Methods for treating metabolic diseases by inhibiting myostatin activation

Abstract

The present invention provides pharmaceutical compositions for treating or preventing metabolic diseases selected from the group consisting of type 1 diabetes, type 2 diabetes, obesity, metabolic syndrome / prediabetes, cardiovascular disease, non-alcoholic steatohepatitis (NASH), spinal cord injury (SCI), hypometabolic state, double diabetes, Cushing's disease, and obesity syndrome. [Solution] A composition 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 who has a metabolic disease or is at risk of developing one, and administering to the human subject an effective amount of the composition comprising the antibody or the antigen-binding fragment.

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 on 6 January 2017; European Priority Application No. 1750586.0, filed on 10 July 2017; and U.S. Provisional Patent Application No. 62 / 530,311, filed on 20 December 2017. The entire contents of these priority applications, including electronically filed electronic sequence listings in ASCII format, are incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing, which is submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on January 5, 2018, is named "SR16-WO-PCT_Sequence_Listing_127036-00220.txt" and has a size of 108,914 bytes. [Background technology]

[0003] Millions of people worldwide suffer from metabolic disorders, and patients with metabolic disorders commonly experience loss of lean body mass or lean muscle mass, excessive increase in body fat mass, a low metabolic rate, insulin resistance, inability to control blood glucose, weight gain, and an increase in 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 ovary syndrome, breast cancer, prostate cancer, and colon cancer, as well as an increased incidence of complications from general anesthesia.

[0004] In addition to the serious health consequences of these metabolic diseases, they also come with substantial economic costs. 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 healthcare cost for obesity-related diseases is a staggering $190.2 billion, or nearly 21% of annual healthcare costs. Not only are there direct healthcare costs for these metabolic diseases, but indirect costs, including loss of productivity due to metabolic disease-related illnesses and premature death, also pose a substantial cost to both society and its citizens.

[0005] Myostatin, also known as growth and 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 Literature 1). Myostatin has primarily been evaluated for its use in treating diseases related to muscle function.

[0006] To date, most metabolic diseases remain inadequately treated. Current treatments do not adequately meet the needs of patients, and there are no effective treatments applicable to the majority of the affected patient population. Therefore, there is still an unmet need for treatment 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 (No. 4): R444-54. [Overview of the project]

[0008] This disclosure includes the recognition that myostatin may act as an important regulator for the function of muscle as an endocrine organ that controls metabolism, including body composition, and for directly mediating its control.

[0009] According to this invention, the regulation of myostatin signaling can influence many metabolic parameters central to the control of energy production and consumption by selectively mobilizing the body's three major energy pools: glucose, lipids, and proteins. While we do not wish to be bound by theory, it is intended that myostatin can play a role in the process both as a molecular sensor of energy consumption and as an effector to influence metabolic normalization. This invention involves the broader role of myostatin in metabolic regulation, including nitrogen mobilization, osmoregulation, calcium metabolism, and acid-base and electrolyte balance.

[0010] Accordingly, the present invention provides methods and compositions for treating or preventing metabolic disorders in human subjects using anti-pro / latent myostatin inhibitors, such as antibodies. The present invention is at least in part based on the discovery that administering myostatin inhibitors, such as antibodies, or antigen-binding fragments thereof, that specifically bind to pro / latent myostatin to subjects with metabolic disorders, such as spinal cord injury (SCI), significantly improves both the physiological and functional characteristics of the injured subjects. In particular, the inventors have surprisingly found that administration of myostatin inhibitors, such as anti-pro / latent myostatin antibodies, significantly increases metabolic rate or energy expenditure in subjects with metabolic disorders, such as spinal cord injury (SCI). Administration of myostatin inhibitors, such as anti-pro / latent myostatin antibodies, also significantly reduces the SCI-induced decline in lesional muscle mass and whole body weight, and simultaneously reduces the amount of undesirable adipose tissue, such as white adipose tissue and visceral adipose tissue. Furthermore, subjects treated with myostatin inhibitors, such as anti-pro / latent myostatin antibodies, showed significant improvements in spontaneous motor 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 myostatin inhibitors, such as anti-pro / latent myostatin antibodies or their antigen-binding fragments, increases not only the bone volume of weight-bearing bone but also the bone volume of non-weight-bearing bone, such as the vertebrae of rodents. It is well known in the art that weight-bearing activity is an important stimulus for bone mass increase, which may potentially explain the increase in bone volume of weight-bearing bone after administration of myostatin inhibitors. 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, that is, myostatin inhibitors not only act to increase bone, for example, through increased muscle stimulation, but also act as important regulators for increasing general metabolic effects, including bone health.

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

[0013] Accordingly, in one embodiment, a composition for use as a pharmaceutical in treating or preventing metabolic diseases in human subjects is disclosed herein, comprising the steps of: selecting a human subject who has a metabolic disease or is at risk of developing one, and a composition comprising a myostatin inhibitor, for example, an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin and blocks the release of mature myostatin; and administering to the human subject an effective amount of the composition comprising the myostatin inhibitor, for example, an antibody or antigen-binding fragment thereof. In one embodiment, the subject is a pediatric subject.

[0014] In some embodiments, subjects are free from myopathy, and optionally, any myopathy is either primary or secondary. In some embodiments, subjects are adult human subjects suffering from growth hormone (GH) deficiency, and optionally, subjects are simultaneously receiving recombinant GH therapy or GH gene therapy.

[0015] In some embodiments, metabolic disorders are selected from the group consisting of type 1 diabetes, type 2 diabetes, obesity, metabolic syndrome / prediabetes, cardiovascular disease, non-alcoholic steatohepatitis (NASH), spinal cord injury (SCI), hypometabolic state, double diabetes, Cushing's disease, and obesity syndrome. In some embodiments, obesity is muscle-depleting obesity.

[0016] In some embodiments, the metabolically impaired state is selected from the group consisting of states associated with prolonged exercise restriction, states associated with bed rest, states associated with casting, states associated with stroke, states associated with amputation, and states after surgery.

[0017] In some embodiments, 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 genetic disorders, and obesity syndrome associated with hypothalamic disorders.

[0019] In some embodiments, the dosage of the composition is as follows: a) To increase the volume and / or function of muscle tissue in human subjects; b) To increase the volume and / or function of fast-twitch muscle tissue in human subjects; c) To increase the volume and / or function of slow-twitch muscle tissue in human subjects; d) To increase the metabolic rate of human subjects; e) To increase insulin sensitivity in human subjects; f) To increase the level of brown adipose tissue in human subjects; g) To increase the level of beige adipose tissue in human subjects; h) To reduce the level of white adipose tissue in human subjects; i) To reduce the level of visceral adipose tissue in human subjects; j) To reduce the fat-to-muscle tissue ratio in human subjects; k) Increase glucose uptake by target tissue in a human subject, where the target tissue is selected from the group consisting of brown adipose tissue, beige adipose tissue, and muscle tissue; 1) Reduce glucose uptake by target tissue in human subjects, where the target tissue is selected from the group consisting of white adipose tissue and liver tissue; m) To reduce protein catabolism in muscle and / or the release of amino acids from muscle in human subjects; n) To increase insulin-dependent blood glucose regulation in human subjects; o) To reduce intramuscular fat infiltration in human subjects; p) Improving standardized quality of life test scores; q) To prevent muscle loss or atrophy in human subjects; r) To reduce bone loss; s) To increase the cross-sectional area of ​​the bone and / or the cortical bone thickness; t) To reduce the frequency or severity of fractures; and / or u) To reduce fluid overload or edema in chronic heart failure (CHF); It causes at least one of the following, for example, two, three, four, five, six, seven, two-four, two-five, two-six, three-five, or three-six.

[0020] In some embodiments, the antibody or its antigen-binding fragment does not bind to GDF11 or activin. In some embodiments, the antibody or its antigen-binding fragment does not bind to mature (fully processed, free, and active) myostatin. In some embodiments, the antibody or its antigen-binding fragment comprises a) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 25 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 31, or b) a heavy chain containing the amino acid sequence of SEQ ID NO: 50 and a light chain containing the amino acid sequence of SEQ ID NO: 51.

[0021] In another embodiment, the Disclosure provides a method for treating or preventing a metabolic disorder in a human subject, comprising the steps of: selecting a human subject who has a metabolic disorder or is at risk of developing one; and administering to the human subject an effective amount of a myostatin inhibitor, for example, 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 a metabolic disorder in the human subject.

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

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

[0024] In one embodiment, metabolic disorders are selected from the group consisting of type 1 diabetes, type 2 diabetes, obesity, metabolic syndrome / prediabetes, cardiovascular disease, non-alcoholic steatohepatitis (NASH), spinal cord injury (SCI), hypometabolic states, bidiabetes, Cushing's disease, and obesity syndromes. In one embodiment, obesity is muscle-depleting obesity. In one embodiment, hypometabolic states are selected from the group consisting of states associated with prolonged exercise restriction, states associated with bed rest, states associated with cast immobilization, states associated with stroke, states associated with amputation, and post-surgical states. In one embodiment, Cushing's disease is selected from the group consisting of corticosteroid-induced Cushing's disease and tumor-induced Cushing's disease. In one embodiment, obesity syndromes are selected from the group consisting of Prader-Willi syndrome, obesity syndromes associated with genetic disorders, and obesity syndromes associated with hypothalamic disorders.

[0025] In some embodiments, the metabolically impaired state is a postoperative condition, such as paraspinal muscle atrophy after lumbar spine surgery. In one embodiment, paraspinal muscle atrophy is nerve injury-dependent muscle atrophy. In one embodiment, the surgery is spinal surgery. In one embodiment, spinal surgery is lumbar surgery or lumbar procedure, such as lumbar fusion procedure, lumbar non-fusion procedure, posterior lumbar fusion procedure, anterior lumbar fusion procedure, minimally invasive (MIS) post-lumbar decompression procedure, minimally invasive (MIS) post-lumbar fusion procedure, non-MIS equivalent procedure, etc.

[0026] In one embodiment, administration of the composition increases the volume and / or function of muscle tissue in a human subject. In one embodiment, administration of the composition increases the volume and / or function of fast-twitch muscle tissue in a human subject. In one embodiment, administration of the composition increases the volume and / or function of slow-twitch muscle tissue in a human subject. In one embodiment, administration of the composition increases the metabolic rate of a human subject. In one embodiment, administration of the composition increases insulin sensitivity in a human subject. In one embodiment, administration of the composition increases the level of brown adipose tissue in a human subject. In one embodiment, administration of the composition increases the level of beige adipose tissue in a human subject. In one embodiment, administration of the composition decreases the level of white adipose tissue in a human subject. In one embodiment, administration of the composition decreases the level of visceral adipose tissue in a human subject. In one embodiment, administration of the composition decreases the fat-to-muscle tissue ratio in a 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 target tissue in a human subject, where the target tissue is selected from the group consisting of brown adipose tissue, beige adipose tissue, and muscle tissue. In another embodiment, administration of the composition decreases glucose uptake by target tissue in a human subject, where the target tissue is selected from the group consisting of white adipose tissue and liver tissue. In yet another embodiment, administration of the composition reduces protein catabolism in muscle and / or release of amino acids from muscle in a human subject. In yet another embodiment, the human subject is a pediatric human subject.

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

[0029] In one embodiment, a method for inhibiting myostatin activation in a subject is provided, wherein the subject is given a composition comprising a myostatin inhibitor, for example, an antibody or antigen-binding fragment that specifically binds to pro / latent myostatin and blocks the release of mature myostatin, and the subject exhibits the following: (a) increased volume and / or function of muscle tissue in the subject; (b) increased metabolic rate in the subject; (c) increased insulin sensitivity in the subject; (d) increased level of brown adipose tissue in the subject; (e) increased level of beige adipose tissue in the subject; (f) decreased level of white adipose tissue in the subject; (g) increased visceral activity in the subject. A method is disclosed herein that includes the step of administering a drug in an amount effective to cause two or more of the following: (h) a decrease in the level of adipose tissue; (i) a decrease in the fat-to-muscle tissue ratio in the subject; (j) an increase in glucose uptake by brown adipose tissue, beige adipose tissue, or muscle tissue in the subject; (k) a decrease in glucose uptake by white adipose tissue or liver tissue; (l) a decrease in protein catabolism in muscle and / or release of amino acids from muscle in the subject; (m) an increase in insulin-dependent blood glucose regulation in the subject; (n) a decrease in intramuscular fat infiltration in the subject; (n) a clinically meaningful improvement in standardized quality of life test scores assessed by a standardized quality of life test (e.g., an increase of at least 8 points on the SF-36 Quality of Life Scoring System); (o) prevention of muscle loss or muscle atrophy in the subject; and / or (p) prevention of the development of metabolic dysregulation associated with muscle dysfunction in the subject, wherein the subject is a human subject for whom reduction of myostatin signaling is beneficial. In one embodiment, the human subject is a child human subject.

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

[0031] In one embodiment, the subject exhibits either i) an elevated level of promyostatin in target muscle compared to a control level of promyostatin, or ii) a decreased level of latent myostatin in 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, muscular atrophy, muscular dystrophy, and nerve injury. In one embodiment, muscular atrophy is associated with a motor neuron defect. In one embodiment, the defect includes a gene mutation. In another embodiment, muscular atrophy is associated with spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), or myasthenia gravis. In one embodiment, nerve injury includes partial denervation of neurons innervating a muscle, or a defect in signaling between motor neurons and target muscles. In one embodiment, nerve injury is SCI. In another embodiment, SCI is partial / incomplete SCI. In one embodiment, SCI in a human subject includes lesions 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 related to SCI. In one embodiment, the metabolic disorder is or includes insulin resistance, inflammation, abnormal lipid metabolism, or increased intramuscular fat infiltration. In one embodiment, muscle atrophy includes glucocorticoid-induced muscle atrophy. In one embodiment, the human subject is a pediatric human subject.

[0033] In one embodiment, the subject has a metabolic disorder selected from the group consisting of type 1 diabetes, type 2 diabetes, obesity, metabolic syndrome / prediabetes, cardiovascular disease, non-alcoholic steatohepatitis (NASH), spinal cord injury (SCI), hypometabolic state, bidiabetes, 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 treatment. In one embodiment, the second treatment includes neuroprotective therapy. In another embodiment, the neuroprotective therapy includes stem cell therapy.

[0035] In another embodiment, a method is disclosed herein for treating or preventing a disease in a human subject that is associated with a defect in nerve signaling between neurons and target tissue, comprising the steps of: selecting a human subject suffering from a disease associated with a defect in nerve signaling between neurons and target tissue; and administering to the human subject in an amount effective to treat or prevent the disease, comprising a composition containing a myostatin inhibitor, for example, 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 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 embodiment, a method for treating a lesion in a subject that causes a defect, though not a complete loss, of signaling between neurons and target muscles is disclosed herein. Such a method includes the step of administering to a subject a composition comprising a myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody, in an amount effective for treating the muscle located beneath the lesion in the subject. In some embodiments, the amount is effective for preventing muscle loss or atrophy beneath the lesion in the subject. In some embodiments, the amount is effective for increasing the volume and / or function of the muscle beneath the lesion in the subject.

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

[0039] In one embodiment, the muscle contains fast-twitch muscle fibers. In another embodiment, the muscle located beneath the lesion is selected from the group consisting of the soleus, gastrocnemius, biceps, and triceps muscles. In one embodiment, the amount is effective in increasing the volume and / or function of the muscle above the lesion in the subject. In another embodiment, a myostatin inhibitor is a drug that blocks, antagonizes, or inhibits myostatin signaling in vivo. In some embodiments, such a drug is an antibody or its antigen-binding moiety, a small molecule, or a 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 compared to mature GDF11. In some embodiments, the antibody specifically binds to mature myostatin but not to mature GDF11. In some embodiments, the antibody binds to and / or blocks the myostatin receptor.

[0040] In one embodiment, the subject has incomplete spinal cord injury (SCI). In one embodiment, incomplete SCI in a human subject includes lesions 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 in treating metabolic conditions in the subject. In one embodiment, the amount is effective in treating (a) increased muscle tissue volume and / or function in the subject; (b) increased metabolic rate in the subject; (c) increased insulin sensitivity in the subject; (d) increased brown adipose tissue level in the subject; (e) increased beige adipose tissue level in the subject; (f) decreased white adipose tissue level in the subject; (g) decreased visceral adipose tissue level in the subject; (h) decreased adipose tissue-to-muscle tissue ratio in the subject; (i) increased glucose uptake by brown adipose tissue, beige adipose tissue, or muscle tissue in the subject; (j) decreased glucose uptake by white adipose tissue or liver tissue; (k) decreased protein catabolism in muscle and / or release of amino acids from muscle in the subject; (l) increased insulin-dependent blood glucose regulation in the subject; (m) decreased intramuscular fat infiltration in the subject; (n) SF-36 Quality of Life Scoring It is effective in causing an increase of at least 8 points in the 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. In one embodiment, the human subject is a pediatric human subject.

[0042] In another embodiment, the present disclosure provides a method for treating or preventing a metabolic disorder in a human subject, comprising the steps of: selecting a human subject suffering from a metabolic disorder; 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 disorder in the human subject. In one embodiment, the human subject is a pediatric human subject.

[0043] In one embodiment, metabolic disorders are selected from the group consisting of type 1 diabetes, type 2 diabetes, obesity, metabolic syndrome / prediabetes, cardiovascular disease, non-alcoholic steatohepatitis (NASH), spinal cord injury (SCI), hypometabolic states, bidiabetes, Cushing's disease, and obesity syndromes. In one embodiment, obesity is muscle-depleting obesity. In one embodiment, hypometabolic states are selected from the group consisting of states associated with prolonged exercise restriction, states associated with bed rest, states associated with cast immobilization, states associated with stroke, states associated with amputation, and post-surgical states. In one embodiment, Cushing's disease is selected from the group consisting of corticosteroid-induced Cushing's disease and tumor-induced Cushing's disease. In one embodiment, obesity syndromes are selected from the group consisting of Prader-Willi syndrome, obesity syndromes associated with genetic disorders, and obesity syndromes associated with hypothalamic disorders.

[0044] In another embodiment, the Disclosure provides a method for treating or preventing a disease associated with a defect in nerve 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 a defect in nerve 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 the defect in nerve 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 a defect in nerve signaling between neurons and target tissue 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 a defect in nerve signaling between neurons and target tissue is spinal cord injury (SCI). In one embodiment, the human subject is in the acute spinal cord injury (SCI) stage. In one embodiment, the human subject is in the subacute spinal cord injury (SCI) stage. In one embodiment, the human subject is in the 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, such as an antibody or its antigen-binding fragment, increases the volume and / or function of muscle tissue in a human subject. In one embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, increases the volume and / or function of fast-twitch muscle tissue in a human subject. In one embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, increases the volume and / or function of slow-twitch 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, such as an antibody or its antigen-binding fragment, increases the metabolic rate of a human subject. In one embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, increases insulin sensitivity in a human subject. In one embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, increases the level of brown adipose tissue in a human subject. In one embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, increases the level of beige adipose tissue in a human subject. In one embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, decreases the level of white adipose tissue in a human subject. In one embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, decreases the level of visceral adipose tissue in a human subject. In one embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, decreases the ratio of adipose tissue to muscle tissue in a human subject. In one embodiment, the human subject is a pediatric human subject.

[0049] In one embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, increases glucose uptake by muscle tissue in a human subject. In another embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, decreases glucose uptake by a target tissue, where the target tissue is selected from the group consisting of white adipose tissue, liver tissue, and vascular tissue. In another embodiment, the human subject is a pediatric human subject.

[0050] In one embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, reduces protein catabolism in muscle and / or release of amino acids from muscle in human subjects. In one embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, increases insulin-dependent blood glucose regulation in human subjects. In one embodiment, the human subjects are pediatric human subjects.

[0051] In another embodiment, a method for increasing the metabolic rate in a human subject is disclosed herein, comprising the steps of: selecting a human subject in which an increase in metabolic rate is deemed beneficial; 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 metabolic rate in the human subject.

[0052] In another embodiment, a method for increasing the level of brown adipose tissue in a human subject is disclosed herein, comprising the steps of: selecting a human subject in which an increase in the level of brown adipose tissue is deemed beneficial; 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 embodiment, a method for increasing the level of beige adipose tissue in a human subject is disclosed herein, comprising the steps of: selecting a human subject in which an increase in the level of beige adipose tissue is deemed beneficial; 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 embodiment, a method for increasing insulin-dependent blood glucose regulation in a human subject is disclosed herein, comprising the steps of: selecting a human subject in which the increase in insulin-dependent blood glucose regulation is deemed beneficial; 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 insulin-dependent blood glucose regulation in the human subject.

[0055] In another embodiment, a method for reducing protein catabolism and / or amino acid release from muscle in a human subject is disclosed herein, comprising the steps of: selecting a human subject in which the reduction of protein catabolism and / or amino acid release from muscle is deemed beneficial; 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 protein catabolism and / or amino acid release from muscle in the human subject.

[0056] In another embodiment, a method for reducing glucose uptake by target tissue in a human subject is disclosed herein, comprising the steps of: selecting a human subject in which a reduction in glucose uptake by target tissue selected from the group consisting of white adipose tissue, liver tissue and vascular tissue is deemed beneficial; 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 target tissue in the human subject.

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

[0058] In another embodiment, a method for treating or preventing a metabolic disorder in a human subject, comprising the steps of: selecting a human subject suffering from a metabolic disorder; and administering to the human subject a myostatin inhibitor, e.g., an antibody or antigen-binding fragment that specifically binds to pro / latent myostatin; in the human subject: (a) an increase in the volume and / or function of muscle tissue in the human subject; (b) an increase in the metabolic rate of 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 white adipose tissue in the human subject. Disclosed herein is a method for treating or preventing a metabolic disorder in a human subject, comprising the step of administering an amount effective in causing at least two or more of the following: (g) a decrease in the level of tissue; (h) a decrease in the level of visceral adipose tissue in a human subject; (i) an increase in glucose uptake by white adipose tissue, hepatic tissue or vascular tissue in a human subject; (j) a decrease in protein catabolism in muscle and / or release of amino acids from muscle in a human subject; and / or (k) an increase in insulin-dependent blood glucose regulation in a human subject. In one embodiment, the human subject is a pediatric human subject.

[0059] In another embodiment, a method for increasing the volume and / or function of muscle located beneath a lesion in a subject suffering from a lesion is disclosed herein, comprising the steps of: selecting a subject suffering from a lesion; and administering to a 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 volume and / or function of muscle located beneath a lesion in the human subject.

[0060] In one embodiment, the lesion is caused by spinal cord injury (SCI). In one embodiment, the human subject is in the acute spinal cord injury (SCI) stage. In one embodiment, the human subject is in the subacute spinal cord injury (SCI) stage. In one embodiment, the human subject is in the chronic spinal cord injury (SCI) stage.

[0061] In one embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, further increases the volume and / or function of muscle tissue above the lesion. In one embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, increases the volume and / or function of fast-switch muscle. In one embodiment, administration of an antibody or its antigen-binding fragment increases the volume and / or function of slow-switch muscle.

[0062] In some embodiments, the amount of muscle tissue 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 amount of muscle tissue 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%.

[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 approximately 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, such as an antibody or its antigen-binding fragment, increases locomotor function in human subjects. In some embodiments, the locomotor function of human subjects 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 locomotor function of human subjects 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 child human subject.

[0065] In one embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, increases motor coordination and equilibrium in a human subject. In some embodiments, motor coordination and equilibrium in a human subject increase 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 equilibrium in a human subject increase 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 child human subject.

[0066] In one embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, increases muscle strength in human subjects. In some embodiments, the muscle strength of human subjects 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 human subjects 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 subjects are pediatric human subjects.

[0067] In one embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, increases grip strength in human subjects. In one embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, reduces the level of white adipose tissue in human subjects. In some embodiments, the level of white adipose tissue decreases 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 decreases 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, such as an antibody or its antigen-binding fragment, increases the total body weight of a human subject. In some embodiments, the level of total body weight 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 level of total body weight 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.

[0069] In one embodiment, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment, increases the metabolic rate of a human subject. In some embodiments, the metabolic rate 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 metabolic rate 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.

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

[0071] In one embodiment, a myostatin inhibitor, such as an antibody or its antigen-binding fragment, is administered to a human subject within 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 60 minutes after the human subject develops a lesion. In another embodiment, a myostatin inhibitor, such as an antibody or its antigen-binding fragment, is administered to a human subject at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, or 24 hours after the human subject develops a lesion. In one embodiment, a myostatin inhibitor, such as an antibody or its antigen-binding fragment, is administered to a 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 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, a myostatin inhibitor, such as an antibody or its antigen-binding fragment, is administered to a human subject over a period of approximately 1 to 30 days, approximately 1 to 50 days, approximately 1 to 100 days, approximately 1 to 200 days, or approximately 1 to 300 days.

[0072] In one embodiment, a myostatin inhibitor, such as an antibody or its antigen-binding fragment, is administered to a human subject over a long period of time. In one embodiment, a myostatin inhibitor, such as an antibody or its antigen-binding fragment, is administered to a human subject in doses ranging from 0.01 mg / kg to 100 mg / kg. In one embodiment, a myostatin inhibitor, such as an antibody or its antigen-binding fragment, is administered to a human subject intraperitoneally, intravenously, intramuscularly, topically, or subcutaneously.

[0073] In one embodiment, the method disclosed herein further includes the step of administering a second treatment to a human subject. In one embodiment, the second treatment is insulin, insulin sensitivity enhancers, alpha-glucosidase inhibitors, biguanides, sulfonylurea, insulin secretagogues, amylin agonists, phosphotyrosine phosphatase inhibitors, aldose reductase inhibitors, neurotrophic factors, PKC inhibitors, advanced glycation end product (AGE) inhibitors, active oxygen quenching agents, statins, squalene tetase inhibitors, fibrates, niacin, PCSK9 inhibitors, triglyceride reducers, cholesterol sequestering agents, angiotensin-converting enzyme inhibitors, angiotensin II antagonists, calcium channel blockers, ursodiol, pioglitazone, orlistat, betaine, rosiglitazone, central anti-obesity agents. The following group of agents is selected: gastrointestinal lipase inhibitors, beta-3 adrenergic receptor agonists, peptide-based appetite suppressants, cholecystokinin agonists, dopamine agonists, DPP-4 inhibitors, glucagon-like peptides, meglitinide, sulfonylurea, sodium glucose transporter (SGLT)2 inhibitors, cyclooxygenase inhibitors, progesterone derivatives, metoclopramide-based drugs, tetrahydrocannabinol-based drugs, and lipid metabolism improvers.

[0074] In one embodiment, a myostatin inhibitor, such as an antibody or its antigen-binding fragment, is administered in a dose of approximately 0.01 mg / kg to approximately 30 mg / kg. In one embodiment, the myostatin inhibitor, such as an antibody or its antigen-binding fragment, is administered intraperitoneally, intravenously, intramuscularly, or subcutaneously.

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

[0076] In one embodiment, the antibody or its antigen-binding fragment cross-reacts with human and mouse pro / latent myostatins. In one embodiment, the antibody or its antigen-binding fragment inhibits the formation of mature myostatin via proteolysis by toroidal proteases. In one embodiment, the formation of mature myostatin via proteolysis by toroidal proteases is inhibited with an IC50 of less than 1 μM.

[0077] In one embodiment, the antibody or its antigen-binding fragment includes a heavy chain variable domain containing a complementarity-determining region 3 (CDRH3) containing the sequence described in any one of SEQ ID NOs. 10-11 and 66. In another embodiment, the antibody or its antigen-binding fragment includes a light chain variable domain containing a complementarity-determining region 3 (CDRL3) containing the sequence described in any one of SEQ ID NOs. 22-23 and 67. In one embodiment, the antibody or its antigen-binding fragment comprises six complementarity-determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises the sequence described in any one of SEQ ID NOs: 1 to 3, CDRH2 comprises the sequence described in any one of SEQ ID NOs: 4 to 9, CDRH3 comprises the sequence described in any one of SEQ ID NOs: 10 to 11 and 66, CDRL1 comprises the sequence described in any one of SEQ ID NOs: 12 to 17, CDRL2 comprises the sequence described in any one of SEQ ID NOs: 18 to 21, and CDRL3 comprises the sequence described in any one of SEQ ID NOs: 22 to 23 and 67.

[0078] In one embodiment, CDRH1 includes the sequence described in SEQ ID NO: 1 or 2, CDRH2 includes the sequence described in SEQ ID NO: 4 or 5, CDRH3 includes the sequence described in SEQ ID NO: 10, CDRL1 includes the sequence described in SEQ ID NO: 12 or 13, CDRL2 includes the sequence described in SEQ ID NO: 18 or 19, and CDRL3 includes the sequence described in SEQ ID NO: 22.

[0079] In one embodiment, CDRH1 includes the sequence described in SEQ ID NO: 1 or 3, CDRH2 includes the sequence described in SEQ ID NO: 6 or 7, CDRH3 includes the sequence described in SEQ ID NO: 11, CDRL1 includes the sequence described in SEQ ID NO: 14 or 15, CDRL2 includes the sequence described in SEQ ID NO: 20 or 21, and CDRL3 includes the sequence described in SEQ ID NO: 23.

[0080] In one embodiment, CDRH1 includes the sequence described in SEQ ID NO: 1 or 2, CDRH2 includes the sequence described in SEQ ID NO: 4 or 5, CDRH3 includes the sequence described in SEQ ID NO: 66, CDRL1 includes the sequence described in SEQ ID NO: 12 or 13, CDRL2 includes the sequence described in SEQ ID NO: 18 or 19, and CDRL3 includes the sequence described in SEQ ID NO: 67.

[0081] In one embodiment, CDRH1 includes the sequence described in SEQ ID NO: 1 or 3, CDRH2 includes the sequence described in SEQ ID NO: 8 or 9, CDRH3 includes the sequence described in SEQ ID NO: 11, CDRL1 includes the sequence described in SEQ ID NO: 16 or 17, CDRL2 includes the sequence described in SEQ ID NO: 20 or 21, and CDRL3 includes the sequence described in SEQ ID NO: 23.

[0082] In one embodiment, the antibody or its antigen-binding fragment includes a heavy chain variable domain sequence described in any one of SEQ ID NOs: 24-29. In one embodiment, the antibody or its antigen-binding fragment includes a light chain variable domain sequence described in any one of SEQ ID NOs: 30-35. In one embodiment, the antibody or its antigen-binding fragment includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 25 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 31.

[0083] In one embodiment, the antibody or its antigen-binding fragment includes a heavy chain containing the amino acid sequence of SEQ ID NO: 50. In another embodiment, the antibody or its antigen-binding fragment includes a light chain containing the amino acid sequence of SEQ ID NO: 51.

[0084] In one embodiment, the antibody or its antigen-binding fragment competes with any other antibody described herein for binding to pro / latent myostatin. In one embodiment, the antibody or its antigen-binding fragment binds to pro / latent myostatin at the same epitope as the antibody described herein.

[0085] In one embodiment, the antibody or its antigen-binding fragment is 10 -6 Antibodies with a minimum M value compete for binding to pro / latent myostatin at the equilibrium dissociation constant Kd between them. In one embodiment, Kd is 10 -11 M~10 -6 It is within the range of M.

[0086] In one embodiment, the antibody or its antigen-binding fragment is a human antibody, a humanized antibody, a diabody, a chimeric antibody, a Fab fragment, an 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 its antigen-binding fragment includes a framework having a human germline sequence.

[0087] In one embodiment, the antibody or its antigen-binding fragment includes 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 includes a constant domain of IgG4. In one embodiment, the antibody includes a constant domain of IgG4 having a Ser-to-Pro scaffold substitution that generates an IgG1-like hinge and allows for the formation of interchain disulfide bonds. In one embodiment, the antibody or its antigen-binding moiety does not bind to GDF11. In one embodiment, the antibody or its antigen-binding moiety does not bind to mature (fully processed, free / soluble, active) myostatin. In one embodiment, the antibody or its antigen-binding moiety selectively or preferentially binds to tissue-bound myostatin (e.g., pro-forms of myostatin, i.e., promyostatin or pro-myostatin). In one embodiment, the antibody or its antigen-binding moiety binds to both the pro-form and latent forms of myostatin (promyostatin and latent myostatin), but not to mature myostatin. [Brief explanation of the drawing]

[0088] [Figure 1] Figures 1A and 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 present as a disulfide-linked dimer. Figure 1B shows the precursor protein assembled in an inactive conformation in which the prodomain (dark gray) encapsulates the growth factor (light gray) by a "straight jacket" assembly. This figure is a modification of the structure of latent TGFβ1 (Shi et al., Nature, 2011). [Figure 2]Figure 2 shows that myostatin activation involves two distinct protease events, generating three major myostatin species. The biosynthetic precursor protein, promyostatin, is processed by two distinct proteases. Cleavage of promyostatin (and pro-GDF11) is carried out by proprotein convertases such as furin / PACE3 (Paired Basic Amino Acid Cleaving Enzyme 3) or PCSK5 (Proprotein Convertase Subtilisin / Kexin type 5), which cleave at a conserved RXXR site between the prodomain and the mature growth factor. This cleavage produces a potential complex in which the mature growth factor is shielded from binding to its receptor by the prodomain. Activation and release of the active growth factor are achieved after further cleavage by BMP / toroid family proteases such as TLL-2 (Toroid-like Protein 2) or BMP1 (Bone Morphogenic Protein 1). These cleavage events result in a mature form of myostatin, which may be called active myostatin or mature myostatin. [Figure 3] Figure 3 shows the body weight of naive mice and the sham, SCI-veh, SCI-IgG, and SCI-Ab1 treatment groups one and two weeks after SCI. The asterisks* above the bar reflect a significant difference from the sham group, and the asterisks* below the bar reflect a significant difference from the SCI-Ab1 group. [Figure 4] Figure 4 shows the wet weight (mass) of the sham, SCI-veh, SCI-IgG, and SCI-Ab1 treatment groups two weeks after SCI. The resected muscles include the sublesional soleus and gastrocnemius muscles, as well as the supralesional biceps and triceps muscles. [Figure 5] Figure 5 shows the analysis of total fat-free (lean) mass and fat mass in the sham, SCI-veh, SCI-IgG, and SCI-Ab1 treatment groups two weeks after SCI. [Figure 6]Figure 6 shows lean body mass as a percentage of body weight in the sham, SCI-veh, SCI-IgG, and SCI-Ab1 treatment 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 treatment groups 2 weeks after SCI. In the graph below, the SCI / treatment control group represents the combination of the SCI / veh + SCI / IgG groups from the graph above. [Figure 8] Figure 8 shows the BMS spontaneous motion assessment 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. The statistical comparison between 1 week and 2 weeks after SCI reflects the combination of 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), one week after SCI, and two weeks after SCI. [Figure 10] Figure 10 shows the grip strength in the sham, SCI-veh, SCI-IgG, and SCI-Ab1 groups after preparatory training (PT), one week after SCI, and two weeks after SCI. [Figure 11]Figures 11A–11D show the effect of Ab2 treatment on changes in lean body mass in healthy cynomolgus monkeys. Healthy male cynomolgus monkeys were administered Ab2 intravenously at three different doses, 3 mg / kg, 10 mg / kg, and 30 mg / kg, once a week for 8 weeks, with a 4-week recovery period. Control animals were administered a vehicle control (20 mM citrate and 150 mM sodium chloride USP, pH 5.5). Lean body mass was measured by dual-energy X-ray absorptiometry (DEXA). Figure 11A is a graph showing the mean % change in lean body mass from all limbs of Ab2-treated animals and control animals, measured at day 0, week 4, week 8, and week 12. Figure 11B is a graph showing the mean % change in lean body mass from all limbs of Ab2-treated animals and vehicle control animals, measured at week 4. Figure 11C is a graph showing the mean percentage change in lean body mass in the limb muscles of animals treated with Ab2 and vehicle control animals, as measured at week 8. Figure 11D is a graph showing the mean percentage change in lean body mass in the limb muscles of animals treated with Ab2 and vehicle control animals, as measured at week 12. [Figure 12] Figures 12A-12B are graphs showing the effect of Ab2 treatment on muscle weight in the biceps brachii 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 were administered a vehicle control (20 mM citrate and 150 mM sodium chloride USP, pH 5.5). Muscle weight was measured by tissue weight at week 12. [Figure 13] Figure 13 shows the mean percentage change in lean body mass from baseline (day 0) and the percentage difference in muscle weight compared to vehicle controls in healthy cynomolgus monkeys treated with Ab2. [Figure 14]Figures 14A and 14B show latent myostatin levels in serum samples from healthy cynomolgus monkeys and control animals treated with Ab2, as measured using quantitative fluorescence Western blotting. Healthy male cynomolgus monkeys were administered three different doses, 3 mg / kg, 10 mg / kg, and 30 mg / kg, intravenously once weekly for 8 weeks, with a 4-week recovery period. Control animals were administered a vehicle control (20 mM citrate 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 fluorescence Western blotting. [Figure 15] Figure 15 shows the change in lean body mass due to Ab2-mediated myostatin inhibition. [Figure 16] Figure 16 shows the differentially expressed genes (DEGs) in the Ab2 treatment group. [Figure 17] Figure 17 shows the suppression of atrogenes (genes that cause muscle atrophy) after Ab2-mediated myostatin inhibition. [Figure 18] Figure 18 shows the expression of muscle-specific markers after Ab2-mediated myostatin inhibition. [Figure 19] Figure 19 shows the expression of respiratory capacity markers after Ab2-mediated myostatin inhibition. [Figure 20] Figure 20 shows the expression of adipocytes and adipogenesis markers after Ab2-mediated myostatin inhibition. [Figure 21] Figure 21 shows the regulation of pyruvate dehydrogenase. [Figure 22] Figure 22 shows the expression levels of pyruvate dehydrogenase and regulatory factors of fatty acid oxidation. [Figure 23] Figure 23 shows an immunofluorescence assay performed on frozen sections of the tibialis anterior muscle from healthy mice, co-stained with laminin using Ab2. [Figure 24]Figures 24A and 24B show cross-sections of the tibialis anterior muscle probed with anti-pro / latent GDF8 antibodies. Ab10 or nonspecific targeted antibodies are shown in Figure 24A, and HuNeg is shown in Figure 24B. Each figure is counterstained with DAPI. The scale bar is 0.01 cm. [Figure 25] Figures 25A-25C show cross-sections of the tibialis anterior muscle probed using anti-pro / latent GDF8 antibody Ab10 incubated in blocking buffer alone (Figure 25A), anti-pro / latent GDF8 antibody Ab10 incubated in blocking buffer with 10x molar excess recombinant mouse GDF8 (Figure 25B), or anti-pro / latent GDF8 antibody Ab10 incubated in blocking buffer with 10x molar excess recombinant mouse GDF11 (Figure 25C). Figures 25A-25C are counterstained with DAPI. [Figure 26] Figures 26A-26C show cross-sections of the tibialis anterior muscle, probed with anti-pro / latent GDF8 antibody Ab10 and anti-laminin, and counterstained with DAPI. Pro / latent GDF8 and laminin colocalize in the interstitial space at the apex of the muscle fibers (arrows), between muscle fibers (heads of arrows), and around the interstitial nucleus (asterisks). [Figure 27] Figures 27A-27C demonstrate the reduction of SCI-induced intramuscular fat infiltration by monoclonal antibodies that inhibit myostatin activation. [Figure 28] Figures 28A-28B show the effects of monoclonal antibodies that inhibit myostatin activation in a cardiotoxin-induced injury model. [Figure 29] Figure 29 demonstrates that antibody-treated animals showed a statistically significant increase in mean total bone cross-sectional area and cortical bone thickness compared to the control (PBS). [Figure 30] Figure 30 demonstrates that antibody-treated animals showed increased trabecular bone volume, trabecular thickness, and trabecular bone number compared to controls. Furthermore, antibody-treated animals showed decreased trabecular bone spacing compared to controls. [Figure 31]Figure 31 demonstrates that animals treated with myostatin inhibitors showed increased bone volume in non-weight-bearing bones, such as the vertebrae. [Figure 32] Figure 32 demonstrates that mice treated with Abl showed a 14.4% increase in body weight at day 50 compared to control mice (treated with PBS). [Figure 33] Figure 33 shows the weight increase of several muscles after treatment with Abl: gastrocnemius, TA, EDL, soleus, and masseter. [Figure 34] Figure 34A shows that plantar flexion force (maximum torque) increased by 23% after Abl treatment compared to PBS controls, and that plantar flexion force / maximum torque / limb length increased by 20% after Abl treatment compared to PBS controls. Figure 34B shows the masseter muscle strength after Abl treatment compared to controls. [Figure 35] Figure 35 shows histological data from the high-dose SMN-C1 cohort, as well as histograms of total fiber cross-sectional area (CSA) and CSA distribution in control (vehicle) versus Abl-treated animals, demonstrating an increasing trend in fiber CSA. This trend was entirely attributable to type IIb fibers (data not shown).

[0089] [Modes for carrying out the invention]

[0090] This invention is at least in part based on the discovery that administering a myostatin inhibitor, such as an antibody or antigen-binding fragment that specifically binds to pro / latent myostatin, or to a subject with a metabolic disorder, 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 its antigen-binding portion, significantly enhances metabolic rate or energy expenditure in subjects with metabolic disorders or dysfunctions. Administration of a myostatin inhibitor, such as an anti-pro / latent myostatin antibody, significantly reduced the SCI-induced decline in lesional muscle mass and whole body weight, and simultaneously reduced 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] Accordingly, the present invention provides a method for treating or preventing metabolic diseases in human subjects using myostatin inhibitors, such as anti-pro / latent myostatin antibodies or their antigen-binding moieties. The present invention also provides a method for treating or preventing diseases in human subjects associated with defects in nerve signaling, increased metabolic rate, increased levels of brown adipose tissue, increased levels of beige adipose tissue, increased insulin-dependent blood glucose regulation, decreased protein catabolism in muscle and / or decreased release of amino acids from muscle, and decreased glucose uptake by target tissues, using myostatin inhibitors, such as anti-pro / latent myostatin antibodies or their antigen-binding moieties. The present invention further provides a method for increasing the amount and / or function of muscle located beneath a lesion in a diseased subject using myostatin inhibitors, such as anti-pro / latent myostatin antibodies or their antigen-binding moieties.

[0092] Accordingly, the present invention includes the use of an antibody and its antigen-binding moiety that specifically binds to promyostatin and / or latent myostatin in vivo and blocks the activation of mature myostatin in subjects where reduction of myostatin signaling is beneficial, such as human subjects. The present invention also includes methods for treating or preventing conditions associated with myostatin dysregulation by using a myostatin inhibitor, such as an antibody or its antigen-binding moiety that specifically binds to promyostatin and / or latent myostatin and blocks the 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 more (i.e., at least one) of the grammatical objects of the article. For example, “an element” means one element or more than one element.

[0094] Unless otherwise indicated in the operating examples or otherwise, any figures representing the quantities of components or reaction conditions used herein should be understood to be modified in all cases by the term “approximately.” When used in relation to percentages, the term “approximately” may mean within ±1% of the mean. Furthermore, the term “approximately” may mean within ±1% of the value.

[0095] The terms “administer,” “administering,” or “administration” include antibodies or antigen-binding fragments thereof, for example, pharmaceutical compositions comprising such antibodies or antigen-binding fragments, or any method of delivering a drug to a system of a subject or to a specific area within or on a subject (systemic administration and topical administration, respectively).

[0096] When used herein, the term “antibody” is intended to refer to an immunoglobulin molecule consisting of four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (hereinafter abbreviated as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (hereinafter abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The antibodies of the present invention are described in further detail in International Patent Application WO2016073853A1 and International Patent Application PCT / US2016 / 052014, filed on 15 September 2016, the entire contents of which are incorporated herein by reference. Antibody variants known in the art are also included in the present invention.

[0097] The terms “antigen-binding fragment,” “antigen-binding part,” or “antigen-binding moiety” (or simply “antibody fragment” or “antibody moiety”) of an antibody, 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 that fall within the scope of the term “antigen-binding fragment” of an antibody include (i) Fab fragments, which are monovalent fragments consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide crosslinks in the hinge region; (iii) Fd fragments consisting of a VH domain and a CH1 domain; (iv) Fv fragments consisting of the VL domain and VH domain of a single arm of the antibody; (v) dAb fragments consisting of a VH domain (Ward et al., (1989), Nature, vol. 341: pp. 544-546); and (vi) isolated complementarity-determining regions (CDRs). Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but can be joined by a synthetic linker using recombination methods, which allows the VL and VH regions to pair up to form a single protein chain that forms a monovalent molecule (known as single-stranded Fv (scFv)); see, for example, Bird et al. (1988), Science, vol. 242: pp. 423-426; and Huston et al. (1988), Proc. Natl. Acad. Sci. USA, vol. 85: pp. 5879-5883). Such single-stranded antibodies are also intended to be included within the scope of the term “antigen-binding portion” of an antibody. Other forms of single-stranded antibodies, such as diabodies, are also included.Diabody is a bivalent, bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between the two domains on the same chain forces the domains to pair with complementary domains on another chain, thereby creating two antigen-binding sites (see, for example, Holliger, P. et al. (1993), Proc. Natl. Acad. Sci. USA, Vol. 90: pp. 6444-6448; Poljak, RJ et al. (1994), Structure, Vol. 2: pp. 1121-1123).

[0098] As used herein, the terms “comprising” or “comprises” are used in reference to compositions, methods, and their respective components that are essential to the present invention; however, the inclusion of unspecified elements, whether essential or not, is still acceptable.

[0099] The term "consisting of" refers to the compositions, methods, and their respective components described herein, and is exclusive of any elements not listed in the description of embodiments.

[0100] The terms “control” or “control sample” as used herein refer to any clinically or scientifically relevant comparative sample or counterpart, including, for example, a sample from a healthy subject, a sample from a subject having a deficiency that causes or makes a subject susceptible to a particular disease or condition, a subject having the disease or condition of interest, a sample from a subject treated with a pharmaceutical carrier, a sample from a subject before treatment, a subject or sample treated with a placebo or buffer, or an untreated subject or sample.

[0101] The term "control level" refers to an acceptable or predetermined level of a biological marker, e.g., the level of the marker obtained before treatment or onset of a disease, or before administration of a drug, e.g., an antibody or its antigen-binding moiety. It also refers to the level of a biological marker present in a subject or population of subjects having one or more specific characteristics, e.g., the presence or absence of a particular disease or condition.

[0102] When used herein, the term “decrease” refers to a statistically significant reduction in the level of a disease symptom. A decrease may 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. A decrease may 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, a reduction is a decrease to a level acceptable as within the normal range for an individual without such disorder, and may also be referred to as normalization of the level.

[0103] As used herein, the term “derene” 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 occlusion of nerves. Denervation may be partial denervation (also called incomplete denervation) or complete denervation. Partial denervation may be, for example, a 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 involves loss or disruption of approximately 1–10%, 10–20%, 1–30%, 20–50%, 30–60%, 40–70%, 50–80%, or 60–90% of nerve supply or neuronal input to target tissue.

[0104] When used herein, “determining” is understood to mean performing an assay or using a method to confirm the presence, absence, level, or degree of any condition of someone or something, such as a particular state, biomarker, pathology, or physiological condition.

[0105] The “onset” or “progression” of a disease means the initial manifestation and / or subsequent progression of the disease. The 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 the purposes of this disclosure, onset or progression means the biological course of symptoms. “Onset” includes appearance, recurrence, and disease onset. As used herein, “onset” or “appearance” of a disease / disorder associated with myopathy includes the initial onset and / or recurrence.

[0106] Methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this disclosure, but preferred methods and materials are listed below. The abbreviation “e.g.” is derived from the Latin “exempli gratia” and is used herein to indicate non-limiting examples. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

[0107] The term “epitope” includes any polypeptide determinant that can specifically bind to an immunoglobulin or T cell receptor. In certain embodiments, the epitope determinant comprises a group of chemically active surfaces of molecules such as amino acids, sugar side chains, phosphoryls, or sulfonyls, and in certain embodiments may have specific three-dimensional structural properties and / or specific charge properties. An epitope is a region of an antigen to which an antibody binds. In certain embodiments, an antibody is said to bind specifically to an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. Epitopes can be linear epitopes or conformational epitopes.

[0108] As used herein, the terms “effective amount” and “effective dose” refer to any amount or dose of a compound or composition sufficient to satisfy its intended purpose, 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 present invention, the intended purpose may be to inhibit myostatin activation in vivo to achieve a clinically meaningful outcome associated with myostatin inhibition.

[0109] The measure of the relevant intended purpose may be objective (i.e., measurable by several assays or markers) or subjective (i.e., the subject shows signs of or feels an effect). In some embodiments, the therapeutically effective dose is the amount administered to a patient population that meets certain clinical criteria for the disease, disorder, or condition (determined by manifested symptoms, disease progression / stage, genetic profile, etc.) and produces a statistically significant therapeutic response in that population.

[0110] In some embodiments, an effective dose 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, and therefore, if adverse effects are present, is well tolerable for the patient to continue the treatment regimen, with the therapeutic benefits outweighing the risks of toxicity. In some embodiments of the present invention, it will be understood by those skilled in the art that a unit dose may be considered to contain an effective dose if it contains an amount suitable for administration with respect to a dosage regimen that correlates with a positive outcome.

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

[0112] When used herein, the term “human antibody” is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences and fragments thereof. The human antibodies of this disclosure may include, for example, amino acid residues not encoded by human germline immunoglobulin sequences in CDRs, particularly CDR3 (mutations introduced, for example, by random or site-directed mutagenesis in vitro, or by somatic mutations in vivo). However, when used herein, the term “human antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, are grafted onto a human framework sequence.

[0113] For example, the term “increase” in relation to the symptoms of a disease, such as a loss of function or quantity associated with the disease, such as a loss of muscle mass, refers to a statistically significant increase in such a level. An increase may 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 detected by the detection method. An increase may 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 detected by the detection method. In certain embodiments, an increase is an increase to a level that is acceptable as being within the normal range for an individual without such a disorder, and may also be referred to as normalization of the level. In certain embodiments, the increase is a normalization of the level of disease signs or symptoms, or an increase in the difference between the subject's level of disease signs and the normal level of disease signs. In certain embodiments, the method includes an increase in the volume and / or function of muscle tissue after treating the subject with an antibody that specifically binds to pro / latent myostatin. In certain embodiments, the method includes an increase in the level of promyostatin in 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 substantially does not contain other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to pro / latent myostatin substantially does not contain antibodies that specifically bind to antigens other than pro / latent myostatin). However, an isolated antibody that specifically binds to pro / latent myostatin may have cross-reactivity to other antigens, such as pro / latent myostatin molecules from other species. Furthermore, an isolated antibody does not necessarily have to substantially contain other cellular material and / or chemicals.

[0115] Unless otherwise specified, the term “mature myostatin” refers to the fully processed, biologically active form of myostatin. The biologically active form of myostatin is capable of binding to and / or activating myostatin receptors. The wild-type sequence of mature myostatin is provided as SEQ ID NO: 52. In some cases, mature myostatin may contain one or more mutations that may result in structural / functional or stability alterations.

[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 may be an antibody (including, for example, a fragment thereof such as the domain antibody (dAb) described in U.S. Patent Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; and 6,696,245), a small molecule inhibitor, adnectin, affibody, DARPin, anticalin, avimer, versabody, or gene therapy. The antibody or its antigen-binding fragment may bind to mature myostatin, myostatin receptor, and / or GDF11. In some embodiments, the myostatin inhibitor is a small molecule inhibitor. In other embodiments, the myostatin inhibitor refers to gene therapy. In one embodiment, the myostatin inhibitor specifically binds to myostatin but not to GDF11. In one embodiment, the myostatin inhibitor may be used to treat metabolic disorders, muscle conditions or disorders, disorders or disorders related to defects in nerve signaling or partial denervation, or other conditions described herein. In another embodiment, the myostatin inhibitor may be used to treat diseases involving fast-twitch muscle fibers as described herein. In yet another embodiment, the myostatin inhibitor may be used to produce a therapeutic effect under 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 the interaction 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 fragment thereof reactive with an antigen) to specifically bind to an antigen (or fragment thereof) and not to specifically bind 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 non-specific antigen, epitope, receptor ligand, or binding partner. "Specific binding", as used herein, can also refer to binding pairs based on binding kinetics such as K on , K off , and K D . For example, a ligand has a Koff of 10 -2 sec -1 or less, 10 -3 sec -1 or less, 10 -4 sec -1 or less, 10 -5 sec -1 or less, or 10 -6 sec -1 or less; and / or a K D of 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 -12If the ligand is M or less, it can be understood that it binds specifically to the target site. It is understood that various proteins may share common epitopes or other binding sites (e.g., kinase-reactive sites). In certain embodiments, a binding site may be bound to more than one ligand, but it can still be considered specific based on its binding preference compared to nonspecific antigens and / or by having certain binding kinetic parameters. Methods for selecting appropriate nonspecific controls are within the capabilities of those 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 I muscle,” or “Type I muscle” refer to muscles rich in Type I muscle fibers, which are frequently used, more postural, and help enable long-distance endurance such as in long-distance running. As used herein, the terms “fast-twitch muscle,” “fast-twitch muscle,” “Type II muscle,” or “Type II muscle” provide higher energy output and strength and are used for powerful, sudden movements such as in sprinting, but such muscles fatigue quickly and cannot be used repeatedly. Fast-twitch muscles are classified into two fiber type categories: moderate-fast-twitch fibers (Type IIA) and fast-twitch fibers (Type IIB or Type IIx). Moderate-fast-twitch fibers are thicker, contract faster, and also deplete more rapidly than slow-twitch fibers. Fast-twitch fibers, which are the strongest and have the lowest endurance, are activated when the body approaches maximum exertion. The majority of muscles tend to be composed of a mixture of various fiber types, but different muscles contain different ratios of fiber types. During development or in response to certain events (e.g., exercise, disease, injury), the fibrous types within a muscle or muscle group may undergo a change in fibrous type, resulting in a alteration of the phenotype 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, in particular a mammal requiring treatment, such as a companion animal (e.g., a dog, a cat), livestock (e.g., a cattle, a pig, a horse, a sheep, a goat, a poultry), and an experimental animal (e.g., a rat, a mouse, a guinea pig). In some embodiments, a subject is a human being for whom treatment is beneficial or who requires 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” means, with respect to an increase in muscle mass, an increase in muscle mass over a specified time period following the administration of a therapeutically effective dose of a myostatin inhibitor, such as an anti-pro / latent myostatin antibody as described herein. The sustained increase may be continuous or discontinuous, but as a whole, results in an increase in muscle mass over the specified time period.

[0123] "Treatment" or "preventing" a disease or disorder means delaying or preventing the onset of such disease or disorder, reversing, alleviating, restoring, inhibiting, slowing or stopping the progression, exacerbation, or worsening of conditions associated with such disease or disorder, but not necessarily requiring 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 includes two members: myostatin and GDF11. Like other members of the TGFβ superfamily, both myostatin and GDF11 are initially expressed as inactive precursor polypeptides (referred to as promyostatin and pro-GDF11, respectively). The domain structure and nomenclature are shown in Figure 1A. Figure 1B illustrates a schematic model of the overall structure of promyostatin, in which the mature growth factor is confined to a cage consisting of two alpha helices linked by a loop called a "latency lasso."

[0125] Myostatin is a well-characterized negative regulator of skeletal muscle mass, released via two separate protease cleavage steps from its autoinhibitory N-terminal prodomain. These cleavage events in the muscle fiber microenvironment can be referred to, for example, as cellular activation. Following activation, mature myostatin signals by binding to complexes of type I and type II cell surface receptors (Alk4 / 5 and ActRIIB), whose downstream signaling induces muscle atrophy. Myostatin has been of interest as a target for the treatment of muscle wasting. Several therapeutic agents targeting the ActRIIB signaling pathway have completed early to mid-stage clinical trials for muscle wasting conditions including muscular dystrophy, muscular dystrophy, cachexia, and total hip replacement / hip fracture. To date, primary clinical strategies have focused on blocking the interaction between mature myostatin and cell surface receptors. However, several treatment programs have been discontinued due to lack of specificity (leading to unacceptable toxicity) and / or lack of efficacy. In vivo, myostatin primarily forms a complex with its inhibitory prodomain.

[0126] Aspects of the disclosure provided herein relate to the assessment of the extent to which means are available for specifically blocking myostatin pathway signaling by blocking the on-cellular activation of myostatin from these inhibitory prodomain complexes. Another aspect of the disclosure relates to the assessment of a panel of human monoclonal antibodies that selectively bind to myostatin precursor morphologies, including a subset that inhibits proteolytic activation in vitro. In some embodiments, antibodies that block activation have been found to protect mice from dexamethasone-induced muscle atrophy. Assessment of serum and muscle samples from healthy animals and animals undergoing dexamethasone-induced atrophy has demonstrated alterations in the in vivo distribution of precursor morphologies during atrophy, which is a unique finding of significant importance in understanding muscle wasting pathogenesis. Furthermore, treatment of healthy mice with a mouse 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 insights into the importance of myostatin processing in skeletal muscle protein homeostasis. Furthermore, blocking the cellular activation of growth factors from their precursor forms is a potent method for preventing myostatin signaling, and this technique offers a novel therapeutic strategy that can be applied to other members of the TGFβ superfamily.

[0127] The activation and release of mature myostatin are achieved by several distinct protease cleavage events. The first cleavage step of promyostatin and proGDF11 is carried out by a proprotein convertase, which cleaves at the conserved RXXR site between the prodomain and the mature growth factor. This cleavage produces “latent myostatin,” in which mature myostatin is shielded from binding to its receptor by the prodomain. The activation and release of the mature, active myostatin growth factor is achieved after the latent myostatin is cleaved by a further protease of the BMP / toroid family, such as mTLL-2. As used herein, the term “mature myostatin” may refer to both full-length mature myostatin and 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, including a disulfide-linked homodimer, where each molecule of the homodimer contains an amino-terminal prodomain covalently bonded to a carboxyl-terminal mature myostatin domain. In one embodiment, “promyostatin” is not cleaved by either a proprotein convertase or a BMP / toroid family protease. Exemplary promyostatin sequences, their variants, 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, including a disulfide-linked homodimer, where each molecule of the homodimer contains an amino-terminal prodomain non-covalently bonded 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 / toroid family protease. In another embodiment, “latent myostatin” can be produced by combining the prodomain and the carboxyl-terminal mature myostatin domain in vitro and allowing them to fold appropriately. See, for example, Sengle et al., J. Biol. Chem., vol. 286(no. 7): pp. 5087-5099, 2011. Exemplary latent myostatin sequences, their variants, 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 humans, rats, mice, and cynomolgus monkeys are provided below. In these pro-GDF8 sequences, proprotein convertase cleavage sites are shown in bold, and toroidal protease sites are shown in underline. In some embodiments, the proprotein convertase cleavage sites include amino acid residues 240-243 of SEQ ID NOs. 52-55. In some embodiments, the toroidal protease sites include amino acid residues 74-75 of SEQ ID NOs. 52-55. It should be understood that the exemplary pro-GDF8 sequences provided herein are not intended to be limiting, and additional pro-GDF8 sequences from other species, including any isoforms thereof, are within the scope of this disclosure.

[0131] ProGDF8 (human):

[0132] [ka]

[0133] ProGDF8 (Rat):

[0134] [ka]

[0135] Pro GDF8 (mouse):

[0136] [ka]

[0137] ProGDF8 (Cynomolgus macaque):

[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, the Fingers region 1, the 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. JPEG2026136188000005.jpg1251 (SEQ ID NO: 118) contains at least one amino acid residue derived from the polypeptide stretch. In some embodiments, the amino acid residue in the arm region of the prodomain that contacts the antibody when bound to the antigen is a residue that is not conserved between myostatin and GDF11. In some embodiments, such residues 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, exhibiting 90 percent identity; however, their prodomain regions are not well-conserved, sharing less than 50 percent amino acid identity between the two. Both myostatin and GDF11 bind to the same receptor, consisting of type I receptors (ALK4 / 5) associated with type II receptors (ACTRIIA / B), and signal transduction through it. Myostatin engagement with type I and type II receptors initiates a signaling cascade that leads to SMAD phosphorylation and transcriptional activation of muscular atrophy genes. The relatively high degree of conservation in mature growth factors makes it difficult to identify reagents, such as monoclonal antibodies, that can distinguish between mature myostatin and GDF11.

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

[0142] >GDF11Arm8 (Sequence ID 65)

[0143] [ka]

[0144] The role of myostatin in muscle homeostasis and metabolic regulation Skeletal muscle accounts for approximately 40% of body weight and is a dynamic organ that turns over at a rate of 1-2% per day. Myostatin is thought to play a central role in maintaining muscle homeostasis in both healthy and diseased 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 observed in numerous situations that cause atrophy, including injury, diseases such as cachexia, disuse, and space travel, demonstrating the importance of the myostatin signaling mechanism. Based on this central role, significant research has been driven to inhibit the action of myostatin in vivo. Indeed, antagonism of myostatin signaling has been shown to be beneficial for muscle growth / enlargement.

[0145] Furthermore, it is well known that muscle is the body's major protein reservoir and therefore contributes to amino acid homeostasis / metabolism. Along with glucose (produced and stored as glycogen, mainly in the liver and muscles) and lipids (stored in adipose tissue), proteins in muscle can function as energy sources (i.e., broken down to produce energy). Deficiencies or imbalances in the utilization or mobilization of these energy sinks in the body can underlie, at least in part, various types of metabolic dysregulation. Therefore, myostatin is intended to play a direct role in metabolic regulation by coordinating the equilibrium between the breakdown and synthesis / storage of glucose, fat, and / or muscle in the body. Indeed, while myostatin has been considered primarily an important regulator of muscle growth / loss since its discovery in 1997, the findings presented in more detail herein suggest a broader role of myostatin as a metabolic regulator.

[0146] Since muscle homeostasis correlates with amino acid / protein metabolism, myostatin inhibition is further intended to control nitrogen metabolism and nitrogen mobilization in the body. In muscle catabolism, muscle tissue is broken down into amino acids, which are its building blocks, and these 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 human urea. If nitrogen metabolism is dysregulated, possible consequences include fluid imbalances that can manifest as systemic or localized edema (e.g., congestion; fluid overload). For example, pulmonary edema and renal congestion are frequently observed in patients with heart failure associated with decreased cardiac output. Pulmonary congestion is, in fact, the most common cause of hospitalization in this clinical background and correlates with a poor prognosis.

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

[0148] Therefore, current guidelines suggest that diuretic therapy should be used to attempt decongestion in subjects with osmoregulatory deficiencies and subjects with fluid retention or volume overload, such as those with heart failure, for example, chronic heart failure (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 this disclosure may provide clinical benefit to such patients. Specifically, the method of the present invention is suitable for enhancing the response in subjects who are refractory to diuretic treatment or who have an insufficient response to diuretic treatment.

[0149] For example, administration of myostatin inhibitors reduces the required diuretic dose and / or improves the 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 hepatic disease, the myostatin inhibitors disclosed herein reduce the required diuretic dose, internally improve the control of symptoms such as peripheral edema or internal congestion (including intraocular volume overload that can lead to pleural effusion, ascites, hepatic congestion, or retinal detachment), and / or reduce the risk of pulmonary edema.

[0151] Subjects at higher risk of developing acute pulmonary edema, such as those receiving intravenous fluids, blood transfusions, or fluid transfers, may be prophylactically administered with the myostatin inhibitors disclosed herein. For example, a myostatin inhibitor can be prophylactically administered during a blood transfusion to a subject with congestive heart failure who requires a blood 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 as a result of the volume overload itself or from diuretics used to treat the volume overload, if the administration of diuretics is limited by hyponatremia as a side effect, myostatin inhibitors disclosed herein may be administered to treat the hyponatremia and / or to enable the use of higher doses of diuretics. However, generally speaking, 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 moieties, and gene therapies, are at 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. In particular, the majority of these antagonists are not myostatin-specific and therefore antagonize signaling of multiple TGFβ family members. For example, several current clinical candidates block activin A, GDF11, and further growth factors such as BMP9 and BMP10, which are regulators of reproductive biology, wound healing, erythrogenesis, and angiogenesis, respectively. Aspects of this disclosure relate to the recognition that, due to the lack of specificity observed in these myostatin antagonists described elsewhere, these myostatin antagonists block further biological pathways, such as those listed above, in addition to myostatin, and therefore may pose a greater risk to certain patient populations. Therefore, this potentially limits the patient population that can be safely treated due to unacceptable adverse effects such as abnormal bleeding, wound healing, or reproductive problems caused by off-target antibody binding (Campbell et al., Muscle Nerve, (2016); David, L., Blood, Vol. 109, pp. 1953-1961 (2007)). For example, activin A is involved in both wound healing and reproductive biology, and therefore, binding to activin A limits its use in patients who have recently undergone surgery or injury, or in women of reproductive age. The increased risk of such adverse effects or toxicity is of particular concern when i) patient populations requiring long-term treatment (e.g., chronic conditions); and / or ii) patient populations are or include pediatric patients who may be more susceptible to such adverse effects and / or toxicity. Therefore, the present invention includes novel methods for inhibiting myostatin signaling in vivo with a potentially safer profile.

[0154] Accordingly, myostatin inhibitors, such as antibodies or their antigen-binding fragments, that can bind to promyostatin and / or latent myostatin and thereby inhibit myostatin activation, as well as their use for treating diseases and disorders associated with myopathy, are provided herein. In some embodiments, taking into account the prevalence of circulating latent complexes, treatments that specifically target myostatin precursors, such as promyostatin and latent myostatin, which are more abundant and longer-existing than mature growth factors, are provided herein. Without wishing to be bound to any particular theory, myostatin inhibitors provided herein, such as antibodies or their antigen-binding fragments, may prevent the activation of promyostatin and / or latent myostatin via proteolysis to mature myostatin, which is considered the "active" form of myostatin that can activate the myostatin pathway, by binding to type I (ALK4 / 5) and type II (ACTRIIA / B) receptors, for example.

[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 its antigen-binding fragment specifically binds to promyostatin. In some embodiments, an anti-pro / latent myostatin antibody or its antigen-binding fragment specifically binds to latent myostatin. In some embodiments, an anti-pro / latent myostatin antibody or its antigen-binding fragment specifically binds to both latent myostatin and promyostatin. In preferred embodiments, an anti-pro / latent myostatin antibody or its antigen-binding fragment 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 its antigen-binding fragment that specifically binds to promyostatin and / or latent myostatin does not bind to pro / latent GDF11 or mature GDF11.

[0156] Antipro / latent myostatin antibodies or their antigen-binding fragments, and their production This disclosure is based, at least in part, on the surprising discovery that a more advantageous method of selectively inhibiting myostatin signaling in vivo may be achieved by blocking the myostatin activation step rather than targeting already active myostatin. Therefore, 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, not only can increased muscle mass be achieved, but also enhanced muscle function and prevention of metabolic dysregulation. Unexpectedly, the advantageous therapeutic effect can also be achieved under lesions in subjects with a defect, though not a complete loss, of signaling between neurons and target tissues such as target muscle.

[0157] An antibody (used interchangeably with the plural) is an immunoglobulin molecule that can specifically bind to targets such as carbohydrates, polynucleotides, lipids, and polypeptides through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Antibodies include antibodies of any class (or subclass thereof), such as IgD, IgE, IgG, IgA, or IgM, and an antibody does not have to be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chain, immunoglobulins can be assigned to various classes. There are five 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 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 described herein can bind to pro / latent myostatin, thereby inhibiting the activation of pro / latent myostatin to mature myostatin via proteolysis. In some cases, the antibodies or antigen-binding fragments described herein can inhibit the activation of pro / latent myostatin via proteolysis 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 the cleavage of promyostatin via proprotein convertase (e.g., furin) via proteolysis 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 described herein can inhibit the cleavage of promyostatin or latent myostatin via proteolysis by toroid proteases (e.g., mTLL2) by at least 20%, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

[0159] In some embodiments, inhibition of the proteolytic cleavage of promyostatin or latent myostatin by toroidal proteases results in a progressive increase in muscle mass. In some embodiments, subjects exhibit a progressive increase in muscle mass over at least 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 weeks (or any range that can be aggregated into any of these values). The inhibitory activity of anti-pro / latent myostatin antibodies can be measured by routine methods, for example, by Western blot analysis as described in Example 1 and Figure 3, disclosed in WO2016 / 073853, the entire contents of which are explicitly incorporated herein by reference. However, it should be understood that further methods may be used to measure the inhibitory activity of anti-pro / latent myostatin antibodies against the proteolytic cleavage of pro / latent myostatin. In some embodiments, inhibition of pro / latent myostatin cleavage (e.g., by proprotein convertase and / or toroidal proteases) provides a measure of inhibitory potency, which is the concentration of the inhibitor (e.g., an anti-pro / latent myostatin antibody) required to reduce the protease (e.g., proprotein convertase or toroidal protease) activity by half, and can be reflected as an inhibition constant (Ki) that is independent of both enzyme and 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 having a proprotein convertase cleavage site. Examples of proprotein convertases for use according to this disclosure include, but are not limited to, PCSK5 / 6, PACE4, PACE7, and PACE3 (e.g., furin). In some embodiments, the proprotein convertase is obtained from any mammal, including, but is not limited to, humans, monkeys, or rodents (e.g., mice, rats, hamsters), and is, for example, purified. In another embodiment, the proprotein convertase is produced recombinantly.

[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%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least about 99.9% identical to PCSK5 / 6, PACE4, PACE7, or PACE3 (e.g., furin).

[0162] In some embodiments, the proprotein convertase cleavage site is an amino acid sequence that can be cleaved by a proprotein convertase (e.g., PCSK5 / 6, PACE4, PACE7, and PACE3). In some embodiments, the proprotein convertase cleavage site comprises the amino acid sequence RXXR, where R is arginine and X is any amino acid. In some embodiments, the proprotein convertase cleavage site comprises the amino acid sequence RX-(K / R)-R, where R is arginine, K is lysine, and X is any amino acid. In some embodiments, the proprotein convertase cleavage site comprises the amino acid sequence RVRR (SEQ ID NO: 57), where R is arginine and V is valine. Exemplary proprotein convertase cleavage sites for human, rat, mouse, and cynomolgus myostatin are shown in bold in SEQ ID NOs: 52-55. In some embodiments, the proprotein convertase cleavage site comprises the amino acid sequence RSRR (SEQ ID NO: 56).

[0163] In some embodiments, the toroidal proteases for use according to this disclosure include, but are not limited to, BMP-1, mTLL-1, and mTLL-2. The toroidal proteases can be obtained from any mammal, including, but are not limited to, humans, monkeys, or rodents (e.g., mice, rats, hamsters). In some embodiments, the toroidal proteases are homologous to toroidal proteases selected from the group consisting of BMP-1, mTLL-1, and mTLL-2. For example, the toroidal proteases 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] In some embodiments, the toroidal protease cleavage site is an amino sequence that can be cleaved by a toroid (e.g., BMP-1, mTLL-1, and mTLL-2). Exemplary toroidal protease cleavage sites for human, rat, mouse, and cynomolgus myostatin are underlined in SEQ ID NOs. 52-55. In some embodiments, the toroidal 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 described herein can bind to pro / latent myostatin, thereby inhibiting myostatin activity. In some cases, the antibodies or antigen-binding fragments described herein can inhibit myostatin signaling by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, inhibition of myostatin signaling can be measured by routine methods, for example, using the myostatin activation assay described in Example 1 disclosed in WO2016 / 073853 (the entire content of which is expressly incorporated herein by reference). However, it should be understood that additional methods may be used to measure myostatin signaling activity.

[0166] It should be understood that the degree of myostatin cleavage via proteolysis, for example by proprotein convertases and / or toroidal proteases, can be measured and / or quantified using any suitable method. In some embodiments, the degree of myostatin cleavage via proteolysis is measured and / or quantified using enzyme-linked immunosorbent assay (ELISA). For example, ELISA can be used to measure the level of released growth factors (e.g., mature myostatin). As another example, antibodies or antigen-binding fragments that specifically bind to promyostatin, latent myostatin, and / or mature myostatin can be used in ELISA to quantify the degree of myostatin cleavage via proteolysis in order to measure the level of specific forms of myostatin (e.g., pro / latent / mature myostatin). In some embodiments, the degree of myostatin cleavage via proteolysis is measured and / or quantified using immunoprecipitation followed by trypsin peptide SDS-PAGE or mass spectrometry, 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, may be found in antibodies. Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins may be assigned to five main classes: A, D, E, G, and M, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Each light chain has an N-terminal variable (V) domain (V L ) and steady-state (C) domain (C L Each heavy chain typically contains an N-terminal V domain (V H ), 3 or 4 C domains (C H 1-3) and typically include the hinge region. V H The C closest to H The domain is C H It is named 1. V H and V LThe 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 the hypervariable sequence (complementarity-determining region, CDR). The CDR contains most of the residues involved in the specific interaction between the antibody and the antigen. The CDR is referred to as CDR1, CDR2, and CDR3. Thus, the CDR components on the heavy chain are referred to as CDRH1, CDRH2, and CDRH3, while the CDR components on the light chain are referred to as CDRL1, CDRL2, and CDRL3. The CDR typically refers to the Kabat CDR 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 the antigen-binding site is the hypervariable loop described by Chothia. See, for example, Chothia, D. et al., (1992) J.Mol.Biol. 227: pp. 799-817 and Tomlinson et al., (1995) EMBO J. 14: pp. 4628-4638. Another criterion is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains in the Antibody Engineering Lab Manual (edited by Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). Embodiments described with respect to Kabat CDR can be performed alternatively using the relationships similarly described with respect to the Chothia hypervariable loop or the AbM-defined loop, or any combination of these methods.

[0168] Suitable anti-pro / latent myostatin antibodies or their antigen-binding fragments for use in the methods of the present invention include those described in International Patent Applications PCT / US15 / 59468 and PCT / US16 / 52014. The entire contents of each of the aforementioned applications are incorporated herein by reference.

[0169] In some embodiments, the antipro / latent myostatin antibody or its antigen-binding fragment, as well as the nucleic acid molecule of the Disclosure encoding the antibody or its antigen-binding fragment, include 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 antipro / latent myostatin antibody of this disclosure, or its antigen-binding moiety, includes any antibody or its antigen-binding fragment containing CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3 or a combination thereof, provided to any one of the antibodies shown in Tables 1-3. In some embodiments, the antipro / latent myostatin antibody, or its antigen-binding moiety, includes CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, provided to any one of the antibodies shown in Tables 1-3. This disclosure also includes any nucleic acid sequences encoding molecules containing CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, provided to any one of the antibodies shown in Tables 1-3. The antibody heavy chain and light chain CDR3 domains may play a particularly important role in the antibody's binding specificity / affinity to the antigen. Therefore, the antipro / latent myostatin antibodies of this disclosure or their antigen-binding moieties, or their nucleic acid molecules, may include at least the heavy chain and / or light chain CDR3 of the antibodies shown in Tables 1-3.

[0180] Aspects of this disclosure relate to monoclonal antibodies or antigen-binding fragments that bind to pro / latent myostatin proteins and contain six complementarity-determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3.

[0181] In some embodiments, CDRH1 includes the sequence described in any one of sequence numbers 1 to 3. In some embodiments, CDRH2 includes the sequence described in any one of sequence numbers 4 to 9. In some embodiments, CDRH3 includes the sequence described in any one of sequence numbers 10 to 11, 66, 71, 76, 81, 86, 91, 96, 101, 106, and 111. CDRL1 includes the sequence described in any one of sequence numbers 12 to 17. In some embodiments, CDRL2 includes the sequence described in any one of sequence numbers 18 to 21. In some embodiments, CDRL3 includes the sequence described in any one of sequence numbers 22 to 23, 67, 72, 77, 82, 87, 92, 97, 102, 107, and 112.

[0182] In some embodiments (for example, for the anti-pro / latent myostatin antibody Ab1 shown in Table 1), CDRH1 contains the sequence described in SEQ ID NO: 1 or 2, CDRH2 contains the sequence described in SEQ ID NO: 4 or 5, CDRH3 contains the sequence described in SEQ ID NO: 10, CDRL1 contains the sequence described in SEQ ID NO: 12 or 13, CDRL2 contains the sequence described in SEQ ID NO: 18 or 19, CDRL3 contains the sequence described in SEQ ID NO: 22, and the antibody binds to pro / latent myostatin.

[0183] In some embodiments (for example, with respect to the anti-pro / latent myostatin antibody Ab2 or its antigen-binding moiety shown in Table 1), CDRH1 comprises the sequence described in SEQ ID NO: 1 or 2, CDRH2 comprises the sequence described in SEQ ID NO: 4 or 5, CDRH3 comprises the sequence described in SEQ ID NO: 66, CDRL1 comprises the sequence described in SEQ ID NO: 12 or 13, CDRL2 comprises the sequence described in SEQ ID NO: 18 or 19, and CDRL3 comprises the sequence described in SEQ ID NO: 67, and the antibody or its antigen-binding moiety binds to pro / latent myostatin.

[0184] In some embodiments (for example, with respect to the anti-pro / latent myostatin antibody Ab3 or its antigen-binding moiety shown in Table 1), CDRH1 comprises the sequence described in SEQ ID NO: 1 or 3, CDRH2 comprises the sequence described in SEQ ID NO: 6 or 7, CDRH3 comprises the sequence described in SEQ ID NO: 11, CDRL1 comprises the sequence described in SEQ ID NO: 14 or 15, CDRL2 comprises the sequence described in SEQ ID NO: 20 or 21, and CDRL3 comprises the sequence described in SEQ ID NO: 23, and the antibody or its antigen-binding moiety binds to pro / latent myostatin.

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

[0186] In some examples, the antipro / latent myostatin antibody of this disclosure or any of its antigen-binding moieties comprises 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 contain one or more CDR sequences shown 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 one of 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 chain and light chain variable regions of the antibodies listed in Table 1 are provided below.

[0187] In some embodiments, the antipro / latent myostatin antibody of this disclosure or its antigen-binding moiety comprises any antibody comprising one heavy chain variable domain of any of SEQ ID NOs: 24-29, 73, 78, 83, 88, 93, 98, 103, 108, and 113 or one light chain variable domain of any of SEQ ID NOs: 30-35, 74, 79, 84, 89, 94, 99, 104, 109, and 114. In some embodiments, the antipro / latent myostatin antibody of this disclosure or its antigen-binding moiety comprises any antibody comprising a heavy chain variable and light chain variable pair of SEQ ID NOs: 24 and 30; 25 and 31; 26 and 32; 27 and 33; 28 and 34; or 29 and 35).

[0188] Aspects of this disclosure provide antipro / latent myostatin antibodies or their antigen-binding moieties having heavy-chain variable and / or light-chain variable amino acid sequences homologous to any of those described herein. In some embodiments, the antipro / latent myostatin antibody or its antigen-binding moiety includes 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 any one of the light-chain variable sequences 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 from 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 heavy-chain variable 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, Vol. 87:2264-68, 1990, modified as described in Karlin and Altschul, Proc.Natl.Acad.Sci.USA, Vol. 90:pp. 5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J.Mol.Biol., Vol. 215:pp. 403-4010, 1990. BLAST protein search can be performed using the XBLAST program, score=50, word length=3, to obtain homologous amino acid sequences for the target protein molecule. If a gap exists between two sequences, gap BLAST can be used as described in Altschul et al., Nucleic Acids Res., Vol. 25 (No. 17):pp. 3389-3402, 1997. When using BLAST and gap BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) may be used.

[0190] In some embodiments, conserved mutations may be introduced into the CDR or framework sequence at positions where the residue is unlikely to be involved in interactions with pro / latent myostatin, as determined based on the crystal structure. As used herein, “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein being substituted. Modified proteins may be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, as found in references summarizing such methods, e.g., 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 amino acid substitutions include substitutions made between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0191] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein include mutations that confer desirable properties to the antibody or antigen-binding fragment. For example, to avoid difficult situations that can occur due to Fab arm exchange, which is known to occur with native IgG4 mAbs, the antibodies or antigen-binding portions thereof provided herein may include a stabilizing “Adair” mutation in which serine 228 (EU numbering, Kabat numbering residue 241) is converted to proline, resulting in 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, pp. 105-108; 1993). Thus, any of the antibodies may include 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 a portion thereof. For example, the V L domain can be attached at its C-terminus to a light chain constant domain such as Cκ or Cλ. Similarly, the V H domain or a portion thereof can be attached to the whole or a part of a heavy chain such as IgA, IgD, IgE, IgG and IgM and any isotype subclass. The antibody may include a suitable constant region (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within this scope may include V H and V L domains, or antigen-binding portions thereof, combined with any suitable constant region.

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

[0194] IgHV3-30

[0195]

Chemical formula

[0196] IgLV1-44

[0197]

Chemical formula

[0198] In some embodiments, the anti-pro / latent myostatin antibody or antigen-binding fragment may or may not contain the framework regions of the antibodies shown in SEQ ID NOs: 24 to 35. In some embodiments, the anti-pro / latent myostatin antibody is a murine antibody and contains murine framework region sequences.

[0199] In some embodiments, the anti-pro / latent myostatin antibody or its antigen-binding fragment has a relatively high affinity, for example 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11The antibody can bind to pro / latent myostatin at a Kd of less than or lower than M. For example, an anti-pro / latent myostatin antibody or its antigen-binding fragment can bind to pro / latent myostatin with affinities between 5 pM and 500 nM, for example between 50 pM and 100 nM, or for example 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 affinities of 50 nM or lower (e.g., 20 nM or lower, 10 nM or lower, 500 pM or lower, 50 pM or lower, or 5 pM or lower). The affinity and binding kinetics of the anti-pro / latent myostatin antibody may be tested using any preferred method, including but not limited to biosensor technologies (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, antibodies or antigen-binding fragments thereof that specifically bind to pro / latent myostatin are disclosed herein. In some embodiments, any of the antibodies or antigen-binding fragments provided herein bind to or near the toroidal cleavage site or to the toroidal docking site of pro / latent myostatin. In some embodiments, the antibody binds near the toroidal cleavage site or to the toroidal docking site if it binds to 15 or fewer amino acid residues of the toroidal cleavage site or toroidal docking site. In some embodiments, any of the antibodies or antigen-binding fragments provided herein bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the toroidal cleavage site or toroidal docking site. In some embodiments, the antibody binds to or near the toroidal cleavage site of GDF8. For example, the antibody can bind to the amino sequence described 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 of pro / latent myostatin, or to or near the proprotein convertase docking site. In some embodiments, the antibody binds to or 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 the proprotein convertase docking site. In some embodiments, any of the antibodies or antigen-binding fragments provided herein bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the proprotein convertase cleavage site or the proprotein convertase docking site. In some embodiments, the antibody binds to or near the proprotein convertase cleavage site of GDF8. For example, the antibody can bind to the amino acid sequence described in SEQ ID NO: 63 (GLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRC).

[0201] For example, the anti-pro / latent myostatin antibodies or their antigen-binding fragments described herein bind specifically to pro / latent myostatin compared to other forms of myostatin and / or other members of the TGFβ family of growth factors. Members of the TGFβ family of growth factors include, but are not limited to, AMH, ARTN, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDNF, INHA, INHBA, INHBB, INHBC, INHBE, LEFTY1, LEFTY2, NODAL, NRTN, PSPN, TGFβ1, TGFβ2, and TGFβ3 proteins. Such antibodies or their antigen-binding fragments can bind to pro / latent myostatin with much higher affinity (e.g., at least 2x, 5x, 10x, 50x, 100x, 200x, 500x, or 1,000x higher) compared to other members of the TGFβ family of growth factors. In some embodiments, such antibodies or their antigen-binding fragments can bind to pro / latent myostatin with at least 000x higher affinity compared to other members of the TGFβ family of growth factors. In some embodiments, antibodies or their antigen-binding fragments provided herein can bind to pro / latent myostatin with much higher affinity (e.g., at least 2x, 5x, 10x, 50x, 100x, 200x, 500x, or 1,000x higher) compared to 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 myostatins with at least 1,000-fold higher affinity compared to one or more forms of GDF11 (e.g., pro-GDF11, latent GDF11, or mature GDF11) or mature myostatin.Alternatively or further, antibodies or their antigen-binding fragments may exhibit much higher inhibitory activity (e.g., at least 2x, 5x, 10x, 50x, 100x, 200x, 500x, 1,000x higher) against the cleavage of pro / latent myostatin via proteolysis (e.g., by proprotein convertases or toroidal proteases) compared to other members of the TGFβ family, such as pro / latent GDF11. In another embodiment, the antibodies or their antigen-binding fragments disclosed herein do not bind to GDF11. This avoids the potential toxicity issues associated with antibodies that cross-react to both myostatin and GDF11.

[0202] In some embodiments, antibodies bind to antigens but fail to effectively remove them from plasma. Therefore, in some embodiments, the concentration of antigen in plasma can be increased by reducing antigen clearance. However, in some embodiments, the antibodies provided herein (e.g., sweeping antibodies) have pH-sensitive affinity for antigens. Such pH-sensitive antibodies can bind to antigens in plasma at neutral pH, dissociate from the antigen in acidic endosomes, thereby reducing antibody-mediated antigen accumulation and / or promoting antigen clearance from plasma.

[0203] Aspects of this disclosure relate to sweeping antibodies. As used herein, “sweeping antibody” or its antigen-binding fragment refers to an antibody or its antigen-binding fragment having both pH-sensitive antigen binding and binding to cell surface neonatal Fc receptors (FcRn) at a neutral or physiological pH at least at a threshold level. In some embodiments, the sweeping antibody or its antigen-binding moiety binds to neonatal Fc receptors FcRn at a neutral pH. For example, a sweeping antibody can bind to FcRn at a pH in the range of 7.0 to 7.6. In some embodiments, the sweeping antibody or its antigen-binding moiety can bind to an antigen at its antigen-binding site and can bind to cellular FcRn via the Fc portion of the antibody. In some embodiments, the sweeping antibody or its antigen-binding moiety can then be translocated internally and release the antigen into acidic endosomes, which may be degraded. In some embodiments, the sweeping antibody or its antigen-binding moiety no longer binds to an antigen and can then be released back into the serum by cells (e.g., by exocytosis).

[0204] In some embodiments, FcRn in the vascular endothelium (e.g., of the subject) extends the half-life of the sweeping antibody or its antigen-binding moiety. In some embodiments, the sweeping antibody or its antigen-binding moiety, which binds to an antigen such as myostatin (e.g., promyostatin, latent myostatin, or primed myostatin), is internalized by vascular endothelial cells. In some embodiments, the sweeping antibody or its antigen-binding moiety is returned to the bloodstream and recirculated. In some embodiments, the sweeping antibody or its antigen-binding moiety has an increased half-life (e.g., in the subject's serum) compared to its conventional counterpart. In some embodiments, the conventional counterpart of the sweeping antibody refers to the antibody or its antigen-binding moiety from which the sweeping antibody or its antigen-binding moiety originates (e.g., before manipulating the Fc moiety of the conventional antibody to bind to FcRn with greater affinity at pH 7). In some embodiments, the sweeping antibody or its antigen-binding moiety has a half-life in the subject's serum that is at least 1%, 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 100%, 150%, 200%, or 250% longer than its conventional counterpart.

[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 at a pH of 7.4. -3 M to 10 -8 It binds to Kd in the M range. In some embodiments, the sweeping antibody is 10 to FcRn 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 Binding occurs at Kd in the M range. In some embodiments, FcRn binds to the CH2-CH3 hinge region of the sweeping antibody. In some embodiments, FcRn binds to the same region as protein A or protein G. In some embodiments, FcRn binds to a different binding site than FcγR. In some embodiments, amino acid residue AA of the sweeping antibody Fc region is required for binding to FcRn. In some embodiments, amino acid residue AA of the sweeping antibody Fc region affects binding to FcRn.

[0206] In some embodiments, any of the antibodies or antigen-binding fragments provided herein are engineered to bind to FcRn with higher affinity. In some embodiments, any of the antibodies or antigen-binding fragments provided herein are engineered to bind to FcRn with higher affinity at pH 7.4. In some embodiments, the affinity of the antibody or antigen-binding fragment to FcRn is increased to extend their pharmacokinetic (PK) properties compared to their conventional counterparts. For example, in some embodiments, the sweeping antibody or its antigen-binding moiety induces fewer adverse reactions due to efficacy at lower doses. In some embodiments, the sweeping antibody or its antigen-binding moiety is administered at a lower frequency. In some embodiments, transcellular transport of the sweeping antibody or its antigen-binding moiety to certain tissue types is increased. In some embodiments, the sweeping antibody or its antigen-binding moiety enhances the efficiency of transplacental delivery. In some embodiments, the sweeping antibody or its antigen-binding moiety is less expensive to produce.

[0207] In some embodiments, any of the antibodies or antigen-binding fragments provided herein are engineered to bind to FcRn with low affinity. In some embodiments, any of the antibodies or antigen-binding fragments provided herein are engineered to bind to FcRn with low affinity at pH 7.4. In some embodiments, the affinity of the sweeping antibody or its antigen-binding moiety to FcRn is reduced to shorten their pharmacokinetic (PK) properties compared to their conventional counterparts. For example, in some embodiments, the sweeping antibody or its antigen-binding moiety is eliminated more rapidly with respect to imaging and / or radioimmunotherapy. In some embodiments, the sweeping antibody or its antigen-binding moiety facilitates the clearance of endogenous pathogenic antibodies as a treatment for autoimmune diseases. In some embodiments, the sweeping antibody or its antigen-binding moiety reduces 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 its antigen-binding moiety exhibits decreased affinity for the antigen at pH levels lower than neutral or physiological pH (e.g., pH 7.4). In some embodiments, the sweeping antibody or its antigen-binding moiety exhibits decreased affinity for the antigen at acidic pH levels (e.g., pH levels ranging from 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 provided herein may be engineered to dissociate from the antigen in response to pH changes (e.g., pH-sensitive antibodies). In some embodiments, the sweeping antibody or its antigen-binding moiety provided herein is engineered to bind to the antigen in a pH-dependent manner. In some embodiments, the sweeping antibody or its antigen-binding moiety provided herein is engineered to bind to FcRn in a pH-dependent manner. In some embodiments, the sweeping antibody or its antigen-binding moiety provided herein is internally transported by endocytosis. In some embodiments, the sweeping antibody or its antigen-binding moiety provided herein is internally transported by FcRn binding. In some embodiments, the endocytized sweeping antibody or its antigen-binding moiety releases the antigen into the endosome. In some embodiments, the sweeping antibody or its antigen-binding moiety is returned to the cell surface and recirculated. In some embodiments, the sweeping antibody remains bound to the cell. In some embodiments, the endocytized sweeping antibody or its antigen-binding moiety is returned to the plasma and recirculated. It should be understood that the Fc portion of any of the antibodies or antigen-binding fragments provided herein may be engineered to have different FcRn binding activities. In some embodiments, the FcRn binding activity affects the antigen clearance time by the sweeping antibody. In some embodiments, the sweeping antibody may be a long-acting or rapid-acting sweeping antibody.

[0210] In some embodiments, converting a conventional therapeutic antibody or its antigen-binding portion to a sweeping antibody or its antigen-binding portion reduces the effective dose. In some embodiments, converting a conventional therapeutic antibody or its antigen-binding portion to a sweeping antibody or its antigen-binding portion 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 its antigen-binding portion to a sweeping antibody or its antigen-binding portion reduces the effective dose by at least 1 / 1.5, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 8, 1 / 10, 1 / 15, 1 / 20, 1 / 50, or 1 / 100.

[0211] In some embodiments, selecting an appropriate dose of the sweeping antibody or its antigen-binding moiety for therapeutic purposes may be done empirically. In some embodiments, high doses of the sweeping antibody or its antigen-binding moiety may saturate the FcRn, producing an antibody that stabilizes the antigen in the serum without internal migration. In some embodiments, low doses of the sweeping antibody or its antigen-binding moiety may not be therapeutically effective. In some embodiments, the sweeping antibody or its antigen-binding moiety is administered once daily, once weekly, 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 be sweeping antibodies. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein may be converted to sweeping antibodies using any suitable method. For example, suitable methods for generating sweeping antibodies or antigen-binding portions thereof are described previously in Igawa et al., (2013) "Engineered Monoclonal Antibody with Novel Antigen-Sweeping Activity In Vivo", PLoS ONE 8(5):e63236; and Igawa et al., "pH-dependent antigen-binding antibodies as a novel therapeutic modality", Biochimica et Biophysica Acta 1844(2014)1943-1950, the contents of each of which are incorporated herein by reference. However, it should be understood that the methods for generating the sweeping antibodies or antigen-binding portions thereof provided herein are not meant to be limiting. Thus, additional methods for generating sweeping antibodies or antigen-binding portions thereof are within the scope of the present 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 a relatively low pH (e.g., a pH in the range of 4.0-6.5) compared to a relatively high pH (e.g., a pH in the range of 7.0-7.4). In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have a Kd for binding to pro / latent myostatin that is increased by a factor of 10 when the pH is between 4.0 and 6.5 -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10-8 It has a Kd for binding to pro / latent myostatin in the range of M. 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 -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 at a pH between 4.0 and 6.5 compared to a pH between 7.0 and 7.4.

[0214] In some embodiments, pro / latent myostatin antibodies or antigen-binding fragments thereof that do not specifically bind to the epitopes in the amino acid sequences described in (SEQ ID NO: 64) are provided herein. In some embodiments, the pro / latent myostatin antibodies or antigen-binding fragments provided herein do not specifically bind to the same epitopes as the antibodies described in Tables 2a, 11a, 11b, or 13 of International Patent Application Publication WO2016 / 098357, published on June 23, 2016, based on International Patent Application PCT / JP2015 / 006323 filed on December 18, 2015. In some embodiments, the pro / latent myostatin antibodies or their antigen-binding fragments provided herein do not compete or cross-compete with the antibodies described in Tables 2a, 11a, 11b, or 13 of International Patent Application Publication WO2016 / 098357, published on June 23, 2016, based on International Patent Application PCT / JP2015 / 006323, filed on December 18, 2015, for binding to the same epitopes. In some embodiments, the pro / latent myostatin antibodies or their antigen-binding fragments provided herein do not specifically bind to the same epitopes as the antibodies containing the VH and VL pairs described in Tables 2a, 11a, 11b, or 13 of International Patent Application Publication WO2016 / 098357, published on June 23, 2016, based on International Patent Application PCT / JP2015 / 006323, filed on December 18, 2015. In some embodiments, the pro / latent myostatin antibodies or their antigen-binding fragments provided herein do not compete or cross-compete with the antibodies containing the VH and VL pairs described in Tables 2a, 11a, 11b, or 13 of International Patent Application Publication WO2016 / 098357, published on June 23, 2016, based on International Patent Application PCT / JP2015 / 006323 filed on December 18, 2015, for binding to the same epitopes.

[0215] Polypeptide Some aspects of this disclosure relate to polypeptides having sequences selected from the group consisting of SEQ ID NOs: 24, 25, 26, 27, 28, and 29. In some embodiments, the polypeptide is a variable heavy chain domain. In some embodiments, the polypeptide is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of the amino acid sequences described in SEQ ID NOs: 24, 25, 26, 27, 28, or 29.

[0216] Some aspects of this disclosure relate to polypeptides having sequences selected from the group consisting of SEQ ID NOs: 30, 31, 32, 33, 34, and 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 described in SEQ ID NOs: 30, 31, 32, 33, 34, or 35.

[0217] Antibodies or antigen-binding fragments that compete with antipro / latent myostatin antibodies or their antigen-binding fragments Aspects of this disclosure relate to antibodies or antigen-binding fragments thereof that compete with or cross-compete with any of the antibodies or antigen-binding fragments provided herein. The term “compete” as used herein with respect to antibodies means that the first antibody binds to a protein epitope (e.g., latent myostatin) in a manner sufficiently similar to the binding of the second antibody, and the result of the binding of the first antibody to that epitope is detectably reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. Alternatively, the binding of the second antibody to its epitope may also be detectably reduced in the presence of the first antibody, but this is not required. That is, the first antibody can inhibit the binding of the second antibody to its epitope, and the second antibody can inhibit the binding of the first antibody to its respective epitope without inhibiting the binding of the first antibody to its respective epitope. However, if each antibody detectably inhibits the binding of other antibodies to its epitope or ligand, then the antibodies can be said to “cross-compete” with each other for the binding of their respective epitopes, whether to the same, greater, or lesser degree. Both competing and cross-competing antibodies are within the scope of this disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), those skilled in the art will understand that such competing and / or cross-competing antibodies are included and may be useful in the methods and / or compositions provided herein.

[0218] Aspects of this disclosure relate to antibodies or antigen-binding moieties thereof that compete or cross-compete with any of the antibodies or antigen-binding fragments provided herein. In some embodiments, the antibody or antigen-binding moiety binds to the same epitope as or near any of the antibodies provided herein. In some embodiments, the antibody or antigen-binding moiety 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 provided herein bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the epitope to which any of the antibodies or antigen-binding fragments provided herein bind.

[0219] In another embodiment, the antibody or its antigen-binding portion is 10 -6 The antibody and protein compete or cross-compete for binding to any of the antigens provided herein (e.g., pro / latent myostatin) with an equilibrium dissociation constant Kd of less than M. In other embodiments, the antibody or its antigen-binding moiety is 10 -11 M to 10 -6 Kd in the M range compete or cross-compete for binding to any of the antigens provided herein.

[0220] Aspects of the present 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 antibody or antigen-binding portion thereof binds pro / latent myostatin at the same epitope as any of the antibodies or antigen-binding portions thereof provided herein. For example, in some embodiments, any of the antibodies provided herein binds to or near the toroid cleavage site of pro / latent myostatin or to or near the toroid docking site. In other embodiments, any of the antibodies or antigen-binding fragments thereof provided herein binds to or near the proprotein convertase cleavage site of pro / latent myostatin or to or near the proprotein convertase docking site. In another embodiment, the antibody or antigen-binding portion thereof competes for binding to pro / latent myostatin with an equilibrium dissociation constant Kd between the antibody or antigen-binding portion thereof less than 10 -6 M. In other embodiments, the antibody or antigen-binding portion thereof that competes with any of the antibodies or antigen-binding portions thereof provided herein binds pro / latent myostatin with a Kd in the range of 10 -11 M to 10 -6 M.

[0221] Any antibody or antigen-binding fragment provided herein may be characterized using any preferred method. One such method is to identify the epitope to which the antigen binds, i.e., “epitope mapping.” There are numerous preferred methods for mapping and characterizing the location of epitopes on proteins, including analyzing the crystal structure of antibody-antigen complexes, competitive assays, gene fragment expression assays, and synthetic peptide-based assays, as described in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In additional examples, epitope mapping may be used to determine the sequence to which an antibody or its antigen-binding moiety binds. Epitopes may be linear epitopes, i.e., they may be contained in a single stretch of amino acids, or they may be conformational epitopes formed by three-dimensional interactions of amino acids, which do not necessarily have to be contained in a single stretch (a linear primary structure sequence). Peptides of varying lengths (e.g., at least 4-6 amino acids) may be isolated or synthesized (e.g., recombinantly) and used for antibody binding assays. In another example, the epitope to which an antibody or its antigen-binding moiety binds may be determined in a systematic screening by using duplicate peptides derived from the target antigen sequence and determining antibody binding. In gene fragment expression assays, the open reading frame encoding the target antigen is fragmented either randomly or by specific genetic construction, and the reactivity of the expressed antigen fragment with the antibody being tested is determined. The gene fragment may be produced, for example, by PCR, and then transcribed and translated into a protein in vitro in the presence of radioactive amino acids. Binding of the antibody or its antigen-binding moiety to the radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes may also be identified using a large library of random peptide sequences presented on the surface of phage particles (phage libraries).Alternatively, a defined library of duplicate peptide fragments can be tested for binding to a test antibody or its antigen-binding moiety in a simple binding assay. In additional examples, antigen-binding domain mutagenesis, domain exchange experiments, and alanine scanning mutagenesis may be performed to identify sufficient and / or required residues for epitope binding. For example, domain exchange experiments may be performed using variants of target antigens in which various fragments of pro / latent myostatin polypeptides are replaced (exchanged) with sequences derived from closely related but antigenically different proteins, such as another member of the TGFβ protein family (e.g., GDF11). The importance of a particular antigen fragment to the binding of the antibody or its antigen-binding moiety can be assessed by evaluating the binding of the variant pro / latent myostatin to the antibody or its antigen-binding moiety.

[0222] Alternatively, a competitive assay may be performed using another antibody known to bind to the same antigen to determine whether an antibody or its antigen-binding moiety binds to the same epitope as another antibody or its antigen-binding moiety. Competitive assays are well known to those skilled in the art.

[0223] Any preferred method, such as the epitope mapping method described herein, may be applied to determine whether an anti-pro / latent myostatin antibody or its antigen-binding moiety binds to one or more specific residues / segments in pro / latent myostatin as described herein. Furthermore, the interaction between the antibody or its antigen-binding moiety and one or more of these defined residues in pro / latent myostatin may be determined by routine techniques. For example, the crystal structure can be determined, and the distance between the residues in pro / latent myostatin and one or more residues in the antibody or its antigen-binding moiety can be determined accordingly. Based on such distances, it may be determined whether a particular residue in pro / latent myostatin interacts with one or more residues in the antibody or its antigen-binding moiety. In addition, preferred methods such as competitive assays and targeted mutagenesis assays may be applied to determine the preferential binding of a candidate anti-pro / latent myostatin antibody or its antigen-binding moiety to pro / latent myostatin compared to another target, such as a mutant pro / latent myostatin.

[0224] Production of anti-pro / latent myostatin antibodies or their antigen-binding fragments Numerous methods can be used to obtain the antibodies or antigen-binding fragments thereof of this disclosure. For example, antibodies and their antigen-binding fragments can be produced using recombinant DNA methods. Monoclonal antibodies and their antigen-binding fragments can also be produced by hybridoma generation by known methods (see, e.g., Kohler and Milstein (1975) Nature, 256: pp. 495-499). Hybridomas formed in this manner are then screened using standard methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (e.g., OCTET or BIACORE) analysis to identify one or more hybridomas that produce antibodies or antigen-binding moieties that specifically bind to a particular antigen. Any form of a particular antigen, e.g., recombinant antigen, naturally occurring form, any variant or fragment thereof, and its antigenic peptide (e.g., any of the epitopes described herein in the scaffold, either as a linear epitope or a conformational epitope) can be used as immunogens. One exemplary method for producing antibodies and their antigen-binding moieties involves screening protein expression libraries, such as phages or ribosome display libraries, that express antibodies or fragments thereof (e.g., scFv). Phage displays are described, for example, by Ladner et al., U.S. Patent No. 5,223,409; Smith (1985), Science Vol. 228: pp. 1315-1317; Clackson et al., (1991), Nature, Vol. 352: pp. 624-628; Marks et al., (1991), J.Mol.Biol., Vol. 222: pp. 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, specific antigens (e.g., promyostatin) may be used to immunize non-human animals, such as rodents, such as mice, hamsters, or rats. 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 preferred recombinant DNA techniques (e.g., chimeric). Various approaches for producing chimeric antibodies have been described. See, for example, Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent Publication No. EP171496, European Patent Publication No. 0173494, and UK Patent No. GB2177096B.

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

[0228] Some aspects of this disclosure relate to host cells transformed with polynucleotides or vectors. The host cells may be prokaryotic or eukaryotic cells. The polynucleotides or vectors present in the host cells may be integrated into the host cell's genome or maintained extrachromosomally. The host cells may be any prokaryotic or eukaryotic cells, such as bacterial, insect, fungal, plant, animal, or human cells. In some embodiments, fungal cells are, for example, those of the genus Saccharomyces, specifically the species S. cerevisiae. The term “prokaryote” includes all bacteria that can be transformed or transfected with DNA or RNA molecules for the expression of antibodies or corresponding immunoglobulin chains. Hosts of prokaryotes may include Gram-negative and Gram-positive bacteria, such as E. coli, S. typhimurium, Serratia marcescens, and Bacillus subtilis. The term “eukaryote” includes yeast, higher plants, insects, and vertebrate cells, such as mammalian cells, such as NSO and CHO cells. Depending on the host used in the recombinant production procedure, the antibody or immunoglobulin chain encoded by the polynucleotide may or may not be glycosylated. The antibody or corresponding immunoglobulin chain may also contain an initiating methionine amino acid residue.

[0229] In some embodiments, once the vector is incorporated into a suitable host, the host may be maintained under conditions favorable for high-level expression of the nucleotide sequence, and optionally, recovery and purification of immunoglobulin light chains, heavy chains, light / heavy chain dimers or intact antibodies, antigen-binding fragments or other immunoglobulin forms may follow; see Beychok, Cells of Immunoglobulin Synthesis, Academic Press, NY, (1979). Thus, the polynucleotide or vector is then introduced into cells that produce antibodies or antigen-binding fragments. Furthermore, transgenic animals, preferably mammals, including the aforementioned host cells, may be used for large-scale production of antibodies or antibody fragments.

[0230] Transformed host cells may be grown in a fermenter and cultured using any suitable technique to achieve optimal cell growth. Once expressed, the 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, and gel electrophoresis; 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. For example, the isolation and purification of antibodies or antigen-binding fragments expressed in microorganisms may be by any conventional means, such as chromatographic separation for preparation and immunological separation, such as using monoclonal or polyclonal antibodies directed against the constant region of the antibody.

[0231] Aspects of this 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 manipulation. The rearranged antibody gene may be reverse transcribed from appropriate mRNA to produce cDNA. In some embodiments, the heavy chain constant region may be replaced with one of a different isotype or removed entirely. The variable region may be ligated to encode a single-stranded Fv region. Multiple Fv regions may be ligated to confer the ability to bind to more than one target, or chimeric heavy and light chain combinations may be used. Any suitable method may be used for cloning the antibody variable region and generating recombinant antibodies and their antigen-binding portions.

[0232] In some embodiments, suitable nucleic acids encoding the variable regions of the heavy and / or light chains are obtained and inserted into an expression vector that can be transfected into a standard recombinant host cell. Various such host cells may be used. In some embodiments, mammalian host cells may be advantageous for efficient processing and production. Typical mammalian cell lines useful for this purpose include CHO cells, 293 cells, or NSO cells. Production of antibodies or antigen-binding fragments may be carried out by culturing the modified recombinant host under culture conditions suitable for host cell proliferation and expression of the coding sequence. Antibodies or antigen-binding fragments can be recovered by isolating them from the culture. The expression system may be designed to include a signal peptide so that the resulting antibody is secreted into the culture medium; intracellular production is also possible.

[0233] This disclosure also includes polynucleotides encoding at least the variable region of the immunoglobulin chain of the antibodies described herein. In some embodiments, the variable region encoded by the polynucleotide includes at least one complementarity-determining region (CDR) of the VH and / or VL of the variable region of the antibody 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 a recombinantly produced chimeric nucleic acid molecule containing any of these polynucleotides, either alone or in combination, synthetically produced DNA or RNA. In some embodiments, the polynucleotide is part of the vector. Such a vector may contain further genes, such as marker genes, that enable vector selection in a suitable host cell and under suitable conditions.

[0235] In some embodiments, polynucleotides are operably ligated to expression regulatory sequences that enable expression in prokaryotic or eukaryotic cells. Polynucleotide expression involves transcription of the polynucleotide into translatable mRNA. Regulatory elements that ensure expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They may include regulatory sequences that promote transcription initiation and, optionally, polyA signals that promote transcription termination and transcript stabilization. Additional regulatory elements may include transcription and translation enhancers, and / or naturally occurring related or heterologous promoter regions. Regulatory elements that may enable expression in prokaryotic host cells include, for example, the PL, Lac, Trp, or Tac promoters in E. coli, while examples of regulatory elements that enable expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV promoter, SV40 promoter, RSV promoter (Roussarcoma virus), CMV enhancer, SV40 enhancer, or globin intron in mammalian and other animal cells.

[0236] Such regulatory elements other than those involved in transcription initiation may also include transcription termination signals downstream of the polynucleotide, such as the SV40 polyA site or the tk polyA site. Furthermore, depending on the expression system used, a leader sequence capable of directing the polypeptide into a cellular compartment or secreting it into the culture medium may be added to the polynucleotide coding sequence, as previously described. The leader sequence(s) are assembled with translation, start, and stop sequences in appropriate phases, and preferably the leader sequence can lead to the secretion of the translated protein or a portion thereof into, for example, an extracellular culture medium. Heterogeneous polynucleotide sequences encoding a fusion protein containing a C or N-terminal discriminant peptide that provides desirable features, such as stabilization of the recombinant product being expressed or simplification of purification, may be optionally used.

[0237] In some embodiments, the polynucleotide encoding at least one variable domain of the light chain and / or heavy chain may encode both immunoglobulin chains or just one variable domain. Similarly, the polynucleotides may be under the regulation of the same promoter or may be regulated separately for expression. Further embodiments relate to vectors conventionally used in genetic engineering, particularly plasmids, cosmids, viruses and bacteriophages, comprising a polynucleotide encoding a variable domain of an immunoglobulin chain of an antibody or antigen-binding fragment in combination with a polynucleotide optionally encoding a variable domain of another immunoglobulin chain of the antibody.

[0238] In some embodiments, the regulatory expression sequence is provided as a eukaryotic promoter system in a vector that can transform or transfect eukaryotic host cells, although regulatory sequences for prokaryotic hosts may also be used. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpesviruses, or bovine papillomavirus may be used to deliver polynucleotides or vectors to a targeted cell population (e.g., to manipulate cells to express antibodies or antigen-binding fragments). Various suitable methods may be used to construct recombinant viral vectors. In some embodiments, polynucleotides and vectors may be reconstituted into liposomes for delivery to target cells. Vectors containing polynucleotides (e.g., heavy and / or light chain variable domains of immunoglobulin chains encoding sequences and regulatory expression sequences) may be transferred to host cells by a preferred method that varies depending on the type of cell host.

[0239] qualification The antibodies and antigen-binding fragments of this disclosure may be modified with detectable labels, including but not limited to enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, non-radioactive paramagnetic metal ions, and affinity labels, for the detection and isolation of pro / latent myostatins. The detectable substances may be coupled or conjugated either directly to the polypeptides of this disclosure or indirectly through intermediates (e.g., linkers) using preferred 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; examples of luminescent materials include luminol; non-limiting examples of bioluminescent materials include luciferase, luciferin, and aequorin; examples of suitable radioactive materials include, 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 Enjoy, 57 Co, 65 Zn, 85 Sr, 32P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se and tin ( 113 Sn, 117 The detectable substance may be directly coupled or conjugated to the antipro / latent myostatin antibody or its antigen-binding moiety, or indirectly through an intermediate (e.g., a linker) using a preferred technique. The antipro / latent myostatin antibody or its antigen-binding moiety conjugated to the detectable substance may be used for the diagnostic assays described herein.

[0240] The biological effects of myostatin inhibitors, such as anti-pro / latent myostatin antibodies and their antigen-binding fragments. Myostatin inhibitors included in this disclosure, such as antibodies and their antigen-binding fragments, may be used as pharmaceuticals to produce beneficial effects (e.g., therapeutic effects) in a subject when administered in an effective amount to the subject. Exemplary such biologically beneficial effects are provided herein. Beneficial biological effects in a subject can be achieved by administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment described herein that specifically binds to pro / latent myostatin. In some embodiments, the antibody or its antigen-binding moiety is administered in an effective amount to produce two or more of the following biological effects. In some embodiments, the myostatin inhibitor, such as an antibody or its antigen-binding moiety, is administered in an effective amount to produce three or more of the following biological effects. In some embodiments, the myostatin inhibitor, such as an antibody or its antigen-binding moiety, is administered in an effective amount to produce four or more of the following biological effects. In some embodiments, the myostatin inhibitor, such as an antibody or its antigen-binding moiety, is administered in an effective amount to produce five or more of the following biological effects. In some embodiments, a myostatin inhibitor, such as an antibody or its antigen-binding moiety, is administered in an amount effective to produce six or more of the following biological effects. In some embodiments, a myostatin inhibitor, such as an antibody or its antigen-binding moiety, is administered in an amount effective to produce seven or more of the following biological effects. In some embodiments, a myostatin inhibitor, such as an antibody or its antigen-binding moiety, is administered in an amount effective to produce eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen of the following biological effects.

[0241] A. Effects on muscle tissue volume and / or function in human subjects Administration of myostatin inhibitors, such as antibodies or antigen-binding fragments that specifically bind to pro / latent myostatin, increases the volume and / or function of muscle tissue in human subjects. In some embodiments, muscle tissue is selected from the group consisting of smooth muscle tissue, skeletal muscle tissue, and cardiomyocyte tissue. Smooth muscle tissue consists of long, tapering cells, is generally involuntary, and differs from striated muscle by having a much higher actin / myosin ratio, the absence of prominent sarcomeres, and the ability to contract to only a fraction of its resting length. Smooth muscle cells are found particularly in the walls of blood vessels, around the intestines, and in the uterus. Cardiomyocyte tissue is striated muscle, but is an involuntary tissue involved in the pumping activity of the heart in vertebrates. Individual cardiomyocytes are in striated muscle tissue and do not fuse to form a multinucleated structure. Skeletal muscle tissue is under voluntary control. Muscle fibers are syncytia and contain tandem arrays of myofibrils and sarcomeres. Skeletal muscle fibers are classified into two general types, slow-twitch (Type I) and fast-twitch (Type II), according to the expression of their specific myosin heavy chain (MHC) isoforms. Slow-twitch fibers are better equipped to work aerobically and help enable long-distance endurance, such as in long-distance running, while fast-twitch fibers fatigue more quickly but are better equipped to work anaerobically and are used for powerful, sudden movements such as in sprinting. The distinction between slow-twitch and fast-twitch fibers is based on histochemical staining for myosin-adenosine triphosphatase (ATPase) and the type of myosin heavy chain. Slow-twitch fibers (Type I fibers) have MHC isoform I, and the three fast-twitch isoforms (Type II fibers) are MHC isoform IIa, MHC isoform IId, and MHC isoform IIb (S. Schiaffino, J. Muscle Res. Cell. Motil., Vol. 10 (1989), pp. 197-205). In some embodiments, the volume and / or function of fast-twitch muscle tissue in human subjects is increased. In other embodiments, the volume and / or function of slow-twitch muscle tissue in human subjects is increased.

[0242] The biological effects of effective amounts of the pharmaceutical compositions provided herein may be related to a change in the phenotype of muscle fibers, a process referred to as fiber type switching. In some embodiments, fiber type switching is induced by events such as injury and starvation.

[0243] In one embodiment, the present disclosure provides a method for promoting fibrillation switching in a subject. The method comprises administering to a subject a composition comprising a myostatin inhibitor, for example, an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin and blocks the release of mature myostatin, in an amount effective to promote fibrillation switching, thereby promoting fibrillation switching in the subject.

[0244] In another aspect, the present disclosure provides a method for preferentially increasing type II or fast-twitch muscle fibers over type I or slow-twitch muscle fibers in a subject. The method comprises administering to a subject a composition comprising a myostatin inhibitor, for example, an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin and blocks the release of mature myostatin, in an amount effective to preferentially increase type II or fast-twitch muscle fibers over type I or slow-twitch muscle fibers, thereby preferentially increasing type II or fast-twitch muscle fibers over type I or slow-twitch muscle fibers in the subject.

[0245] In some embodiments, an increase in muscle mass can be induced by administering an effective dose to a subject of a myostatin inhibitor, such as an antibody or antigen-binding fragment described herein. Preferably, such an increase in muscle mass is clinically significant for benefiting or otherwise improving the subject's health. For example, a clinically significant change in muscle mass may improve a patient's mobility, self-care, metabolism, etc. In some embodiments, the increase in muscle mass is an increase in lean muscle(s). In some embodiments, such an increase in muscle mass is a systemic effect, and therefore the whole or substantially whole muscle exhibits a measurable effect. In other embodiments, the effect is localized to a specific group / type of muscle. In some embodiments, the amount of muscle tissue, such as lean muscle tissue, 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 amount of muscle tissue, such as lean muscle tissue, 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%. Such increases in muscle mass can be estimated or measured by any preferred known method, including measurement of cross-sectional area by MRI (e.g., forearm cross-section), circumference, diaphragm width (e.g., by ultrasound).

[0246] In some embodiments, enhancement of muscle function can be induced by administering an effective amount of the antibody or antigen-binding fragment described herein to a subject. Muscle function can be assessed by various measures, not limited to force generation, grip strength (e.g., maximum grip strength), endurance, oxidative capacity of muscle, and dynamic grip endurance. In some embodiments, serum creatinine levels are used as a validated biomarker indicating muscle mass, although with limited sensitivity.

[0247] In some embodiments, muscle tissue function 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, muscle tissue function 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 some embodiments, the increase in muscle function includes, for example, an improvement in the score from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.

[0248] In some embodiments, myostatin inhibitors for use in the methods of the present invention, such as anti-pro / latent myostatin antibodies or their antigen-binding fragments, may increase the volume and / or function of muscle tissue in subjects suffering from lesions resulting from spinal cord injury, for example. In some embodiments, the subject is in the acute spinal cord injury phase immediately after injury, which is generally difficult to diagnose between complete and incomplete injury. In other embodiments, the subject is in the subacute spinal cord injury phase, where there is a difference between complete and incomplete spinal cord injury and recovery is possible with continued rehabilitation. In yet another embodiment, the subject is in the chronic spinal cord injury phase. The chronic SCI phase occurs approximately four or six months from the date of injury, when the patient shows a substantial decrease in the rate of recovery or when attempts at rehabilitation reach a plateau despite continued attempts at standard treatment.

[0249] In some embodiments, the volume and / or function of muscle tissue beneath a lesion is increased in a subject suffering from a lesion, such as a spinal cord injury. In other embodiments, the volume and / or function of muscle tissue above a lesion is increased in a subject suffering from a lesion, such as a spinal cord injury. In some embodiments, the muscle is selected from the group consisting of the soleus, gastrocnemius, biceps, and triceps. In some embodiments, the volume 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 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 some embodiments, the function of the muscle tissue 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 function 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%.

[0250] In some embodiments, spontaneous motor function is increased in human subjects, for example, subjects suffering from a lesion, by administration of a myostatin inhibitor, such as an antibody or antigen-binding fragment that specifically binds to pro / latent myostatin. In some embodiments, spontaneous motor function in human subjects 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 spontaneous motor function of human subjects is increased by at least approximately 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, in human subjects, for example, subjects suffering from a lesion, administration of a myostatin inhibitor, such as an antibody or antigen-binding fragment that specifically binds to pro / latent myostatin, increases motor coordination and equilibrium. In some embodiments, motor coordination and equilibrium in human subjects increase 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 equilibrium of human subjects 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%.

[0252] In another embodiment, muscle strength is increased in human subjects, for example, subjects suffering from a lesion, by administration of a myostatin inhibitor, such as an antibody or antigen-binding fragment that specifically binds to pro / latent myostatin. In some embodiments, the muscle strength of human subjects 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 human subjects 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%.

[0253] In some embodiments, administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment that specifically binds to pro / latent myostatin, may induce clinically meaningful changes in muscle function corresponding to enhanced functionality in the patient. In some embodiments, enhanced functionality includes improvements in the patient's mobility, self-care, metabolism, etc. In some embodiments, administration of an effective dose of a myostatin inhibitor, such as an antibody or its antigen-binding fragment that specifically binds to pro / latent myostatin, may facilitate or accelerate recovery from conditions such as injury, surgery, and other medical procedures. Appropriate such conditions may include conditions related to neurological disorders (whether resulting from injury or from surgery or other medical procedures).

[0254] For example, suitable subjects include generally healthy individuals, e.g., i) patients with persistent acute injury accompanied by nerve damage 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 involving immobilization of specific muscles or muscle groups (e.g., cast immobilization); and v) patients in a state of 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, antibodies may be administered before or immediately after a surgical procedure that may cause nerve damage and associated muscle dysfunction to prevent muscle dysfunction. Prevention includes mitigating or reducing the severity of such dysfunction. In these embodiments, administration may be a local administration to or near the affected area, e.g., injury, surgery, etc.

[0255] B. Effects on the metabolic rate of human subjects Administration of a myostatin inhibitor, such as an antibody or its antigen-binding fragment that specifically binds to pro / latent myostatin, increases the metabolic rate of a human subject. In some embodiments, administering an effective dose of such a myostatin inhibitor, such as an antibody or its antigen-binding fragment, can increase the basal metabolic rate of a subject. The metabolic rate can be calculated by any method known in the art, for example, by examining oxygen input and carbon dioxide output, or by an indirect calorimetry method demonstrated in Example 11 of this application. In some embodiments, the metabolic rate 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 metabolic rate 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%.

[0256] C. Effects on insulin sensitivity in human subjects Insulin sensitivity in human subjects is increased by the administration of myostatin inhibitors, such as antibodies or antigen-binding fragments that specifically bind to pro / latent myostatin. Methods for measuring insulin sensitivity are known in the art, and include, for example, glucose tolerance tests and fasting insulin or glucose tests. During a glucose tolerance test, a fasted patient ingests 75 grams of oral glucose, and then blood glucose levels are measured over the following two hours. A blood glucose level of less than 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. Regarding fasting insulin tests, a fasting serum insulin level greater than 25 mIU / L or 174 pmol / L is considered insulin resistance. In some embodiments, the metabolic rate 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 metabolic rate 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%.

[0257] D. Effects on adipose tissue levels in human subjects Administration of myostatin inhibitors, such as antibodies or antigen-binding fragments that specifically bind to pro / latent myostatin, affects the level of adipose tissue in human subjects. As used herein, the term “adipose tissue” refers to fat, including connective tissue that stores fat. Adipose tissue originates from preadipocytes. Its primary role is to store energy in the form of lipids, but it also cushions and isolates the body from impact. There are two types of adipose tissue: 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 temperatures or excessive feeding, and also possesses the ability to regulate energy equilibrium. Activation of brown adipose tissue has been shown to improve glucose homeostasis and insulin sensitivity in humans, suggesting that BAT activation may be beneficial for anyone with insulin dysfunction (Stanford et al., J Clin Invest., 2013, Vol. 123 (No. 1): pp. 215-223).

[0259] Beige adipose tissue, also known as beiging, arises as a result of browning of WAT. This occurs when BAT characteristics develop in adipocytes within the WAT storage area. Beige adipocytes take on a multilocular 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, Vol. 19 (No. 10): pp. 1252-1263).

[0260] Visceral fat, or abdominal fat (also known as organ fat or intraperitoneal fat), is located inside the abdominal cavity and fills the spaces between organs (such as the stomach, liver, intestines, and kidneys). Visceral fat is distinct from subcutaneous fat, which is located beneath the skin, and intramuscular fat, which is scattered within skeletal muscle. Fat in the lower body, such as the thighs and buttocks, is subcutaneous fat and is not consistently spaced tissue, while abdominal fat is mostly visceral fat and is semi-fluid. Excessive visceral fat is known as central obesity, or "belly fat," characterized by an excessively protruding abdomen, and newer developments such as the Body Volume Index (BVI) have been specifically designed to measure abdominal volume and abdominal fat. Excessive visceral fat is also associated with type 2 diabetes, insulin resistance, inflammatory diseases, and other obesity-related conditions (Mokdad et al., JAMA: The Journal of the American Medical Association, 2001, Vol. 289 (No. 1): pp. 76-79).

[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 absorptometry (DXA), as demonstrated in Example 11 of this application.

[0262] Administration of myostatin inhibitors, such as antibodies that specifically bind to pro / latent myostatin or their antigen-binding fragments, increases the levels of brown adipose tissue and / or beige adipose tissue in human subjects. Conversely, administration of myostatin inhibitors, such as anti-pro / latent myostatin antibodies or their antigen-binding moieties, decreases the levels of white adipose tissue and visceral adipose tissue in human subjects.

[0263] In some embodiments, the level of brown adipose tissue or beige adipose tissue 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 level of brown adipose tissue or beige adipose tissue 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%.

[0264] In some embodiments, the level of white adipose tissue or visceral adipose tissue decreases 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 or visceral adipose tissue decreases 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 the fat-to-muscle tissue ratio in human subjects Administration of a myostatin inhibitor, such as an antibody or antigen-binding fragment that specifically binds to pro / latent myostatin, reduces the fat-to-muscle ratio in human subjects. In some embodiments, the fat-to-muscle ratio 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 fat-to-muscle ratio 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 myostatin inhibitors, such as antibodies or antigen-binding fragments that specifically bind to pro / latent myostatin, also increases the muscle-to-fat ratio in human subjects. In some embodiments, the muscle-to-fat ratio 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-to-fat ratio 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%.

[0267] F. Effects on glucose uptake in human subjects Administration of myostatin inhibitors, such as antibodies or antigen-binding fragments that specifically bind to pro / latent myostatin, affects glucose uptake by tissues in human subjects. In some embodiments, glucose uptake by muscle tissue increases. For example, glucose uptake by muscle tissue 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 some embodiments, glucose uptake by muscle tissue 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%.

[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 protein catabolism in muscle and / or release of amino acids from muscle in human subjects Administration of myostatin inhibitors, such as antibodies or antigen-binding fragments that specifically bind to pro / latent myostatin, reduces protein catabolism and / or release of amino acids from muscle in human subjects. In some embodiments, protein catabolism and / or release of amino acids from muscle 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, protein catabolism in muscle and / or release of amino acids from muscle 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%.

[0270] H. Effects on insulin-dependent blood glucose regulation in human subjects Administration of myostatin inhibitors, such as antibodies or antigen-binding fragments that specifically bind to pro / latent myostatin, increases insulin-dependent blood glucose regulation in human subjects. In some embodiments, insulin-dependent blood glucose regulation 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, insulin-dependent blood glucose regulation 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%.

[0271] I. Effects on intramuscular fat infiltration in human subjects Administration of myostatin inhibitors, such as antibodies or antigen-binding fragments that specifically bind to pro / latent myostatin, reduces intramuscular fat infiltration in human subjects. 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 the quality of life of human subjects Assessment of quality of life in patients with severe or chronic conditions, such as SCI patients, may involve a comprehensive approach to assess various aspects of physical, mental, social, and other parameters. Generally, a higher degree of quality of life is associated with factors such as the availability of assistive technologies; reintegration into the community; lower limb function and walking and / or wheelchair mobility; mental health; severity of neurological and autonomic disorders; pain management; functional independence and self-care; upper limb strength; and spasticity regulation. Administration of antibodies or antigen-binding fragments that specifically bind to pro / latent myostatin increases the quality of life in human subjects to achieve clinically meaningful improvements measured by standardized quality of life tests / systems. Several suitable tests for assessing the quality of life in patients are known in the art, 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 (SIP 68); and World Health Organization Quality of Life-BREF (WHOQOL-BREF).

[0273] In some embodiments, quality of life is assessed according to the validated scoring system, the SF-36 Quality of Life Scoring System, where an 8-point change is considered clinically significant. Generally, for SCI patients, the value is in the lower 50s. In some embodiments, a clinically significant improvement in the standardized quality of life test score is achieved by administering an effective dose of an antibody or its antigen-binding fragment that specifically binds to pro / latent myostatin. As used herein, the term “clinically significant improvement” refers to a significant improvement over the 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 dose of the antibody or its antigen-binding fragment 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, for example, an increase of at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 points in the SF-36 Quality of Life Scoring System score. In other embodiments, the SF-36 Quality of Life Scoring System score increases by at least approximately 8-10, 10-15, 15-20, 20-30, 30-40, 40-50, 8-20, 8-30, 8-40, or 8-50 points.

[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 myostatin signaling inhibitors disclosed herein. The advantages of this test include i) ease of administration; ii) assessment of both physical function and mental health; and iii) high validation for several clinical indications.

[0275] K. Effects on preventing muscle loss or atrophy in human subjects By administering an effective dose of a myostatin inhibitor, such as an antibody or antigen-binding fragment that specifically binds to pro / latent myostatin, muscle loss or atrophy can be prevented in human subjects 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, preferred subjects are those who have not yet developed atrophy but are considered to be at risk of developing it. In some embodiments, subjects have a disease or condition associated with a neurological deficit that impairs motor neuron function. In some embodiments, such a condition is caused by muscular dystrophy or atrophy. In some embodiments, the neurological deficit is caused by nerve injury. In some embodiments, the nerve injury involves partial denervation of motor neurons, resulting in a partial functional deficit in the affected muscle. In some embodiments, such a condition is caused by SCI. In some embodiments, subjects with SCI are in the acute or subacute phase of SCI (e.g., have not yet reached the chronic phase).

[0277] In some embodiments, when a composition comprising an effective amount of the myostatin signaling inhibitor described herein is administered to a group of patients at risk of developing muscle atrophy associated with partial denervation of motor neurons, the composition results in: i) prevention of the onset or exacerbation of muscle atrophy in a statistically significant proportion of the patient group, or ii) a reduction in the severity of muscle atrophy in a statistically significant proportion of the patient group.

[0278] The prevention of muscle loss or atrophy by using myostatin inhibitors, such as the antibodies or antigen-binding fragments described herein, can be readily monitored or evaluated by any suitable method for assessing motor function involving the affected muscles.

[0279] In some embodiments, administering such antibodies in an effective dose also prevents or reduces early-onset axonal polyneuropathy in the affected limb.

[0280] L. Effect on preventing the onset of metabolic diseases in test subjects By administering an effective dose of a myostatin inhibitor, such as an antibody or antigen-binding fragment that specifically binds to pro / latent myostatin, the development of metabolic disease in a subject, such as a human subject, is prevented. 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, preferred subjects are those who have not yet fully developed a metabolic disease but are considered to be at risk of developing such a condition. In some embodiments, subjects have a disease or condition related to muscle dysfunction. In some embodiments, the muscle dysfunction is related to partial denervation of motor neurons, resulting in a partial defect of function in the affected muscle. 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, subjects with SCI are in the acute or subacute phase of SCI (e.g., have not yet reached the chronic phase).

[0282] In some embodiments, when a composition comprising an effective amount of the myostatin signaling inhibitor described herein is administered to a group of patients at risk of developing metabolic disorders associated with muscle dysfunction, the composition may: i) prevent the manifestation or exacerbation of metabolic disorders in a statistically significant proportion of the patient group, or ii) reduce the severity of metabolic disorders in a statistically significant proportion of the patient group.

[0283] In some embodiments, the effects on metabolism may be monitored or measured by insulin resistance, lipid panel / markers (e.g., leptin), and, but not limited to, inflammatory and oxidative stress markers including IL-6, TNF, CRP, total plasma antioxidant status, lipid oxidation, and erythrocyte glutathione peroxidase activity.

[0284] Pharmaceutical composition Myostatin inhibitors, such as the antibodies or antigen-binding fragments described herein, can be formulated into pharmaceutical compositions suitable for administration to human or non-human subjects. Such pharmaceutical compositions may be intended for therapeutic or prophylactic use. One or more myostatin inhibitors, such as anti-pro / latent myostatin antibodies, can be mixed with a pharmaceutically acceptable carrier (excipient), including a buffer, to form a pharmaceutical composition for administration to patients for whom reduction of myostatin signaling in vivo may be beneficial. "Pharmacologically acceptable" means that the carrier must be compatible with the active ingredient of the composition (and preferably able to stabilize the active ingredient) and not harmful to the subject being treated. Examples of pharmaceutically acceptable excipients (carriers), including a buffer, will be apparent to those skilled in the art and have been previously described. See, for example, Remington: The Science and Practice of Pharmacy, 20th edition (2000), Lippincott Williams and Wilkins, eds., KE. Hoover.Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used and include buffers such as phosphates, citrates, or other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than approximately 10 residues) polypeptides; and serum albumin. The excipients may include 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 nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0285] In one embodiment, the pharmaceutical composition described herein contains more than one myostatin inhibitor, for example, more than one antipro / latent myostatin antibody or its antigen-binding moiety that recognizes different epitopes / residues of a target antigen.

[0286] In some embodiments, the pharmaceutical compositions described herein include emulsion-based or lipid-based formulations, such as liposomes containing myostatin inhibitors, for example, anti-pro / latent myostatin antibodies or their antigen-binding moieties, which can be prepared by any suitable method, including those described in Epstein et al., Proc. Natl. Acad. Sci. USA, Vol. 82:p. 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, Vol. 77:p. 4030 (1980); and U.S. Patents No. 4,485,045 and No. 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be produced by reverse-phase evaporation in lipid compositions containing phosphatidylcholine, cholesterol, and PEG-derivativeized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of a specified pore size to obtain liposomes with a desired diameter.

[0287] Myostatin inhibitors, such as anti-pro / latent myostatin antibodies or their antigen-binding moieties, can also be captured in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in microcapsules prepared by, for example, coacervation techniques or interfacial polymerization, such as hydroxymethyl cereal or gelatin microcapsules and poly-(methyl methacrylate (methacylate)) microcapsules, respectively. 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 a sustained-release form. A preferred example of a sustained-release preparation comprises a semipermeable matrix of a solid hydrophobic polymer containing an antibody or its antigen-binding moiety, the matrix being in the form of a molded article, e.g., a film or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol (v nylalcohol)), polylactide lactic acid (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable glycolic acid copolymers such as LUPRON DEPOT (an injectable microsphere composed of glycolic acid lactic 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 can be easily achieved, for example, by filtration through a sterile filtration membrane. Therapeutic antibody compositions are generally placed in containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be pierced by a subcutaneous needle.

[0290] The pharmaceutical compositions described herein may be in the form of tablets, pills, capsules, powders, granules, solutions or suspensions or suppositories for oral, parenteral, rectal, or inhalation or inhalation administration.

[0291] To prepare solid compositions such as tablets, the main active ingredient may be mixed with other pharmaceutical diluents, such as water or non-toxic, pharmaceutically acceptable salts thereof, to form a solid pre-formulation composition containing a conventional tableting component, such as a pharmaceutical carrier, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gum, and a homogeneous mixture of the compound of the Disclosure. When these pre-formulation compositions are referred to as homogeneous, it means that the active ingredient is homogeneously dispersed throughout the composition, thereby allowing the composition to be easily divided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid pre-formulation composition is then divided into the above-described unit dosage forms containing 0.1 mg to about 500 mg of the active ingredient of the Disclosure. Tablets or pills of the novel compositions may be coated or otherwise formulated to provide a dosage form that offers the benefit of long-term action. For example, a tablet or pill may contain an inner dosage and an outer dosage component, the latter in the form of an envelope covering the former. The two components may be separated by an enteric layer, which helps to resist disintegration in the stomach and allows the internal components to pass through the duodenum intact or delay their release. Various materials may be used for such an enteric layer or coating, and such materials include several polymer acids as well as mixtures of polymer acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0292] Suitable surfactants specifically include nonionic 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). The composition containing the surfactant may conveniently contain 0.05 to 5% surfactant, and may be 0.1 to 2.5%. It is understood that other components, such as mannitol or other pharmaceutically acceptable vehicles, may be added as needed.

[0293] Suitable emulsions may be prepared using commercially available lipid emulsions such as Intralipid®, Liposyn®, Infonutrol®, Lipofandin®, and Lipiphysan®. The active ingredient may be dissolved in a pre-mixed 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 phospholipid, soybean phospholipid, or soybean lecithin) with water. It is understood that other components, 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%.

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

[0295] Pharmaceutical compositions for inhalation or inhalation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, as well as powders. Liquid or solid compositions may contain the preferred pharmaceutically acceptable excipients described above. In some embodiments, compositions are administered orally or via nasal respiratory routes for topical or systemic effects.

[0296] Preferably, the composition in a sterile, pharmaceutically acceptable solvent may be sprayed using a gas. The sprayed solution may be inhaled directly from a spraying device, or the spraying device may be attached to a face mask, tent, or intermittent positive airway pressure (PAP) respirator. The solution, suspension, or powder composition may be administered preferably orally or nasally from a device that delivers the formulation in an appropriate manner.

[0297] Subject The pharmaceutical compositions described herein are suitable for administration to human or non-human subjects. Therefore, myostatin inhibitors, such as the anti-pro / latent myostatin antibodies and their antigen-binding moieties described herein, are useful as pharmaceuticals for administration to subjects in whom reduction of myostatin signaling may be beneficial. In some embodiments, suitable subjects may include individuals who, despite being healthy, may benefit from increased muscle mass / function and improved metabolism. In some embodiments, suitable subjects have pre-existing muscle conditions and / or associated metabolic disorders. In some embodiments, suitable subjects are at risk of developing such conditions. In some embodiments, suitable subjects are subjects receiving treatment involving other therapeutic agents intended to address muscle / metabolic conditions but which may have adverse or toxic effects. In some embodiments, subjects are pediatric subjects, e.g., human patients from birth to under 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 involves fast-twitch muscle fibers or muscles rich in fast-twitch muscle fibers; and iii) the subject retains anabolic capacity (e.g., generally a healthy adult with injury) and / or is in a growth phase (e.g., a young child).

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

[0300] The pediatric populations requiring myostatin inhibitors, such as the anti-pro / latent myostatin antibody and its antigen-binding portion described herein, may span the following age ranges: 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, and 12 to 72 months. In some embodiments, the pediatric populations for whom receiving myostatin inhibitors, such as the antibody or antigen-binding fragment described herein, may be suitable and potentially beneficial, may span the following age ranges: 0 to 6 years, 0 to 12 years, 3 to 12 years, and 0 to 17 years. In some embodiments, the age of the group is at least 5 years, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 years. In some embodiments, the children's group may be under 18 years. In some embodiments, the children's group may be (a) at least 5 years and (b) under 18 years.

[0301] The age of the adult population requiring myostatin inhibitors, such as the anti-pro / latent myostatin antibodies and their antigen-binding moieties described herein, may be at least 18 years, for example, at least 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65 years. In some embodiments, the adult population may be under 65 years. In some embodiments, the adult population may be (a) at least 18 years and (b) under 65 years.

[0302] The age of the elderly population requiring myostatin inhibitors, such as the anti-pro / latent myostatin antibodies and their antigen-binding moieties described herein, may be 65 years or older (i.e., ≥65 years), for example, at least 70, 75, or 80 years.

[0303] Human subjects who may benefit from treatment may be human patients who have, are at risk of developing, or are suspected of having, metabolic disorders / disorders related to defects in nerve signaling, such as those listed below. Subjects with pro / latent myostatin-related disorders or disorders may be identified by routine medical examinations, e.g., clinical tests, organ function tests, CT scans, or ultrasound. Subjects suspected of having any of such disorders / disorders may exhibit one or more symptoms of the disorder / disorder. Subjects at risk of a disorder / disorder may have one or more risk factors for that disorder / disorder.

[0304] A control subject as described herein is a subject that provides an appropriate reference for evaluating the effects of a particular treatment or intervention on a test subject or a subject. A 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 requiring treatment with anti-pro / latent myostatin antibodies. Methods for assaying myostatin can be found in Lakshman et al., Molecular and Cell Endocrinology, (2009) Vol. 302: pp. 26-32 (myostatin ELISA), both of which are incorporated herein by reference, and Bergen et al., Skeletal Muscle, (2015) Vol. 5: p. 21 (liquid chromatography with tandem mass spectrometry).

[0306] In some embodiments, methods are provided for improving muscle performance in a subject. The subject may have, may not have, may be at risk of having, 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 the ability of a muscle to contract and / or to apply force (e.g., to an external object). In some embodiments, muscle performance may relate to the ability of a muscle to expend energy. For example, in some embodiments, muscle performance may relate to the ability of a muscle to produce and / or expend adenosine triphosphate (ATP) molecules to facilitate muscle contraction. In some embodiments, muscle performance refers to the ability of a muscle to repeatedly contract over a specific duration. In some embodiments, muscle performance refers to the ability of a muscle to apply force to an object, e.g., to move an object over a measurable distance. In some embodiments, muscle performance refers to the ability of a muscle to apply force to an object over a specific duration (e.g., to move an object over a measurable distance over a specific duration).

[0307] In some embodiments, a myostatin inhibitor, such as the anti-pro / latent myostatin antibody and its antigen-binding moiety described herein, is administered to a subject requiring treatment in an amount sufficient to inhibit in vivo the activation of pro / latent myostatin to active myostatin via proteolysis by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more). In other embodiments, a myostatin inhibitor, such as the antibody or its antigen-binding moiety, 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 the anti-pro / latent myostatin antibody or its antigen-binding moiety described herein, is administered to a subject in whom an increase in muscle mass would be beneficial. In some embodiments, a myostatin inhibitor, such as the anti-pro / latent myostatin antibody or its antigen-binding moiety described herein, is administered to a subject in whom an increase in muscle-to-fat ratio would be beneficial. In some embodiments, a myostatin inhibitor, such as the anti-pro / latent myostatin antibody or its antigen-binding moiety described herein, is administered to a subject in whom an increase in muscle function would be beneficial. In some embodiments, the subject may have, or may not have, a condition associated with decreased muscle mass and / or decreased muscle function, or may be at risk of having, or may not have. In some embodiments, the subject has, or is at risk of having, a condition associated with decreased muscle mass and / or decreased muscle function.

[0309] The method of the present invention further includes the step of selecting a subject. In some embodiments, the subject has or is at risk of developing a muscle condition or disorder. In some embodiments, the subject has or is at risk of developing a metabolic disorder. In some embodiments, the subject has or is at risk of developing a disease or disorder related to defects in nerve signaling.

[0310] Route of administration For carrying out the methods described herein, an effective amount of the pharmaceutical composition described above may be administered to a subject in need of treatment (e.g., a human) via a suitable route such as intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrabursal, intrathecal, oral, inhalation, or topical route, for example, by intravenous administration, e.g., as a bolus or by continuous infusion over a period of time. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations may be sprayed directly, and lyophilized powders may be sprayed after reconstitution. Alternatively, anti-pro / latent myostatin antibodies may be aerosolized using fluorocarbon formulations and metered-dose inhalers, or inhaled as lyophilized and pulverized 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 being treated or the site of the disease. The composition may also be administered via other conventional routes, for example, by oral, parenteral, or inhalation spray, or via topical, rectal, nasal, buccal, vaginal, or implanted reservoir. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-bursal, intrasternal, intrathecal, intrafocal, and intracranial injection or infusion techniques. Furthermore, the composition may be administered to the subject via an injectable depot route of administration, such as using 1, 3, or 6-month depot-injectable or biodegradable materials and methods.

[0312] The injectable composition may contain various carriers such as vegetable oil, dimethylacetamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies may be administered by infusion, in which a pharmaceutical preparation containing the antibody and physiologically acceptable excipients is injected. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations, such as sterile preparations in a suitable soluble salt form of the antibody, may be dissolved in a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% glucose solution and administered.

[0313] In one embodiment, a myostatin inhibitor, such as an anti-pro / latent myostatin antibody or its antigen-binding moiety, is administered via site-specific or targeted local delivery technology. Examples of site-specific or targeted local delivery technologies include various implantable depot sources of the myostatin inhibitor, such as an anti-pro / latent myostatin antibody or its antigen-binding moiety, or local delivery catheters such as infusion catheters, indwelling catheters or needle catheters, synthetic grafts, outer membrane wraps, shunts and stents or other implantable devices, site-specific carriers, direct injection, or direct application. See, for example, PCT Publication WO00 / 53211 and U.S. Patent No. 5,981,568.

[0314] The specific dosing regimens used in the methods described herein, such as dosage, timing, and repetitions, depend on the specific subject and the subject's medical history.

[0315] The efficacy of treatment for myopathic diseases / disorders may be evaluated using any preferred method. For example, the efficacy of treatment for myopathic diseases / disorders may be evaluated by assessing muscle weakness (e.g., assessing the pattern and severity of weakness), electromyography, blood chemistry (e.g., assessing electrolytes, assessing endocrine causes, measuring creatinine kinase levels, determining erythrocyte sedimentation rate, and performing antinuclear antibody assays), and biopsy (e.g., by histological, histochemical, electron microscopy, biochemical, and genetic analysis).

[0316] "Effective dose," as used herein, refers to the amount of each activator necessary to impart a therapeutic effect to a subject, either alone or in combination with one or more other activators. For example, an effective dose may result in biological effects such as: an increase in muscle mass or muscle fiber diameter, switching of muscle fiber types, an increase in the amount of force generated by muscles, an increase in the volume and / or function of muscle tissue in the subject; an increase in the metabolic rate of the subject; an increase in insulin sensitivity in the subject; an increase in the level of brown adipose tissue in the subject; an increase in the level of beige adipose tissue in the subject; a decrease in the level of white adipose tissue in the subject; a decrease in the level of visceral adipose tissue in the subject; a decrease in the adipose tissue-to-muscle tissue ratio in the subject; an increase in glucose uptake by brown adipose tissue, beige adipose tissue, or muscle tissue in the subject; a decrease in glucose uptake by white adipose tissue or liver tissue; a decrease in protein catabolism in muscle and / or release of amino acids from muscle in the subject; an increase in insulin-dependent blood glucose regulation in the subject; or a decrease in intramuscular fat infiltration in the subject; or a clinically significant outcome such as partial or complete recovery of the ability to perform physical tasks after injury; SF-36 Quality of Life Scoring This refers to a sufficient amount of myostatin inhibitor, such as the antibody or antigen-binding fragment of this disclosure, to achieve clinically meaningful improvements in quality of life, as assessed by a standardized system such as the System; prevention of muscle loss or atrophy in the subject; and / or prevention of the development of metabolic disease in the subject.

[0317] As is recognized by those skilled in the art, the effective dose varies depending on the specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex, and weight, the duration of treatment, the nature of any concurrent treatments, the specific route of administration, and similar factors within the knowledge and expertise of healthcare professionals. These factors are well known to those skilled in the art and can be addressed through routine experimentation. It is generally preferable to use the maximum dose of an individual component or combination thereof, i.e., the safest dose based on sound medical judgment. However, it is understood by those skilled in the art that a patient may advocate for a lower or tolerable dose for medical reasons, psychological reasons, or virtually any other reason.

[0318] In some embodiments, with respect to an increase in the level of promyostatin in the target muscle, the increase is at least 1, 1.2, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 times compared to the control level of promyostatin, or greater (or any range that falls into any of these values). In one embodiment, the increase in the level of promyostatin in the target muscle is an increase in the range of 1 to 3 times, 1.2 to 10 times, 2 to 9 times, 3 to 8 times, 4 to 7 times, 2 to 7 times, etc., compared to the control level of promyostatin.

[0319] In some embodiments, with respect to the increase in latent myostatin in target muscle after the administration step, the increase is detectable within 4 hours, 24 hours, 48 ​​hours, 7 days, 14 days, 21 days, 28 days, or 30 days after the administration step (or within any range of time grouped into any of the listed durations). In one embodiment, the increase in latent myostatin in target muscle after the administration step is detectable over at least 5 days, 7 days, 14 days, 21 days, 28 days, or 30 days after the administration step (or within any range of time grouped into any of the listed durations). In one embodiment, the increase in the level of latent myostatin in the target muscle after the administration step is at least 1, 1.2, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 times (or any range that falls into any of these values) compared to the level of latent myostatin in the target muscle before the administration step. In one embodiment, the increase in the level of latent myostatin in the target muscle after the administration step is an increase in the range of 1 to 3 times, 1.2 to 10 times, 2 to 9 times, 3 to 8 times, 4 to 7 times, 2 to 7 times, etc., compared to the level of latent myostatin in the target muscle before the administration step.

[0320] In some embodiments, with respect to the increase in circulating latent myostatin after the administration step, the increase is detectable within 4 hours, 24 hours, 48 ​​hours, 7 days, 14 days, 21 days, 28 days, or 30 days after the administration step (or within any range of time grouped into any of the listed durations). In one embodiment, the increase in circulating latent myostatin after the administration step is detectable over at least 5 days, 7 days, 14 days, 21 days, 28 days, or 30 days after the administration step (or within any range of time grouped into any of the listed durations). In one embodiment, the increase in the level of circulating latent myostatin after the administration step is at least 1x, 2x, 3x, 5x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, or 50x or more compared to the level of circulating latent myostatin before the administration step (or within any range of any of these values). 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 to 3 times, 1.2 to 10 times, 2 to 9 times, 3 to 8 times, 4 to 7 times, 2 to 7 times, etc., compared to the level of latent myostatin in the target muscle before the administration step.

[0321] In some embodiments, with respect to the reduction in the level of circulating latent myostatin, the reduction is at most 1 / 1, 1.2 / 1, 1.5 / 1, 1 / 2 / 1, 1 / 3 / 1, 1 / 4 / 1, 1 / 5 / 1, 1 / 6 / 1 / 7 / 1, 1 / 8 / 1 / 9 / 1, or 1 / 10 / 10 or less (or any range that falls into any of these values) compared to the control level of latent myostatin. In one embodiment, the reduction in the level of circulating latent myostatin is in the range of 1 / 1 to 1 / 3, 1.2 to 1 / 10, 1 / 2 to 1 / 9, 1 / 3 to 1 / 8, 1 / 4 to 1 / 7, 1 / 2 to 1 / 7, etc., compared to the control level of latent myostatin.

[0322] As described above, in some embodiments, with respect to the administration of a myostatin inhibitor, such as a pro / latent myostatin antibody or its antigen-binding fragment, to a subject, the effective dose is an amount effective in increasing the amount of target muscle in the subject compared to the control muscle mass. In some embodiments, muscle treated with an effective dose of antibody increases by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, etc., compared to the control muscle mass not treated with an effective dose of antibody. In some embodiments, such an increase in muscle mass is achieved in a selected group or type of muscle in the subject.

[0323] In some embodiments, with respect to the administration of a myostatin inhibitor, such as a pro / latent myostatin antibody or its antigen-binding fragment, to a subject, the effective dose is an amount effective in switching the fibril type in the subject. In some embodiments, an effective dose of antibody may promote the switching of fibril type from type I to type II. In some embodiments, an effective dose of myostatin inhibitor, such as an antibody or its antigen-binding moiety, may promote the switching of fibril type from type I to type IIB. In some embodiments, an effective dose of myostatin inhibitor, such as an antibody or its antigen-binding moiety, may promote type II fibril compared to other types of fibril. In some embodiments, an effective dose of myostatin inhibitor, such as an antibody or its antigen-binding moiety, may promote type IIB fibril compared to other types of fibril. In some embodiments, such a switching of fibril phenotype may occur without a significant change in overall muscle mass. In other embodiments, such a switching of fibril phenotype may occur simultaneously with an increase in overall muscle mass.

[0324] In some embodiments, with respect to the administration of a myostatin inhibitor, such as a pro / latent myostatin antibody or its antigen-binding fragment, to a subject, an effective dose is an amount effective in increasing the diameter of muscle fibers in the subject compared to control muscle fibers. In some embodiments, the increase in muscle fiber diameter is at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 4 times, at least 5 times or more compared to control muscle fibers. In some embodiments, the increase in muscle fiber diameter is in the range of 1 to 5 times, 2 to 10 times, 1 to 1.5 times, 1 to 2 times, etc., compared to control muscle fibers.

[0325] In some embodiments, with respect to the administration of a myostatin inhibitor, such as a pro / latent myostatin antibody or its antigen-binding fragment, to a subject, an effective dose is an amount effective in increasing the muscle-to-fat ratio in the subject compared to the control muscle mass. In some embodiments, the increase in the muscle-to-fat ratio is at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 4 times, at least 5 times or more compared to the control subject. In some embodiments, the increase in the muscle-to-fat ratio is in the range of 1 to 5 times, 2 to 10 times, 1 to 1.5 times, 1 to 2 times, etc., compared to the control subject.

[0326] In some embodiments, with respect to the administration of a myostatin inhibitor, such as a pro / latent myostatin antibody or its antigen-binding fragment, to a subject, the effective dose is an amount effective in reducing intramuscular fat infiltration in the subject compared to the control muscle mass. In some embodiments, the reduction in intramuscular fat infiltration is a reduction of at most 1 / 1.1, at most 1 / 1.2, at most 1 / 1.3, at most 1 / 1.4, at most 1 / 1.5, at most 1 / 1.6, at most 1 / 1.7, at most 1 / 1.8, at most 1 / 1.9, at most 1 / 2, at most 1 / 4, at most 1 / 5, or less, compared to the control subject. In some embodiments, the reduction in intramuscular fat infiltration is a reduction in the range of 1 / 1 to 1 / 5, 1 / 2 to 1 / 10, 1 / 1 to 1 / 1.5, 1 / 1 to 1 / 2, etc., compared to the control subject.

[0327] In some embodiments, a method for preventing and / or increasing muscle mass in a human subject includes administering a myostatin inhibitor, such as a pro / latent myostatin antibody or its antigen-binding fragment, to the subject, which inhibits the formation of mature myostatin via proteolysis by toroidal proteases. In one embodiment, a gradual increase in muscle mass results from the inhibition of the proteolytic cleavage of promyostatin or latent myostatin by toroidal proteases. In one embodiment, the subject exhibits a gradual increase in muscle mass over at least 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 weeks (or any range that can be grouped into any of these values). In some embodiments, a method for preventing and / or increasing muscle mass in a human subject includes administering a myostatin inhibitor, such as a pro / latent myostatin antibody or its antigen-binding fragment, to the subject in more than two doses. In one embodiment, the step of administering a myostatin inhibitor, such as a pro / latent myostatin antibody or its antigen-binding fragment, comprises at least a first dose and a second dose, the first and second doses being administered to the subject at intervals of at least about two weeks, four weeks, six weeks, eight weeks, or twelve weeks.

[0328] In some embodiments, with respect to the administration of a myostatin inhibitor, such as a pro / latent myostatin antibody or its antigen-binding fragment, to a subject, the effective dose is an amount effective in increasing the function of the target muscle in the subject compared to the function of the control muscle. In some embodiments, the increase in muscle function is at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 4 times, at least 5 times or more compared to the control muscle function. In some embodiments, the increase in muscle function is in the range of 1 to 5 times, 2 to 10 times, 1 to 1.5 times, 1 to 2 times, etc., compared to the control muscle function.

[0329] As used herein, the term “control muscle mass” refers to a reference criterion useful for evaluating the effect of a condition (e.g., treatment with a myostatin inhibitor, e.g., pro / latent myostatin antibody or its antigen-binding fragment) 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 determined experimentally. In some embodiments, the control muscle mass is the target muscle mass in subjects who have not been administered a myostatin inhibitor, e.g., pro / latent myostatin antibody or its antigen-binding fragment. In some embodiments, the control muscle mass is the target muscle mass (e.g., average) in a population of subjects who have not been administered a myostatin inhibitor, e.g., pro / latent myostatin antibody or its antigen-binding fragment. In some embodiments, the control muscle mass is the target muscle mass in subjects before (e.g., immediately before) administration of a myostatin inhibitor, e.g., pro / latent myostatin antibody or its antigen-binding fragment. In some embodiments, the control muscle mass is the target muscle mass in subjects administered with a myostatin inhibitor, e.g., a pro / latent myostatin antibody or its antigen-binding fragment, instead of a conventional antibody (e.g., of the same isotype as the pro / latent myostatin antibody) obtained from an animal not exposed to the antigen targeted by the pro / latent myostatin antibody or its antigen-binding fragment. In some embodiments, the control muscle mass is the target muscle mass in subjects administered with a vehicle, e.g., saline, instead of a myostatin inhibitor, e.g., a pro / latent myostatin antibody or its antigen-binding fragment.

[0330] In some embodiments, the effective dose of a myostatin inhibitor, e.g., a pro / latent myostatin antibody or its antigen-binding fragment, administered to a subject is an effective amount to increase the force-generating capacity of a target muscle in the subject (e.g., maximum force generation determined in vitro using a muscle lever system adapted to a horizontal perfusion bath) compared to the control force-generating capacity. In some embodiments, the increase in force-generating capacity is at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 4 times, at least 5 times or more compared to the control force-generating capacity. In some embodiments, the increase in force-generating capacity is in the range of 1 to 5 times, 2 to 10 times, 1 to 1.5 times, 1 to 2 times, etc., 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 its antigen-binding fragment) on the force generation capacity of a subject’s muscle. In some embodiments, the control force generation capacity is a predetermined value. The control force generation capacity is determined experimentally. In some embodiments, the control force generation capacity is the force generation capacity of a target muscle in a subject that has not been administered a myostatin inhibitor, e.g., a pro / latent myostatin antibody or its antigen-binding fragment. 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 that have not been administered a myostatin inhibitor, e.g., a pro / latent myostatin antibody or its antigen-binding fragment. In some embodiments, the control force generation capacity is the force generation capacity of a target muscle in a subject before (e.g., immediately before) administration of a myostatin inhibitor, e.g., a pro / latent myostatin antibody or its antigen-binding fragment. In some embodiments, the control muscle force generation capacity is the force generation capacity of a target muscle in a subject administered with a myostatin inhibitor, e.g., a normal antibody (e.g., of the same isotype as the pro / latent myostatin antibody) obtained from an animal not exposed to the antigen targeted by the pro / latent myostatin antibody, instead of the pro / latent myostatin antibody. In some embodiments, the control muscle force generation capacity is the force generation capacity of a target muscle in a subject administered with a vehicle, e.g., saline, instead of the myostatin inhibitor, e.g., the pro / latent myostatin antibody or its antigen-binding fragment.

[0332] In some embodiments, the target muscle is the plantar flexor muscle. In some embodiments, the target muscle is a muscle containing type II 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, as a result of administration of a myostatin inhibitor, e.g., a pro / latent myostatin antibody or its antigen-binding fragment, the cross-sectional area of ​​type IIB fibers increases by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% (or any range that can be grouped into any of these values) compared to the cross-sectional area before the administration step.

[0333] Dosage Empirical considerations such as half-life generally contribute to the determination of dosage. For example, antibodies compatible with the human immune system, such as humanized antibodies or fully human antibodies, and their antigen-binding moieties may be used to extend the antibody half-life and prevent the antibody from being attacked by the host 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 recovery and / or delay of the disease / disorder associated with myopathy. Alternatively, myostatin inhibitors, such as sustained-release formulations of anti-pro / latent myostatin antibodies or their antigen-binding moieties, may also be appropriate. Various formulations and devices for achieving sustained release are apparent to those skilled in the art and are within the scope of this disclosure.

[0334] For example, the dosage of a myostatin inhibitor described herein, such as an anti-pro / latent myostatin antibody or its antigen-binding fragment, may be empirically determined in an individual given one or more doses of the myostatin inhibitor, such as an antibody or its antigen-binding fragment. The individual is given an increasing dose of the antagonist. Disease / disability indicators may be tracked to evaluate the efficacy of the antagonist.

[0335] In general, for any administration of the antibodies or antigen-binding fragments described herein, the initial candidate dose may be approximately 2 mg / kg. For the purposes of this disclosure, a typical daily dose may be in the range of approximately 0.1 μg / kg, 3 μg / kg, 30 μg / kg, 300 μg / kg, 3 mg / kg, 30 mg / kg, 100 mg / kg, or greater, depending on the factors described above. For repeated administrations over several days or longer, treatment should be continued, depending on the condition, until the desired suppression of symptoms occurs, or until a sufficient therapeutic level is achieved to alleviate the disease or disorder or symptoms associated with pro / latent myostatin. An exemplary dosing regimen includes an initial dose of approximately 2 mg / kg, followed by weekly maintenance doses of approximately 1 mg / kg of the antibody or antigen-binding fragment, or followed by every-weekly maintenance doses of approximately 1 mg / kg. However, other dosing regimens may be useful depending on the pattern of pharmacodynamic decay that the clinician wishes to achieve. For example, administration 1 to 4 times per week is intended. In some embodiments, doses of approximately 3 μg / mg to 2 mg / kg (e.g., approximately 3 μg / mg, 10 μg / mg, 30 μg / mg, 100 μg / mg, 300 μg / mg, 1 mg / kg, and 2 mg / kg) are used. In some embodiments, the frequency of administration is once weekly, 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 monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 8 months, every 10 months, annually, or at longer intervals. The progression of this treatment can be easily monitored by conventional procedures and assays. The administration regimen (including the antibody used) may be changed over time.

[0336] In some embodiments, the administration of a myostatin inhibitor, such as the antibody or its antigen-binding fragment described herein, comprises a single dose. In some embodiments, the administration of a myostatin inhibitor, such as the antibody or its antigen-binding fragment described herein, comprises multiple doses (e.g., at least two, three, four, five, six, seven, eight, nine, or ten doses). The administration step may comprise more than two doses. In some embodiments, the administration comprises at least a first and a second dose of a therapeutically effective amount of the myostatin inhibitor, such as the antibody or its antigen-binding moiety. In one embodiment, the first and second doses are administered to the subject at intervals of at least about four weeks, six weeks, eight weeks, or twelve weeks.

[0337] In some embodiments, doses ranging from approximately 0.3 to 5.00 mg / kg may be administered to adult patients of normal weight. Specific dosing regimens, such as dose, timing, and repetition, depend on the specific individual, their medical history, and the characteristics of the individual drug (e.g., drug half-life and other relevant considerations).

[0338] For the purposes of this disclosure, the appropriate dosage of a myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody or its antigen-binding fragment, depends on the specific antibody (or its composition) 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 antagonists, and the judgment of the attending physician. In some embodiments, the clinician administers the myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody or its antigen-binding moiety, until a dosage is reached that achieves the desired outcome. The administration of the myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody or its antigen-binding moiety, may be continuous or intermittent, depending on, for example, the recipient's physiological condition and other factors known to a skilled physician, regardless of whether the purpose of administration is therapeutic or prophylactic. Myostatin inhibitors, such as anti-pro / latent myostatin antibodies or their antigen-binding fragments, may be administered essentially continuously over a pre-selected period, or in a series of intervald doses, for example, before, during, or after the onset of a disease or disorder associated with pro / latent myostatin.

[0339] As used herein, the term “to treat” means the application or administration of a composition comprising one or more activators to a subject having a disease / disorder associated with myopathy, symptoms of a disease / disorder, or predisposition to a disease / disorder, for the purpose of curing, healing, alleviating, reducing, altering, improving, restoring, improving, or influencing the disorder, symptoms of a disease, or predisposition to a disease / disorder.

[0340] Alleviating pro / latent myostatin-related disease / disorder includes delaying the onset 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 pro / latent myostatin-related disease / disorder means deferring, hindering, slowing, retarding, stabilizing, and / or postponing the progression of the disease. This retarding may be of varying lengths depending on the medical history 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 of reducing the likelihood of developing one or more symptoms of the disease and / or reducing the severity of symptoms in a given time frame compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to yield statistically significant results.

[0341] Combination therapy The present invention encompasses pharmaceutical compositions and related methods used as combination therapies for treating subjects in which in vivo myostatin inhibition may be beneficial. In any of these embodiments, such a subject may receive combination therapy comprising a first composition comprising at least one myostatin inhibitor, e.g., an antibody or antigen-binding moiety described herein, and a second composition comprising at least one further therapeutic agent intended for the treatment of the same or overlapping disease or clinical condition. The first and second compositions may both act on the same cellular target, or they may both act on distinct cellular targets. In some embodiments, the first and second compositions may treat or alleviate the same or overlapping set of symptoms or appearances of the disease or clinical condition. In some embodiments, the first and second compositions may treat or alleviate distinct sets of symptoms or appearances of the disease or clinical condition. For example, the first composition may treat a disease-related myopathy, and the second composition may treat inflammation or fibrosis associated with the same disease, and so on. Such combination therapies may be administered in combination with each other. The phrase "in conjunction with" means, with respect to combination therapy, that the therapeutic effect of the first therapy temporarily and / or spatially overlaps with the therapeutic effect of the second therapy in a subject receiving combination therapy. Therefore, 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, combination therapy produces a synergistic effect in treating the disease. The term "synergistic" refers to an effect greater than the sum of the individual therapies (e.g., greater efficacy).

[0343] In some embodiments, combination therapy comprising the pharmaceutical compositions described herein produces an overall efficacy equivalent to that produced by another therapy (e.g., monotherapy of the second drug), but with fewer undesirable adverse effects or a lower severity of toxicity compared to monotherapy of the second drug. In some embodiments, such combination therapy makes it possible to use a lower dose of the second drug while maintaining the overall efficacy. Such combination therapy may be particularly suitable for patient populations where long-term treatment is guaranteed and / or involving pediatric patients.

[0344] Accordingly, the present invention provides pharmaceutical compositions and methods for use in combination therapy to enhance muscle mass / function and to treat or prevent metabolic disorders or disorders associated with defects in nerve signaling, including diabetes, obesity, and spinal cord injury. Accordingly, the methods or pharmaceutical compositions further include a second treatment. In some embodiments, the second treatment may be useful in treating or preventing metabolic disorders or disorders associated with defects in nerve signaling. The second treatment can attenuate or treat at least one symptom(s) associated with the targeted disease. The first and second treatments may exert their biological effects by similar or unrelated mechanisms of action, or one or both of the first and second treatments may 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 treatments in the same pharmaceutically acceptable carrier or in different pharmaceutically acceptable carriers for each of the embodiments described. It should be further understood that the first and second treatments may be administered simultaneously or sequentially within the scope of the embodiments described.

[0346] One or more anti-myostatin antibodies or other myostatin inhibitors of the present invention may be used in combination with one or more further therapeutic agents. Examples of further therapeutic agents that may be used with the anti-myostatin antibodies of the present invention include, but are not limited to, diabetic treatment agents, diabetic complication treatment agents, cardiovascular disease treatment agents, antihyperlipidemic agents, antihypertensive or antihypertensive agents, anti-obesity agents, non-alcoholic steatohepatitis (NASH) treatment agents, chemotherapeutic agents, immunotherapeutic agents, and immunosuppressants. Such combination therapies can advantageously utilize lower doses of the therapeutic agents administered and thus avoid potential toxicity or complications associated with various monotherapies.

[0347] Examples of drugs 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 sensitivity enhancers, their pharmaceutically acceptable salts, hydrates, or solvent compounds (e.g., pioglitazone, troglitazone, rosiglitazone, netoglitazone, paraglitazone, riboglitazone, tesaglitazal, farglitazar, CLX-0921, R-483, NIP-221, NIP-223, DRF-2189, GW-7282TAK-559, T-131, RG-12525, LY-510929, LY-519818, BMS-298585, DRF-2725, GW-1536, GI- This includes 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, trazamide, acetohexamide, glyclopyramide, glimepiride, etc.), as well as other insulin secretagogues (e.g., repaglinide, senaglinide, nateglinide, mitiglinide, GLP-1, etc.), amylin agonists (e.g., plumlintide, etc.), phosphotyrosine phosphatase inhibitors (e.g., vanadic acid, etc.).

[0348] Examples of medications used to treat complications of diabetes include, but are not limited to, aldose reductase inhibitors (e.g., torrestat, epalrestat, zenalestat, zopolrestat, minalestat, fidarestat, SK-860, CT-112, etc.), neurotrophic factors (e.g., NGF, NT-3, BDNF, etc.), PKC inhibitors (e.g., LY-333531, etc.), and advanced glycation end product (AGE) inhibitors (e.g., ALT946, pimagedine). This includes pyradoxamine, phenacylthiazolium bromide (ALT766), etc., reactive oxygen species quenchers (e.g., thioctic acid or its derivatives, bioflavonoids, e.g., flavones, isoflavones, flavanones, procyanidins, anthocyanidins, pycnogenol, lutein, lycopene, vitamin E, coenzyme Q, etc.), and cerebral vasodilators (e.g., tiapride, mexiletene, etc.).

[0349] In one embodiment, the alleviation of diabetes can be induced by administering a myostatin inhibitor in combination with a calorie-restricted diet or other dietary restrictions.

[0350] Antihyperlipidemia agents include, for example, statin-based compounds that are cholesterol synthesis inhibitors (e.g., pravastatin, simvastatin, lovastatin, atorvastatin, fluvastatin, rosuvastatin, etc.), squalene synthetase inhibitors or fibrate compounds that have a triglyceride-lowering effect (e.g., fenofibrate, gemfibrozil, bezafibrate, clofibrate, simfibrate, clinofibrate, etc.), niacin, PCSK9 inhibitors, triglyceride-lowering agents, or cholesterol sequestering agents.

[0351] Antihypertensive drugs include, for example, angiotensin-converting enzyme inhibitors (e.g., captopril, enalapril, delapril, benazepril, cilazapril, enalapril, enalaprilat, hosinopril, 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, and rosiglitazone. In one embodiment, steatosis, resulting hepatitis, and fibrosis in NAFLD and / or NASH subjects can be treated by administering myostatin inhibitors in combination with a calorie-restricted diet or other dietary restrictions.

[0353] Anti-obesity agents include, for example, central anti-obesity agents (e.g., dexfenfluramine, fenfluramine, phentermine, sibutramine, amfepramon, 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.), and cholecystokinin agonists (e.g., lintitript, FPL-15849, etc.).

[0354] Chemotherapy 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 fluturon and neofluturon are preferred.

[0355] Immunotherapy drugs include, for example, microbial or bacterial components (e.g., muramyl dipeptide derivatives, picibanil, etc.), polysaccharides with immune-enhancing activity (e.g., lentinan, schizophyllan, krestin, etc.), cytokines obtained by genetic engineering techniques (e.g., interferon, interleukin (IL), etc.), and colony-stimulating factors (e.g., granulocyte colony-stimulating factor, erythropoietin, etc.). Among these, preferred substances include IL-1, IL-2, and IL-12.

[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(R), azathioprine, cyclophosphamide); interleukin antagonists (basiliximab, daclizumab, deoxyspagarin); lymphocyte depletion agents such as antithymocyte globulins (thymoglobulin, lymphoglobulin); and anti-CD3 antibodies (OKT3).

[0357] Furthermore, agents whose cachexia-improving effects have been established in animal models or clinical settings, such as cyclooxygenase inhibitors (e.g., indomethacin), progesterone derivatives (e.g., megestrol acetate), glucosteroids (e.g., dexamethasone), metoclopramide-based agents, tetrahydrocannabinol-based agents, lipid metabolism improvers (e.g., eicosapentaenoic acid), growth hormone, antibodies against IGF-1, TNF-α, LIF, IL-6, and oncostatin M, may also be used concurrently with the anti-myostatin antibody according to the present invention. Further therapeutic agents for use in the treatment of diseases or conditions associated with metabolic disorders and / or defects in nerve signaling are apparent to those skilled in the art and fall within the scope of this disclosure.

[0358] In some embodiments, a second agent suitable for administration as a combination therapy with the antibodies described herein is an antifibrotic agent such as a TGFβ1 inhibitor.

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

[0360] Any of the above-mentioned drugs may be administered in combination with the myostatin antibody of the present invention to treat metabolic disorders or diseases associated with defects in nerve signaling between neurons and target tissues, such as spinal cord injury, muscular atrophy, and muscular dystrophy.

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

[0362] A. Metabolic disorders and diseases The present invention provides methods for treating or preventing metabolic disorders in a subject. As used herein, the term “metabolic disorder” refers to any undesirable condition involving a disruption of the normal physiological state of homeostasis resulting from alterations in metabolism (anabolism and / or catabolism). Metabolic disorders affect how the body processes substances necessary for performing physiological functions and generally involve abnormal glucose, lipid / fat and / or protein / nitrogen metabolism, or osmotic dysregulation, as well as the pathological consequences arising from such conditions. Several metabolic disorders of the present invention share certain characteristics, and are accompanied, for example, by decreased fat-free or lean muscle mass, excess fat, a low metabolic rate, insulin resistance, inability to control blood glucose, weight gain, and / or an increase in body mass index. In some cases, such metabolic conditions may be caused or aggravated by medications the patient receives. As discussed in more detail herein, metabolic disorders may occur secondarily to or as a result of muscle conditions or disorders.

[0363] This invention is at least in part based on the discovery that administering a myostatin inhibitor, such as an antibody or antigen-binding fragment that specifically binds to pro / latent myostatin, to subjects with metabolic disorders significantly improves both the physiological and functional characteristics of the affected subjects. Specifically, the inventors were surprised to find that administering a myostatin inhibitor, such as an anti-myostatin antibody or its antigen-binding moiety, significantly increased metabolic rate or energy expenditure in subjects with metabolic disorders. Furthermore, administration of a myostatin inhibitor, such as an anti-myostatin antibody or its antigen-binding moiety, significantly reduced SCI-induced reduction in lesional muscle mass and total body weight, while simultaneously reducing the amount of undesirable adipose tissue, such as white and visceral adipose tissue. Moreover, subjects treated with a myostatin inhibitor, such as an anti-myostatin antibody or its antigen-binding moiety, showed significant improvements in spontaneous motor function, muscle strength, and motor coordination and balance skills.

[0364] Accordingly, the present invention provides a method for treating or preventing metabolic disorders in human subjects. The method comprises the steps of selecting a human subject suffering from a metabolic disorder, and administering to the human subject an effective amount of a myostatin inhibitor, such as an antibody or its antigen-binding fragment that specifically binds to myostatin, thereby treating or preventing the metabolic disorder in the human subject. Preferably, the antibody or its antigen-binding fragment specifically binds to pro / latent myostatin but not to GDF11. An antibody that specifically recognizes pro / latent myostatin but does not recognize GDF11 is beneficial, as it avoids 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 disorders 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), non-alcoholic steatohepatitis (NASH), spinal cord injury (SCI) (e.g., complete or incomplete / partial SCI), hypometabolic states, bidiabetes, Cushing's disease (also known as Cushing's syndrome), obesity syndromes (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, metabolic disorders include disorders related to defects in nerve signaling or partial denervation. In some embodiments, metabolic disorders include conditions (e.g., side effects) caused by or associated with certain drugs.

[0366] Further diseases or conditions related to metabolic disorders and / or body composition will be obvious to those skilled in the art and fall within the scope of this disclosure.

[0367] Diabetes is a group of metabolic disorders characterized by high blood glucose levels resulting from a deficiency in insulin secretion, action, or both. There are two most common types of diabetes, type 1 and type 2, both of which are caused by the body's inability to control insulin. Insulin is a hormone released by the pancreas in response to an increase in blood glucose levels.

[0368] The term "Type 1 diabetes," as used herein, refers to a chronic disease in which the pancreas produces insufficient insulin to adequately control blood glucose levels. Type 1 diabetes is also known as insulin-dependent diabetes mellitus, IDDM, and juvenile-onset diabetes. People with Type 1 diabetes (insulin-dependent diabetes mellitus) produce little to no insulin. Approximately 6 percent of the US population has some form of diabetes, and only about 10 percent of all diabetic patients have Type 1 diabetes. The vast majority of people with Type 1 diabetes develop the condition before the age of 30. Type 1 diabetes is the result of progressive autoimmune destruction of the pancreatic beta cells, followed by insulin deficiency. More than 90 percent of the pancreatic insulin-producing cells (beta cells) are permanently destroyed. The resulting insulin deficiency is severe, and people with Type 1 diabetes must regularly inject insulin to survive.

[0369] In type 2 diabetes (also known as non-insulin-dependent diabetes mellitus, or NDDM), the pancreas continues to produce insulin, sometimes even at levels higher than normal. However, the body develops resistance to its effects, resulting in a relative insulin deficiency. While type 2 diabetes can occur in children and adolescents, it usually begins after age 30 and becomes increasingly common with age: approximately 15 percent of people over 70 have type 2 diabetes. Obesity is a risk factor for type 2 diabetes, and 80-90 percent of people with this condition are obese.

[0370] In some embodiments, diabetes includes prediabetes. “Prediabetes” refers to one or more early diabetic conditions, including impaired glucose utilization, abnormal or impaired fasting blood glucose levels, impaired glucose tolerance, impaired insulin sensitivity, and insulin resistance. Prediabetes is a major risk factor for type 2 diabetes, cardiovascular disease, and mortality. Much focus has been on developing therapeutic interventions to prevent the development of type 2 diabetes by treating prediabetes.

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

[0372] Diabetes mellitus can be diagnosed by performing a glucose tolerance test. Clinically, diabetes mellitus is often classified into several basic categories. Key examples of these categories include autoimmune diabetes, non-insulin-dependent diabetes mellitus (Type 1 NDDM), insulin-dependent diabetes mellitus (Type 2 IDDM), non-autoimmune diabetes mellitus, non-insulin-dependent diabetes mellitus (Type 2 NIDDM), and juvenile-onset adult-onset diabetes mellitus (MODY). Further categories, often referred to as secondary, refer to diabetes resulting from several identifiable conditions that cause or enable the development of diabetic syndromes. 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, e.g., Harrison's (1996), 14th edition, New York, McGraw-Hill).

[0373] Obesity is another prevalent metabolic disorder that can be treated or prevented by the methods of the present invention. “Obesity” refers to a chronic condition defined by an excess amount of body fat. A normal amount of body fat (expressed as a percentage of body weight) is 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 significant definition, for example, body mass index (BMI, kg / m²) in adults. 2 ) is frequently used as a measure of overweight and obesity, with overweight being defined as a BMI of 25-29.9 kg / m². 2 Obesity is defined as having a BMI of 30 kg / m². 2 Pathological obesity is defined as having a BMI equal to or greater than 40 kg / m². 2Obesity is defined as exceeding a certain threshold. In adults, obesity can also be defined by central adiposity, measured by waist circumference, with waist circumference equal to or greater than 102 cm for men and equal to or greater than 88 cm for women. Subjects with obesity 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. Severe complications of obesity include a very high risk of coronary artery disease and its major risk factors for type 2 diabetes, hyperlipidemia, and hypertension. The majority of obesity-related morbidities are associated with type 2 diabetes, as poorly regulated diabetes and obesity lead to a group of symptoms known together as syndrome X, or metabolic syndrome. In some embodiments, obesity is muscle-depleting obesity. In some embodiments, subjects with obesity are subjected to a calorie-restricted regimen.

[0374] The methods of the present invention are also suitable for treating or preventing metabolic diseases such as metabolic syndrome. As used herein, “metabolic syndrome” refers to the concept of a clustering of metabolic risk factors that occur together in a single individual and lead to a high risk of developing diabetes and / or cardiovascular disease. Key features of metabolic syndrome include insulin resistance, hypertension (high blood pressure), cholesterol abnormalities, dyslipidemia, triglyceride abnormalities, an increased risk of coagulation, particularly in the abdomen, and excess body weight, or obesity. The American Heart Association suggests that metabolic syndrome is diagnosed by the presence of three or more of the following components: (1) high waist circumference (men, equal to or greater than 40 inches (102 cm); women, equal to or greater than 35 inches (88 cm)); (2) high triglycerides (equal to or greater than 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 (equal to or greater than 130 / 85 mmHg); and (5) high fasting blood glucose (equal to or greater than 100 mg / dL).

[0375] In another embodiment, the methods of the present invention are suitable for treating or preventing metabolic disorders such as obesity syndromes. The term “obesity syndrome” refers to any disorder or condition that causes significant fat or excess body weight in a subject. As with other metabolic disorders, persons with obesity syndromes are typically accompanied by decreased fat-free or lean muscle mass, excess body fat, a low metabolic rate, insulin resistance, inability to control blood glucose, weight gain, and an increased body mass index. In some embodiments, obesity syndromes are selected from the group consisting of Prader-Willi syndrome, obesity syndromes associated with genetic disorders, and obesity syndromes associated with hypothalamic disorders.

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

[0377] In another aspect, the methods of the present invention are suitable for treating or preventing metabolic disorders such as Cushing's disease, also known as Cushing's syndrome. The term "Cushing's disease" refers to a set 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 to exogenous causes, such as the use of oral corticosteroids. Characteristic signs and symptoms of Cushing's disease may include progressive obesity, particularly 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, easily bruised, poorly healing cuts, 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 fractures over time. In children, Cushing's disease may result in growth defects (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 treatment for Cushing's disease is designed to lower high cortisol levels in the body, regardless of whether the underlying cause is endogenous overproduction of hormones or medication-induced. The best course of action for a particular patient depends on the cause of the syndrome. Currently available treatment options include, for example, reducing corticosteroid use, surgery, radiation therapy, and medications.

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

[0380] When Cushing's disease is caused by long-term use of corticosteroids, it may be possible to control and reduce the dosage of the drug over a period of time while still appropriately managing the underlying disease or condition that is causing the drug to be administered. Therefore, in some embodiments, patients undergoing corticosteroid treatment have 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 immune system in order to prevent the body from rejecting allografts, such as transplanted organs or tissues.

[0381] In some embodiments, patients receiving corticosteroid treatment include a subpopulation of individuals who are not sufficiently tolerant of other treatment options, such as non-corticosteroid medications, and who respond poorly to or do not respond to such treatment options. In such situations, physicians may continue prescribing corticosteroids. In some embodiments, surgery may be considered as an alternative option.

[0382] If Cushing's syndrome is caused by a tumor, complete surgical resection and / or radiation therapy may 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 performed to provide the body with adequate adrenal hormone production.

[0383] In some embodiments, patients with Cushing's syndrome may not experience a recovery of normal adrenal function and may therefore require lifelong replacement therapy. The myostatin activating inhibitors described herein may be suitable for treating such patients.

[0384] In some embodiments, if surgery and / or radiation therapy are unsuccessful, drugs can be used to control cortisol production. In patients with severe Cushing's syndrome, drugs may be used before surgery. Myostatin activating inhibitors incorporated herein may be used to treat such patients before surgery in order to improve signs and symptoms and minimize the risks 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 type 2 diabetes or Cushing's syndrome with impaired glucose tolerance. Mifepristone does not reduce cortisol production but blocks the effects of cortisol on tissues. Side effects of these drugs may include fatigue, nausea, vomiting, headache, muscle pain, hypertension, hypokalemia, and swelling. Some may have more serious side effects, such as neurological side effects and hepatotoxicity. Myostatin activating inhibitors included in this disclosure may be used alone (instead of these drugs) or in combination with any of these drugs.

[0386] More recently, pasireotide (Signifor) has become available for the treatment of Cushing's disease, which works by reducing ACTH production from pituitary tumors. This drug is administered as an injection twice daily. It is typically recommended when pituitary surgery is unsuccessful or not possible. Side effects associated with this drug are quite common and may include diarrhea, nausea, hyperglycemia, headache, abdominal pain, and fatigue. Myostatin activating inhibitors included in 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, a tumor or its treatment may lead to a deficiency in other hormones produced by the pituitary gland or adrenal gland, which may necessitate hormone replacement therapy. In some embodiments, none of these currently available treatment options may be 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 an option may benefit from the myostatin inhibitor treatments described herein before and / or after adrenalectomy.

[0388] In yet another embodiment, 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 diseases or heart diseases include, but are not limited to, angina pectoris, arrhythmias, coronary artery disease (CAD), coronary heart disease, cardiomyopathy (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 valve stenosis, and the like. Vascular diseases include, but are not limited to, peripheral vascular diseases, arterial diseases, carotid artery diseases, deep vein thrombosis, venous diseases, and atherosclerosis. In some embodiments, subjects with heart failure are resistant to diuretic treatment. In another embodiment, subjects with heart failure have an inadequate response to diuretic treatment.

[0389] Another aspect of this disclosure includes methods for treating subjects having age-related metabolic disorders or conditions. Exemplary age-related disorders and conditions include, but are not limited to, muscle loss (age-related muscle loss), frailty, and androgen deficiency.

[0390] Another aspect of this disclosure includes, for example, methods for treating subjects having metabolic diseases or conditions associated with inactivity or hereditary atrophy / trauma, such as atrophy caused by inactivity, atrophy caused by gene mutation(s), or atrophy caused by injury. 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, hip fracture, stroke, bed rest, SCI, rotator cuff injury, knee replacement, fracture, and burns.

[0391] This disclosure includes the beneficial effects 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 together tissues and organs), muscle and bone homeostasis are closely related. Bone growth in response to muscle growth is a mechanosensitive process regulated by endocrine signaling.

[0392] Similar to muscle, bone homeostasis involves a dynamic process of balancing 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, and bone repair rate. Factors known to play a role in this process (e.g., cytokines, hormones) include, but are not limited to, parathyroid hormone, 1,25-dihydroxyvitamin D3, T4, corticosteroids, prostaglandins such as prostaglandin E2, interleukin-4, interleukin-18, interferon-γ, interleukin-17, interleukin-6, interleukin-1, RANKL, CSF, TGFβ, osteoprotegerin, BMP, IGF, and FGF. Osteoclasts and osteoblasts contribute to bone resorption and bone growth, respectively.

[0393] From a structural standpoint, bone strength is determined by the combination of the structure and composition of the trabeculae and cortical bone. Generally, decreased bone strength, such as osteopenia and osteoporosis, is observed with age. In some situations, bone loss is caused by other factors, such as medications. For example, glucocorticoid treatment can cause bone loss. Medical conditions that may be associated with bone loss include, non-limitingly, cancer and muscle / metabolic disorders accompanied by bone loss. In some embodiments, such conditions are associated with spinal cord injury (SCI), muscular dystrophy such as DMD, obesity, and / or Cushing's disease.

[0394] Bone loss can be measured using assays well known to those skilled in the art (see Shanmugarajan et al., J. Pathol., 2009, Vol. 219 (No. 1): pp. 52-60, and Wasserman et al., Neuromuscular Disorders, 2017, Vol. 27 (No. 4): pp. 331-337). For example, bone mineral density, such as area mineral density, can be measured using DXA scanning of the lumbar spine, whole body, and distal lateral femur, in accordance with ISCD recommendations (Wasserman et al., Neuromuscular Disorders, 2017, Vol. 27 (No. 4): pp. 331-337). After scanning, bone mineral density can be calculated using reference data from Henderson et al., Am.J. Roentgenol, 2002, Vol. 178: pp. 439-443; Kalkwarf et al., J. Bone Miner. Res., 2013, Vol. 28: pp. 206-212; Kelly et al., J. Pediatr. Hematol. Oncol., 2005, Vol. 27: pp. 248-253; and Zemel et al., J. Clin. Endocrinol. Metab., 2011, Vol. 96: pp. 3160-3169. The patient's fracture history, including age at fracture, number of fractures, and fracture location, can also be collected. Osteoporosis is typically measured using the 2013 ISCD criteria, which include vertebral compression fractures in the absence of high-energy trauma or invasive disease; or a BMD Z score ≤ 2.0 SD, and two or more long bone fractures by age 10 or three or more long bone fractures by age 19 (see, e.g., Bishop et al., J. Clin. Densitom, 2014, Vol. 17: pp. 275-280).

[0395] Several therapeutic drugs are currently available to treat bone loss. Drugs used to slow the rate of bone loss include bisphosphonates and denosumab. Bisphosphonates block osteoclast recruitment and induce apoptosis of osteoclasts. These are often used to treat postmenopausal and glucocorticoid-induced osteoporosis, Paget's disease, and malignant hypercalcemia. Developed by Amgen, denosumab 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 promote new bone growth include teriparatide and romosozumab. Forteo is a teriparatide drug marketed by Eli Lilly, which is a recombinant protein fragment containing the first 34 amino acid residues of the thyroid hormone. It is typically used to treat osteoporosis and patients at high risk of fracture, as well as patients intolerant to other treatments. Romosozumab, available from Amgen, is a monoclonal antibody that blocks sclerostin (a Wnt pathway antagonist). To date, there are no drugs that can increase both bone and muscle.

[0397] Several groups are conducting preclinical and clinical trials using drugs that at least partially affect the myostatin pathway. For example, Acceleron has developed ActRIIA and ActRIIB ligand traps (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 drugs appear to be myostatin / GDF8 specific, but they may affect one or more of the other pathways. Eli Lilly's monoclonal antibody LY2495655 (landogrozumab) did not increase bone mass in humans undergoing selective hip replacement when measured by dexa. This antibody binds to both GDF8 and GDF11.

[0398] Unlike these agents that affect myostatin and further pathways, the monoclonal antibodies contained herein bind specifically 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 antibody or fragment binds to inactive myostatin (e.g., promyostatin) in a tethered form (e.g., intramuscular) that has the ability to act locally on tissue-associated myostatin within the disease site. In some embodiments, the antibody or fragment binds to inactive myostatin (e.g., latent myostatin) in a soluble form (e.g., circulating) that has the ability to act on circulating latent myostatin that may have endocrine or systemic effects. In any such embodiment, the preferred inhibitor of myostatin for carrying out the method of the present invention is selective for myostatin that does not antagonize other members of the TGF superfamily of growth factors / cytokines, such as GDF11. Such selectivity is particularly advantageous in pediatric patient populations and / or patient populations requiring long-term care (e.g., long-term treatment), where inhibiting other pathways, such as GDF11, may result in adverse or undesirable side effects or adverse events. The inventors of this disclosure have shown that such antibodies can effectively inhibit myostatin activation and produce beneficial muscle and metabolic effects. Furthermore, the evidence provided herein also shows that such antibodies can produce beneficial biological effects on bone homeostasis in vivo (see Figures 28–31).

[0399] Accordingly, the present invention includes the use of myostatin activating inhibitors to enhance one or more parameters of bone homeostasis, including relative bone volume (e.g., measured by bone volume relative to the total volume of the corresponding tissue or specimen); trabecular bone volume; number...

Claims

1. A composition 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 who has a metabolic disease or is at risk of developing one, and administering to the human subject an effective amount of the composition comprising the antibody or the antigen-binding fragment.

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

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

4. The composition according to claim 1, wherein the metabolic disease is selected from the group consisting of type 1 diabetes, type 2 diabetes, obesity, metabolic syndrome / prediabetes, cardiovascular disease, non-alcoholic steatohepatitis (NASH), spinal cord injury (SCI), scalable muscle atrophy (SMA), hypometabolic state, bidiabetes, metabolic bone disorder, Cushing's disease, and obesity syndrome.

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

6. The composition according to claim 5, wherein the heart failure is CHF including fluid overload.

7. The composition according to claim 6, wherein the excess body fluid includes systemic edema and / or pulmonary edema.

8. The composition according to claim 6, wherein the human subject has an insufficient response to diuretic treatment.

9. The composition according to claim 4, wherein the obesity is muscle-depleting obesity.

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

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

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

13. The composition according to any one of claims 10 to 12, wherein the subject has not undergone an exercise regimen.

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

15. The composition according to claim 4, wherein the metabolically reduced state is selected from the group consisting of a state associated with prolonged exercise restriction, a state associated with bed rest, a state associated with cast immobilization, a state associated with stroke, a state associated with amputation, and a post-surgical state.

16. The composition according to 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. The composition according to claim 4, wherein the obesity syndrome is selected from the group consisting of Prader-Willi syndrome, obesity syndromes associated with hereditary disorders, and obesity syndromes associated with hypothalamic disorders.

18. The administration of the above composition is as follows: a) To increase the volume and / or function of muscle tissue in the human subject; b) To increase the volume and / or function of fast-twitch muscle tissue in the human subject; c) To increase the volume and / or function of slow-twitch muscle tissue in the human subject; d) To increase the metabolic rate of the human subject; e) To increase insulin sensitivity in the human subject; f) To increase the level of brown adipose tissue in the human subject; g) To increase the level of beige adipose tissue in the human subject; h) To reduce the level of white adipose tissue in the human subject; i) To reduce the level of visceral adipose tissue in the human subject; j) Reducing the fat-to-muscle tissue ratio in the human subject; k) To increase glucose uptake by target tissue in the human subject, wherein the target tissue is selected from the group consisting of brown adipose tissue, beige adipose tissue, and muscle tissue; 1) To reduce glucose uptake by target tissue in the human subject, where the target tissue is selected from the group consisting of white adipose tissue and liver tissue; m) Reducing protein catabolism and / or release of amino acids from muscle in the human subject; n) To increase insulin-dependent blood glucose regulation in the human subject; o) To reduce intramuscular fat infiltration in the human subject; p) Improving standardized quality of life test scores; q) To prevent muscle loss or atrophy in the aforementioned human subjects; r) To increase bone density or bone volume; s) To prevent or reduce bone loss or fracture; t) To reduce fluid overload or edema associated with chronic heart failure (CHF); and / or u) Enhancing the subject's response to the treatment; A composition according to any one of claims 1 to 17, which causes at least one of the following:

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

20. The antibody or its antigen-binding fragment a) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 25 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 31, or b) Heavy chain containing the amino acid sequence of SEQ ID NO: 50 and light chain containing the amino acid sequence of SEQ ID NO: 51 A composition according to any one of claims 1 to 19, comprising:

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