0337] 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 that individual's medical history and the characteristics of the individual drug, such as the drug's half-life and other relevant considerations.

[0338] For purposes of this disclosure, the 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, the 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, may be continuous or intermittent, depending, for example, on the physiological condition of the recipient, and other factors known to the skilled physician. Administration of a myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody or antigen-binding fragment thereof, may be essentially continuous over a preselected period of time, or may 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.

[0339] As used herein, the term "treat" refers to the application or administration of a composition containing one or more active agents to a subject having a disease / disorder, a symptom of a disease / disorder, or a predisposition to a disease / disorder involving myopathy to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder, the symptom of the disease, or the predisposition to the disease / disorder.

[0340] 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 to defer, hinder, slow, retard, stabilize and / or postpone the progression of the disease. This delay may be for a variety of 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 number of subjects sufficient to provide statistically significant results.

[0341] Combination therapy The present invention encompasses pharmaceutical compositions and related methods used as combination therapy to treat subjects who may benefit from myostatin inhibition in vivo. In any of these embodiments, such subjects may receive combination therapy that includes a first composition that includes at least one myostatin inhibitor, such as an antibody or antigen-binding portion thereof described herein, in combination with a second composition that includes at least one additional therapeutic agent for treating the same or overlapping disease or clinical condition. The first and second compositions may both act on the same or distinct cellular targets. In some embodiments, the first and second compositions may 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 may treat or alleviate separate sets of symptoms or aspects of a disease or clinical condition. For example, the first composition may treat myopathy associated with a disease, and the second composition may treat inflammation or fibrosis associated with the same disease, etc. Such combination therapies may be administered in conjunction with each other. The phrase "in conjunction with" refers to a combination therapy, meaning that the therapeutic effect of the first therapy overlaps temporally and / or spatially with the therapeutic effect of the 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.

[0342] In a preferred embodiment, the combination therapy produces a synergistic effect in treating a disease. The term "synergistic" refers to an effect (e.g., greater efficacy) that is greater than the additive effect of each monotherapy taken together.

[0343] In some embodiments, the combination therapy comprising the pharmaceutical compositions described herein produces an overall efficacy equivalent to that produced by another therapy (such as, for example, 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 combination therapy allows for lower dosages of the second agent while still maintaining overall efficacy. Such combination therapy may be particularly suitable for patient populations where long-term treatment is warranted and / or involving pediatric patients.

[0344] Thus, the present invention provides pharmaceutical compositions and methods for use in combination therapy to enhance muscle mass / function, and to treat or prevent metabolic disease or disease associated with defective neuronal signaling, including diabetes, obesity, and spinal cord injury. Thus, the method or pharmaceutical composition further comprises a second treatment. In some embodiments, the second treatment can be useful in treating or preventing metabolic disease or disease associated with defective neuronal signaling. The second treatment can attenuate or treat at least one symptom(s) associated with the targeted disease. The first treatment and the second treatment can exert their biological effects by similar or unrelated mechanisms of action, or one or both of the first treatment and the second treatment can exert their biological effects by multiple mechanisms of action.

[0345] 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 pharma- ceutically acceptable carrier or in different pharma-ceutically acceptable carriers. It should be further understood that the first therapeutic agent and the second therapy may be administered simultaneously or sequentially within the described embodiments.

[0346] 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, diabetes treatment agents, diabetes complication treatment agents, cardiovascular disease treatment agents, antihyperlipidemic agents, hypotensive or antihypertensive agents, antiobesity agents, nonalcoholic steatohepatitis (NASH) treatment agents, chemotherapeutic agents, immunotherapeutic agents, immunosuppressants, etc. Such combination therapies can advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with various monotherapies.

[0347] 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, pharma- ceutically acceptable salts, hydrates, or solvates thereof (e.g., pioglitazone, troglitazone, rosiglitazone, netoglitazone, balaglitazone, rivoglitazone, tesaglitazar, farglitazar, CLX-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 (e.g., phenformin, metformin, buformin, etc.) or sulfonylureas (e.g., tolbutamide, glibenclamide, gliclazide, chlorpropamide, tolazamide, acetohexamide, glyclopyramide, glimepiride, etc.), as well as other insulin secretagogues (e.g., repaglinide, senaglinide, nateglinide, mitiglinide, GLP-1, etc.), amylin agonists (e.g., pramlintide, etc.), phosphotyrosine phosphatase inhibitors (e.g., vanadate, etc.), etc.

[0348] 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. , pyradoxamine, 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.).

[0349] In one embodiment, diabetes remission can be induced by administering a myostatin inhibitor in combination with a calorie restricted diet or other dietary restriction.

[0350] 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.

[0351] 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.

[0352] Non-alcoholic steatohepatitis (NASH) treatment agents include, for example, ursodiol, pioglitazone, orlistat, betaine, rosiglitazone. In one embodiment, steatosis and resulting hepatitis, as well as fibrosis in NAFLD and / or NASH subjects, can be treated by administering a myostatin inhibitor in combination with a calorie-restricted diet or other dietary restriction.

[0353] 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.), beta 3-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.

[0354] 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.

[0355] Immunotherapeutic agents include, for example, microbial or bacterial components (e.g., muramyl dipeptide derivatives, picibanil, etc.), polysaccharides with immune-enhancing activity (e.g., lentinan, schizofiran, 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 among these, preferred substances are IL-1, IL-2, IL-12, etc.

[0356] 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.

[0357] Additionally, agents whose cachexia-ameliorating effects have been established in animal models or at clinical stages, 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-ameliorating agents (e.g., eicosapentaenoic acid, etc.), growth hormone, IGF-1, TNF-α, LIF, IL-6, and antibodies against oncostatin M, etc., may also be used in conjunction with the anti-myostatin antibodies of the present invention. Additional therapeutic agents for use in the treatment of diseases or conditions associated with metabolic disorders and / or defective neuronal signaling will be apparent to those of skill in the art and are within the scope of the present disclosure.

[0358] 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.

[0359] In some embodiments, a second agent suitable for administration as a combination therapy with an antibody described herein is a modulator (e.g., agonist and antagonist) of certain members of the TGFβ superfamily of growth factors, such as BMP6, BMP7, GDF11, TGFβ2, TGFβ3, RGMc, etc.

[0360] Any of the above agents may be administered in combination with the myostatin antibodies of the 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.

[0361] Use of anti-pro / latent myostatin antibodies or antigen-binding fragments thereof for treating diseases / disorders The pharmaceutical compositions described herein are suitable for administration to human patients to treat or prevent diseases and conditions in which it is desirable to reduce myostatin signaling.Such diseases and conditions include, but are not limited to, metabolic disorders, and diseases associated with defective neuronal signaling, such as spinal cord injury.Exemplary conditions in which the compositions and methods of the present invention may be useful are further described below.

[0362] A. Metabolic Disorders and Diseases The present invention provides a method for treating or preventing metabolic disease in a subject. As used herein, the term "metabolic disease" refers to any undesirable condition involving a disturbance of the normal physiological state of homeostasis due to alteration of metabolism (anabolism and / or catabolism). Metabolic disorders affect how the body processes substances required to perform physiological functions, and are generally accompanied by abnormal glucose, lipid / fat and / or protein / nitrogen metabolism, or osmoregulation failure, and pathological consequences resulting from such conditions. Some metabolic disorders of the present invention share certain characteristics, such as being accompanied by a loss of fat-free or lean muscle mass, excess fat mass, low metabolic rate, insulin resistance, lack of ability to control blood sugar, weight gain, and / or increased body mass index. In some cases, such metabolic conditions may be caused or exacerbated by medications that patients receive. As discussed in more detail herein, metabolic disorders may occur secondary to or as a result of muscle conditions or disorders.

[0363] The present invention is based at least in part on the discovery that administering a myostatin inhibitor, such as an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, to a subject with a metabolic disease significantly improves both the physiological and functional characteristics of the injured subject. Specifically, the inventors have surprisingly discovered that administering a myostatin inhibitor, such as an anti-myostatin antibody or antigen-binding portion thereof, significantly increases metabolic rate or energy expenditure in subjects with metabolic disease. Administration of a myostatin inhibitor, such as an anti-myostatin antibody or antigen-binding portion thereof, also significantly attenuates SCI-induced reductions in sublesional muscle mass and total body weight, while simultaneously reducing the amount of undesirable adipose tissue, such as white and visceral adipose tissue. Furthermore, subjects treated with a myostatin inhibitor, such as an anti-myostatin antibody or antigen-binding portion thereof, showed significant improvements in locomotor function, muscle strength, and motor coordination and balance skills.

[0364] Thus, the present invention provides a method for treating or preventing metabolic disease in a human subject. The method includes 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, such as an antibody or an antigen-binding fragment thereof that specifically binds to myostatin, thereby treating or preventing the metabolic disease in the human subject. Preferably, the antibody or an antigen-binding fragment thereof specifically binds to pro / latent myostatin but not to GDF11. An antibody that specifically recognizes pro / latent myostatin but not GDF11 is beneficial, avoiding undesirable toxicity caused by off-target binding of the antibody to GDF11 in the subject. In one embodiment, the subject is a pediatric subject.

[0365] Examples of metabolic diseases that can be treated or prevented by the methods of the present invention include, but are not limited to, type 1 diabetes, type 2 diabetes, metabolic syndrome, prediabetes, obesity, cardiovascular disease (e.g., congestive heart failure), nonalcoholic steatohepatitis (NASH), spinal cord injury (SCI) (e.g., complete or incomplete / partial SCI), hypometabolic conditions, double diabetes, Cushing's disease (also called Cushing's syndrome), obesity syndrome (e.g., diet-associated obesity or diet-induced obesity), insulin resistance, insulin deficiency, hyperinsulinemia, impaired glucose tolerance (IGT), abnormal glycogen metabolism, hyperlipidemia, hypoalbuminemia, hypertriglyceridemia, syndrome X, fatty liver disease, and metabolic bone disease. In some embodiments, the metabolic disease includes diseases associated with defective or partial denervation of neural signaling. In some embodiments, the metabolic disease includes conditions (e.g., side effects) caused by or associated with certain drugs.

[0366] Additional diseases or conditions related to metabolic disorders and / or body composition will be apparent to those of skill in the art and are within the scope of the present disclosure.

[0367] Diabetes refers to a group of metabolic diseases characterized by high blood sugar (glucose) levels due to defects in insulin secretion or action, or both. There are two most common types of diabetes, type 1 and type 2, both of which result from the body's inability to control insulin. Insulin is a hormone released by the pancreas in response to rising levels of blood sugar (glucose) in the blood.

[0368] The term "type 1 diabetes" as used herein refers to a chronic disease that occurs when the pancreas produces too little insulin to adequately control blood glucose levels. Type 1 diabetes is also referred to as insulin-dependent diabetes, IDDM, and juvenile-onset diabetes. People with type I diabetes (insulin-dependent diabetes) produce little or no insulin. About 6 percent of the U.S. population has some form of diabetes, and only about 10 percent of all diabetics have type I disease. The majority of people with type I diabetes develop the disease before age 30. Type 1 diabetes is the result of progressive autoimmune destruction of pancreatic beta cells, followed by insulin deficiency. More than 90 percent of the pancreas' insulin-producing cells (beta cells) are permanently destroyed. The resulting insulin deficiency is severe, and people with type I diabetes must regularly inject insulin to survive.

[0369] In type II diabetes (also called non-insulin-dependent diabetes mellitus, NDDM), the pancreas continues to produce insulin, sometimes even at higher than normal levels. However, the body develops resistance to its effects, resulting in relative insulin deficiency. Type II diabetes can occur in children and adolescents, but usually begins after age 30 and becomes progressively more common with age: about 15 percent of people over 70 have type II diabetes. Obesity is a risk factor for type II diabetes, and 80 to 90 percent of people with the disorder are obese.

[0370] In some embodiments, diabetes includes prediabetes. "Prediabetes" refers to one or more early diabetic conditions, including impaired glucose utilization, abnormal or defective fasting blood glucose level, impaired glucose tolerance, impaired insulin sensitivity and insulin resistance.Prediabetes is the main risk factor for type 2 diabetes, cardiovascular disease and mortality.Many focus has been placed on developing therapeutic interventions to prevent the onset of type 2 diabetes by treating prediabetes.

[0371] In some embodiments, diabetes includes double diabetes, which is a combination of type 1 diabetes and type 2 diabetes characterized by insulin resistance.

[0372] Diabetes can be diagnosed by performing a glucose tolerance test. Clinically, diabetes is often classified into several basic categories. The main examples of these categories include autoimmune diabetes, non-insulin-dependent diabetes mellitus (NDDM type 1), insulin-dependent diabetes mellitus (IDDM type 2), non-autoimmune diabetes, non-insulin-dependent diabetes mellitus (NIDDM type 2), and maturity-onset diabetes of the young (MODY). A further category, often referred to as secondary, refers to diabetes that is brought about by some identifiable condition that causes or allows the development of a diabetic syndrome. Examples of secondary categories include diabetes caused by pancreatic disease, hormonal abnormalities, drug or chemical induced diabetes, diabetes caused by insulin receptor abnormalities, diabetes associated with genetic syndromes, and diabetes of other causes (see, for example, Harrison's (1996), 14th ed., New York, McGraw-Hill).

[0373] Obesity is another prevalent metabolic disease that can be treated or prevented by the methods of the present invention. "Obesity" refers to a chronic condition defined by an excessive amount of body fat. Normal amounts of body fat (expressed as a percentage of body weight) are 25-30% for women and 18-23% for men. Women with more than 30% body fat and men with more than 25% body fat are considered obese. Obesity can be defined using any clinically meaningful definition. For example, in adults, the body mass index (BMI, kg / m 2 ) is frequently used as a measure of overweight and obesity, with overweight being defined as a BMI between 25 and 29.9 kg / m 2 Obesity is defined as a BMI of 30 kg / m 2 Morbid obesity is defined as a BMI equal to or exceeding 40 kg / m 2Obesity can also be defined in adults by central adiposity as measured by waist circumference, with high waist circumference defined as equal to or greater than 102 cm in men and equal to or greater than 88 cm in women. Obese subjects may exhibit other symptoms such as increased fasting plasma glucose, increased fasting plasma triglycerides, decreased fasting high density lipoprotein (HDL) levels, and elevated blood pressure. Obesity can also cause various orthopedic problems, skin disorders, and swelling of the feet and ankles. Serious complications of obesity include a very high risk of coronary artery disease and its major risk factors for type II diabetes, hyperlipidemia, and hypertension. Most of the morbidity associated with obesity is associated with type II diabetes, as poorly controlled diabetes and obesity lead to a constellation of symptoms known together as syndrome X, or metabolic syndrome. In some embodiments, the obesity is sarcopenic obesity. In some embodiments, the subject with obesity is undergoing a calorie restriction regimen.

[0374] The method of the present invention is also suitable for treating or preventing metabolic diseases, such as metabolic syndrome.As used herein, "metabolic syndrome" refers to the concept of clustering of metabolic risk factors that occur together in a single individual and lead to high risk of developing diabetes and / or cardiovascular disease.The main characteristics of metabolic syndrome include insulin resistance, hypertension (high blood pressure), cholesterol abnormalities, dyslipidemia, triglyceride abnormalities, increased risk of clotting, especially in the abdomen, and excess weight, or obesity. The American Heart Association suggests that the metabolic syndrome be diagnosed by the presence of three or more of the following components: (1) high waist circumference (men, greater than or equal to 40 inches (102 cm); women, greater than or equal to 35 inches (88 cm)); (2) high triglycerides (greater than or equal to 150 mg / dL); (3) low high-density lipoprotein cholesterol or HDL (men, less than 40 mg / dL; women, less than 50 mg / dL); (4) high blood pressure (greater than or equal to 130 / 85 mmHg); and (5) high fasting glucose (greater than or equal to 100 mg / dL).

[0375] In another aspect, the method of the present invention is suitable for treating or preventing metabolic disorders such as obesity syndrome. The term "obesity syndrome" refers to any disorder or condition that causes a subject to become significantly fat or overweight. As with other metabolic disorders, individuals with obesity syndrome are usually accompanied by reduced fat-free or lean muscle mass, excess fat mass, low metabolic rate, insulin resistance, inability to control blood sugar, weight gain, and increased body mass index. In some embodiments, the obesity syndrome is selected from the group consisting of Prader-Willi syndrome, obesity syndrome associated with genetic disorders, and obesity syndrome associated with hypothalamic disorders.

[0376] The method of the present invention is also suitable for treating or preventing metabolic diseases associated with hypometabolic states. The term "hypometabolic state" refers to a state of reduced metabolism or metabolic activity in which the body does not produce enough energy. Patients with hypometabolic states generally have a low metabolic rate, reduced fat-free or lean muscle mass, excessive fat mass, insulin resistance, lack of ability to control blood glucose, weight gain, and increased body mass index. In some embodiments, the hypometabolic state is selected from the group consisting of conditions associated with prolonged immobilization, conditions associated with bed rest, conditions associated with cast immobilization, conditions associated with stroke, conditions associated with amputation, and post-surgical conditions. In some embodiments, the hypometabolic state is a post-surgical condition, for example, paraspinal muscular atrophy after lumbar surgery. In one embodiment, the paraspinal muscular atrophy is nerve damage-dependent muscular atrophy. In one embodiment, the surgery is spinal surgery. In one embodiment, the spinal surgery is a lumbar surgery or procedure, such as a lumbar fusion procedure, a lumbar non-fusion procedure, a posterior lumbar fusion procedure, an anterior lumbar fusion procedure, a minimally invasive (MIS) posterior lumbar decompression procedure, a minimally invasive (MIS) posterior lumbar fusion procedure, a non-MIS equivalent procedure, etc.

[0377] In another embodiment, the method of the present invention is suitable for treating or preventing metabolic disorders such as Cushing's syndrome or Cushing syndrome. The term "Cushing's syndrome" refers to a collection of signs and symptoms resulting from long-term exposure to cortisol. This can be due to endogenous causes, such as a condition in which the pituitary gland releases excessive amounts of adrenocorticotropic hormone (ACTH), or exogenous causes, such as the use of oral corticosteroid drugs. Characteristic signs and symptoms of Cushing's disease can include progressive obesity, such as weight gain and fatty tissue deposits, especially around the midsection and upper back and between the shoulders (buffalo hump) (upper body obesity above the waist); thin arms and legs, a round, red, round face (moon face); skin changes such as pink or purple stretch marks (striae) on the skin of the abdomen, thighs, chest, and arms, thinning, weak skin that bruises easily, cuts that are difficult to heal, insect bites and infections, and acne. Patients with Cushing's disease may also experience severe fatigue, muscle weakness, depression, anxiety and irritability, loss of emotional control, cognitive difficulties, new or worsening hypertension, headaches, type 2 diabetes, and / or bone loss that may lead to bone fractures over time.In children, Cushing's disease may cause growth deficiencies (slow growth rate).In some embodiments, Cushing's disease is selected from the group consisting of corticosteroid-induced Cushing's disease and tumor-induced Cushing's disease.

[0378] To date, standard treatments for Cushing's disease are designed to lower high cortisol levels in the body, whether the cause is endogenous overproduction of the hormone or due to medications. The best treatment for a particular patient depends on the cause of the syndrome. Currently available treatment options include, for example, reduced use of corticosteroids, surgery, radiation therapy, and medications.

[0379] Thus, the use of the myostatin activation inhibitors described herein provides an alternative or additional treatment option for patients suffering from Cushing's disease.

[0380] If the cause of Cushing's disease is the long-term use of corticosteroid drugs, it may be possible to control and reduce the dosage of the drug over a period of time while still adequately managing the disease or condition that the drug is administered for.Thus, in some embodiments, the patient undergoing corticosteroid treatment has one or more autoimmune or inflammatory diseases, such as rheumatoid arthritis, lupus, and asthma.Corticosteroids may also be prescribed to patients to suppress the body's immunity, to prevent the body from rejecting allograft transplants, such as transplanted organs or tissues.

[0381] In some embodiments, patients undergoing corticosteroid therapy include a subpopulation of individuals who are not well tolerated and respond poorly or non-responsive to other treatment options, such as non-corticosteroid drugs. In such situations, physicians may continue to prescribe corticosteroid drugs. In some embodiments, surgery may be considered as an alternative option.

[0382] If the cause of Cushing's syndrome is a tumor, complete surgical removal and / or radiation therapy can be considered.In some embodiments, the patient has a tumor in the pituitary gland, adrenal gland, lung, or pancreas.After surgery, cortisol replacement therapy is typically administered to provide the body with the appropriate amount of adrenal hormone production.

[0383] In some embodiments, patients with Cushing's syndrome may never experience recovery of normal adrenal function and therefore require lifelong replacement therapy. The myostatin activation inhibitors described herein may be suitable for treating such patients.

[0384] In some embodiments, when surgery and / or radiation therapy fail, drugs can be used to control cortisol production.For patients who become seriously ill with Cushing's syndrome, drugs can be used before surgery.The myostatin activation inhibitors encompassed in the present disclosure can be used to treat such patients before surgery to improve signs and symptoms and minimize the risk of surgery.

[0385] Drugs currently used to control the overproduction of cortisol in the adrenal glands include ketoconazole (Nizoral), mitotane (Lysodren), and metyrapone (Metopirone). Mifepristone (Korlym) is approved for patients with Cushing's syndrome who have type 2 diabetes or glucose intolerance. Mifepristone does not reduce cortisol production, but blocks the effects of cortisol on tissues. Side effects of these drugs can include fatigue, nausea, vomiting, headache, muscle pain, high blood pressure, low potassium, and swelling. Some have more severe side effects such as neurological side effects and hepatotoxicity. The myostatin activation inhibitors encompassed by the present disclosure may be used alone (in place of these therapeutic agents) or in combination with any of these therapeutic agents.

[0386] More recently, pasireotide (Signifor) has become available for the treatment of Cushing's disease, which works by decreasing ACTH production from pituitary tumors. This drug is given as an injection twice a day. It is typically recommended when pituitary surgery is unsuccessful or cannot be performed. Side effects associated with this drug are very common and can include diarrhea, nausea, hyperglycemia, headache, abdominal pain, and fatigue. The myostatin activation inhibitors encompassed by this disclosure may be used alone (in place of such therapeutic agents) or in combination with any of such therapeutic agents.

[0387] In some embodiments, the tumor or its treatment may result in a deficiency of other hormones produced by the pituitary or adrenal glands, which may necessitate hormone replacement therapy. In some embodiments, none of these currently available treatment options are appropriate or effective, and surgical removal of the adrenal glands (bilateral adrenalectomy) may be considered, which requires lifelong replacement therapy. Patients who are candidates for such options may benefit from the myostatin inhibition treatment described herein before and / or after adrenalectomy.

[0388] In yet another aspect, the method of the present invention is suitable for treating or preventing metabolic diseases such as cardiovascular diseases. The term "cardiovascular disease" refers to any disease of the heart or blood vessels. Cardiovascular disease or heart disease includes, but is not limited to, angina pectoris, arrhythmias, coronary artery disease (CAD), coronary heart disease, cardiomyopathies (including dilated cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and diabetic cardiomyopathy), heart attack (myocardial infarction), heart failure (e.g., CHF), hypertrophic cardiomyopathy, mitral regurgitation, mitral valve prolapse, pulmonary stenosis, and the like. Vascular diseases include, but are not limited to, peripheral vascular disease, arterial disease, carotid artery disease, deep vein thrombosis, venous disease, and atherosclerosis. In some embodiments, the subject with heart failure is resistant to diuretic therapy. In another embodiment, the subject with heart failure responds poorly to diuretic therapy.

[0389] Another aspect of the present disclosure includes a method of treating a subject with a metabolic disease or condition associated with aging. Exemplary diseases and conditions associated with aging include, but are not limited to, sarcopenia (age-related muscle loss), frailty, and androgen deficiency.

[0390] Another aspect of the present disclosure includes a method of treating a subject with a metabolic disease or condition associated with disuse or genetic atrophy / trauma, such as atrophy caused by disuse, atrophy caused by gene mutation(s), atrophy resulting from injury, etc. Examples of such diseases and conditions include, but are not limited to, muscle weakness associated with time spent in an intensive care unit (ICU), hip replacement surgery, hip fracture, stroke, bed rest, SCI, rotator cuff injury, knee replacement, fracture, and burn.

[0391] The present disclosure includes the beneficial effect of myostatin inhibition on bone homeostasis.In the musculoskeletal system (defined as the bones, muscles, cartilage, tendons, ligaments, joints, and other connective tissues that support and bind tissues and organs together), muscle and bone homeostasis are closely related.Bone growth in response to muscle growth is a mechanosensitive process that is controlled by endocrine signaling.

[0392] Similar to muscle, bone homeostasis involves a dynamic process that balances bone growth (bone formation) and bone loss (bone resorption). Parameters that can be used to assess bone homeostasis include, but are not limited to, bone mass, bone volume, bone density, bone cross-sectional area, bone strength, fracture frequency, bone repair rate, and the like. Factors (e.g., cytokines, hormones) that are known to play a role in this process include, but are not limited to, parathyroid hormone, 1,25-dihydroxyvitamin D3, T4, corticosteroids, prostaglandins such as prostaglandin E2, interleukin-4, interleukin-18, interferon-gamma, interleukin-17, interleukin-6, interleukin-1, RANKL, CSF, TGFβ, osteoprotegerin, BMP, IGF, and FGF, and the like. Osteoclasts and osteoblasts contribute to bone resorption and bone growth, respectively.

[0393] From a structural standpoint, bone strength is determined by the combination of trabecular and cortical bone structure and composition. In general, a decrease in bone strength, such as osteopenia and osteoporosis, is observed with age. In some situations, bone loss is caused by other factors, such as drugs. For example, glucocorticoid treatment can cause bone loss. Medical conditions that may be associated with bone loss include, but are not limited to, cancer and muscle / metabolic disorders with concomitant bone loss. In some embodiments, such conditions are associated with spinal cord injury (SCI), muscular dystrophies such as DMD, obesity, and / or Cushing's disease.

[0394] Bone loss can be measured using assays well known to those of skill in the art (see Shanmugarajan et al., J. Pathol., 2009, vol. 219(1):52-60; and Wasserman et al., Neuromuscular Disorders, 2017, vol. 27(4):331-337). For example, bone mineral density, e.g., areal bone mineral density, can be measured using DXA scans of the lumbar spine, total body, and distal lateral femur, following ISCD recommendations (Wasserman et al., Neuromuscular Disorders, 2017, vol. 27(4):331-337). After scanning, bone mineral density can be calculated using reference data from Henderson et al., Am. J. Roentgenol, 2002, 178:439-443; Kalkwarf et al., J. Bone Miner. Res., 2013, 28:206-212; Kelly et al., J. Pediatr. Hematol. Oncol., 2005, 27:248-253; and Zemel et al., J. Clin. Endocrinol. Metab., 2011, 96:3160-3169. The patient's fracture history, including age at fracture, number of fractures, and fracture location, may also be collected. Osteoporosis is typically measured using the 2013 ISCD criteria, which include vertebral compression fracture in the absence of high-energy trauma or invasive disease; or BMD Z-score ≦2.0SD, and two or more long bone fractures by age 10 years or three or more long bone fractures by age 19 years (see, e.g., Bishop et al., J. Clin. Densitom, 2014, 17:275-280).

[0395] Several therapeutic agents are currently available for the treatment of bone loss. Agents used to slow the rate of bone loss include bisphosphonates and denosumab. Bisphosphonates block osteoclast recruitment and induce osteoclast apoptosis. They are often used to treat postmenopausal and glucocorticoid-induced osteoporosis, Paget's disease, and hypercalcemia of malignancy. Denosumab, developed by Amgen, is a monoclonal antibody that blocks RANKL (osteoclast development). It is used to treat osteoporosis, bone metastases, and other bone tumors.

[0396] Drugs used to grow new bone include Teriparatide and Romosozumab. Forteo is Teriparatide, marketed by Eli Lilly, which is a recombinant protein fragment that contains the first 34 amino acid residues of thyroid hormone. It is typically used to treat patients with osteoporosis and high risk of fracture, as well as those intolerant to other therapies. Romosozumab, available from Amgen, is a monoclonal antibody that blocks sclerostin, a Wnt pathway antagonist. To date, there is no drug that can increase both bone and muscle.

[0397] Several groups are conducting preclinical and clinical trials with agents that at least partially affect the myostatin pathway. For example, Acceleron has developed ActRIIA and ActRIIB ligand trapping agents (e.g., ACE-011, ACE-536, ACE-031, and ACE-2494), at least some of which are said to increase bone mass when administered in vivo. None of these agents appear to be specific for myostatin / GDF8, but also affect one or more of the other pathways. Eli Lilly's monoclonal antibody LY2495655 (Landogrozumab) did not increase bone mass in humans undergoing elective hip replacement surgery, as measured by DEXA. The antibody binds to both GDF8 and GDF11.

[0398] Unlike these agents that affect myostatin and additional pathway(s), the monoclonal antibodies included in the present disclosure specifically bind to myostatin / GDF8 and inhibit its activation process. In some embodiments, such antibodies bind to promyostatin and / or latent myostatin, thereby inhibiting the activation and subsequent release of mature myostatin, but not to mature myostatin that is not associated with the latent (inactive) complex. In some embodiments, the antibodies or fragments thereof bind to inactive myostatin (e.g., promyostatin) in a tethered form (e.g., in muscle) that has the ability to act locally on tissue-associated myostatin in a disease niche. In some embodiments, the antibodies or fragments thereof bind to inactive myostatin (e.g., latent myostatin) in a soluble form (e.g., in the circulation) that has the ability to act on circulating latent myostatin, which may have endocrine or systemic effects. In any of such embodiments, preferred inhibitors of myostatin for carrying out the methods of the invention are th...

Claims

1. A composition comprising an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin and blocks the release of mature myostatin, for use as a pharmaceutical in the treatment or prevention of metabolic disease in a human subject, comprising the steps of selecting a human subject suffering from or at risk of developing a metabolic disease; and administering to the human subject an effective amount of the composition comprising the antibody or antigen-binding fragment thereof.

2. 2. The composition of claim 1, wherein the subject does not have a myopathy, and optionally, the myopathy is a primary or secondary myopathy.

3. 2. The composition of claim 1, wherein the subject is an adult human subject suffering from growth hormone (GH) deficiency, and optionally, the subject is concurrently receiving recombinant GH therapy or GH gene therapy.

4. 2. The composition of claim 1, wherein the metabolic disease is selected from the group consisting of type I diabetes, type II diabetes, obesity, metabolic syndrome / pre-diabetes, cardiovascular disease, non-alcoholic steatohepatitis (NASH), spinal cord injury (SCI), SMA, hypometabolic states, dual diabetes, metabolic bone disorder, Cushing's disease, and obesity syndrome.

5. The composition of claim 4 , wherein the cardiovascular disease is heart failure.

6. The composition of claim 5, wherein the heart failure is CHF involving fluid overload.

7. The composition of claim 6, wherein the fluid overload comprises generalized edema and / or pulmonary edema.

8. The composition of claim 6, wherein the human subject has an inadequate response to diuretic therapy.

9. The composition of claim 4, wherein the obesity is sarcopenic obesity.

10. The composition of claim 4 , wherein the human subject is on a restricted diet.

11. The composition of claim 10 , wherein the dietary restriction is a calorie restricted diet.

12. The composition of claim 11 , wherein the subject also has limitations in physical activity.

13. The composition of any one of claims 10 to 12, wherein the subject is not on an exercise regimen.

14. The composition of any one of claims 10 to 12, wherein the subject is undergoing an exercise regimen.

15. 5. The composition of claim 4, wherein the hypometabolic condition is selected from the group consisting of conditions associated with prolonged immobilization, conditions associated with bed rest, conditions associated with cast immobilization, conditions associated with stroke, conditions associated with amputation, and post-surgical conditions.

16. 5. The composition of claim 4, wherein the Cushing's disease is selected from the group consisting of corticosteroid-induced Cushing's disease and tumor-induced Cushing's disease.

17. 5. The composition of claim 4, wherein 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.

18. Administering the composition comprises: a) increasing muscle tissue mass and / or function in said human subject; b) increasing fast muscle tissue mass and / or function in said human subject; c) increasing the mass and / or function of slow muscle tissue in said human subject; d) increasing the metabolic rate of said human subject; e) increasing insulin sensitivity in said human subject; f) increasing the level of brown adipose tissue in said human subject; g) increasing the level of beige adipose tissue in said human subject; h) reducing the level of white adipose tissue in said human subject; i) reducing the level of visceral adipose tissue in said human subject; j) reducing the ratio of fat to muscle tissue in said human subject; k) increasing glucose uptake by a target tissue in said human subject, wherein the target tissue is selected from the group consisting of brown adipose tissue, beige adipose tissue, and muscle tissue; 1) decreasing glucose uptake by a target tissue in said human subject, wherein the target tissue is selected from the group consisting of white adipose tissue and liver tissue; m) reducing muscle catabolism of protein and / or muscle release of amino acids in said human subject; n) increasing insulin-dependent glycemic control in said human subject; o) reducing intramuscular fat infiltration in said human subject; p) improving standardized quality of life test scores; q) preventing muscle loss or atrophy in said human subject; r) increasing bone density or volume; s) preventing or reducing bone loss or fracture; t) reducing fluid overload or edema associated with chronic heart failure (CHF); and / or u) enhancing the subject's response to treatment; The composition according to any one of claims 1 to 17, which causes at least one of the following:

19. The composition of any one of claims 1 to 18, wherein the antibody or antigen-binding portion thereof does not bind to mature myostatin, GDF11, or activin.

20. the antibody or antigen-binding fragment thereof a) 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; or b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 50 and a light chain comprising the amino acid sequence of SEQ ID NO: 51 The composition according to any one of claims 1 to 19, comprising:

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