Myostatin pathway inhibitors in combination with GLP-1 pathway activators for use in the treatment of metabolic disorders

Abstract

The present disclosure relates to the treatment of metabolic disorders such as metabolic syndrome, obesity and type 2 diabetes. Adjunctive and combination therapies comprising myostatin pathway inhibitors for use in conjunction with GLP-1 pathway activators are disclosed.

Description

[Technical field]

[0001] This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated by reference herein in its entirety. The ASCII copy, created on Jun. 16, 2022, is named 15094_0048-00304_SL.txt and is 111,218 bytes in size.

[0002] Related Applications This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 214,234, filed June 23, 2021, entitled "COMBINATION THERAPIES FOR WEIGHT MANAGEMENT AND RELATED METABOLIC CONDITIONS" and U.S. Provisional Patent Application No. 63 / 260,136, filed August 10, 2021; No. 63 / 260,254, filed August 13, 2021; No. 63 / 266,348, filed January 3, 2022; and No. 63 / 269,702, filed March 21, 2022, each entitled "THERAPIES FOR WEIGHT MANAGEMENT AND RELATED METABOLIC CONDITIONS," the contents of which are expressly incorporated herein by reference in their entireties.

[0003] The present application relates to the use of myostatin pathway inhibitors and GLP-1 pathway activators for the treatment and management of metabolic disorders, including obesity, metabolic syndrome and type 2 diabetes mellitus. [Background technology]

[0004] Among metabolic diseases, obesity is a chronic condition that affects more than one in three adults and approximately 17 percent of children and adolescents in the United States alone. More than one in three adults are overweight. Being overweight or obese increases the risk of type 2 diabetes, heart disease, stroke, fatty liver disease, kidney disease, and other health problems.

[0005] Attempts to lose weight are often met with difficulties. For example, extreme dieting, such as very low calorie restriction, is impractical, if not impossible, for most patients. For example, when dieting in individuals with obesity syndrome, a 10% weight loss will result in a decline in overall energy expenditure of approximately 20-30%, with a concomitant decline in basal metabolic rate, leptin levels, and may result in a loss of lean mass. It is well known that weight loss through diet / calorie restriction not only causes a loss of adipose (fat) tissue mass, but also results in a loss of skeletal muscle mass. Combined with the body's natural tendency to maintain homeostasis by adapting to low energy supplies, the loss of skeletal muscle causes a decline in metabolic rate, which leads to a decline in energy expenditure. In fact, a loss of 10% of total body weight will result in a 30-40% decrease in the energy expended above resting energy expenditure during physical activity.

[0006] In addition to more invasive procedures such as gastric bypass, several weight loss medications have been approved by regulatory agencies (e.g., FDA) for the treatment of overweight and obesity (see, e.g., Williams. Diabetes Ther (2020) 11:1199-1216). These include orlistat (XENICAL®, ALLI®), phentermine and topiramate (QSYMIA®), naltrexone, HCl / bupropion, HCl (CONTRAVE®), liraglutide (SAXENDA), semaglutide (WEGOVY®, OZEMPIC®, RYBELSUS®), dulaglutide (TRULICITY®), and setomelanotide (IMCIVREE®). Other medications that aim to suppress appetite include phentermine, benzphetamine, diethylpropion, and phendimetrazine. Many side effects have been reported with the use of the medications listed above, some of which come with warnings such as severe liver damage, birth defects, suicidal thoughts, pancreatitis, and thyroid tumors.

[0007] Recently, semaglutide (available as WEGOVY®, 2.4 mg subcutaneous injection once weekly) was approved by the U.S. Food and Drug Administration (FDA) for chronic weight management in adults who are obese or overweight with at least one weight-related condition (e.g., high blood pressure, type 2 diabetes, and high cholesterol) as an addition to a reduced-calorie diet and increased physical activity. The prescribing information for WEGOVY contains a boxed warning to inform health care professionals and patients about the potential risk of thyroid C-cell tumors and pancreatitis, among other risks.

[0008] Myostatin, also known as growth differentiation factor 8 or GDF-8, is a member of the transforming growth factor-β (TGF-β) superfamily. Myostatin is a negative regulator of skeletal muscle growth, although there are suggestions that activin A may play a more prominent role than myostatin in controlling muscle mass in primates (Latres et al., 2017, Nature Communications, 8:15153). Garito et al. (Diabetes Obes Metab. 2018 Jan;20(1):94-102. Epub 2017) and Heymsfield et al. (JAMA Network Open. 2021;4(1):e2033457) reported that bimagrumab, an antibody that binds to activin receptor type IIB, thereby preventing ligand binding, showed clinically beneficial effects in improving body weight, reducing adiposity, and preserving lean mass. Because multiple ligands (including activin, myostatin, GDF11, etc.) signal through this receptor, bimagrumab likely inhibits the activity of all of these growth factors, raising safety concerns: Indeed, clinical trials have found an increase in serious adverse events in bimagrumab-treated patients compared with placebo. Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there remains an unmet need for therapies for subjects suffering from metabolic disorders such as obesity, type 2 diabetes, or obesity associated with type 2 diabetes. [Means for solving the problem]

[0010] The present disclosure provides a novel therapy for improving metabolic pathology, whereby inhibitors of the myostatin signaling pathway and insulin secretagogues (e.g., specifically activators of the glucagon-like peptide-1 (GLP-1) signaling pathway) are used in combination to treat obesity / overweight or for weight management in a subject. In the context of the present disclosure, weight management encompasses not only overall weight loss, but also improvements in body composition, such as enhancing lean mass (muscle) versus fat mass (fat). Combination therapies as well as add-on / adjunctive therapies are contemplated herein.

[0011] The data disclosed herein show that myostatin inhibitors can enhance overall weight loss when used in conjunction with GLP-1 receptor agonists (e.g., GLP-1 analogs). Advantageously, weight loss is achieved while substantially maintaining or enhancing lean mass (e.g., muscle) in diet-induced obese rodent models, demonstrating that body fat tissue is preferentially metabolized relative to muscle tissue, leading to improved body composition (e.g., increased lean mass to fat mass ratio). Surprisingly, these synergistic effects can be attributed to myostatin inhibition alone due to the selectivity of the myostatin inhibitors used, without the contribution of other structurally related growth factors that signal through the same receptor, such as GDF11 and activins. This suggests that GLP-1 pathway activators, when used in conjunction with myostatin pathway inhibitors, can enhance overall weight loss, alter body composition by increasing muscle to fat ratio, and / or otherwise improve the treatment of obese or overweight patients. In addition, the present disclosure examines the effect of treatment with a myostatin inhibitor on metabolic changes associated with weight loss in obese or overweight subjects. It is contemplated that treatment with a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) may reverse metabolic changes associated with weight loss and facilitate maintaining weight loss in obese or overweight subjects, for example, following alterations in diet and / or exercise regimens, alone or in combination with other therapeutic interventions.

[0012] In some embodiments, the disclosure provides an inhibitor of the myostatin signaling pathway (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) for use in treating obesity or overweight in a subject as a monotherapy. In some embodiments, the myostatin signaling pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) is used as an adjunct therapy and the subject has been administered or is being treated with an activator of the GLP-1 signaling pathway. In some embodiments, the myostatin signaling pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) is used in conjunction with an activator of the GLP-1 signaling pathway. In some embodiments, the disclosure encompasses the use of a myostatin signaling pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) to treat a subject with obesity. In some embodiments, the myostatin signaling pathway inhibitor is a myostatin selective inhibitor, such as apitegromab, GYM-329, trevogrumab, or MST1032 mutant as described in PCT / JP2015 / 006323. In some embodiments, the use of a myostatin selective inhibitor may provide a safer treatment option (e.g., less toxicity, improved safety profile, less side effects, and / or better patient tolerability) compared to a non-selective myostatin inhibitor (e.g., a myostatin inhibitor that also inhibits GDF11 and / or activin A). In some embodiments, the use of a myostatin selective inhibitor may provide superior therapeutic efficacy compared to a non-selective myostatin inhibitor (e.g., a myostatin inhibitor that also inhibits GDF11 and / or activin A) (Muramatsu et al. Sci Rep. 2021 Jan 25; 11(1):2160).

[0013] In some embodiments, the myostatin signaling pathway inhibitor is administered intravenously or subcutaneously. In some embodiments, the subject has obesity with type 2 diabetes. In some embodiments, the disclosure encompasses the use of apitegromab to treat an obese subject, the subject has obesity, optionally with type 2 diabetes. In some embodiments, the disclosure encompasses the use of apitegromab in conjunction with a GLP-1 pathway activator to treat an obese subject, the subject has obesity, optionally with type 2 diabetes. In some embodiments, the disclosure encompasses the use of GYM-329 to treat an obese subject, the subject has obesity, optionally with type 2 diabetes. In some embodiments, the disclosure encompasses the use of GYM-329 in conjunction with a GLP-1 pathway activator to treat an obese subject, the subject has obesity, optionally with type 2 diabetes. In some embodiments, the disclosure encompasses the use of MST1032 variants to treat an obese subject, the subject has obesity, optionally with type 2 diabetes. In some embodiments, the present disclosure encompasses the use of an MST1032 variant in conjunction with a GLP-1 pathway activator to treat an obese subject, the subject having obesity, optionally accompanied by type 2 diabetes.

[0014] In some embodiments, the subject to which the myostatin signaling pathway inhibitor is given has previously received a GLP-1 pathway activator (such as a GLP-1 analogue) but failed to achieve a weight loss of at least 5% of baseline body weight after 24 weeks of treatment with a GLP-1 pathway activator and / or was unable to tolerate a therapeutic dose of a GLP-1 pathway activator. In some embodiments, the subject has received a GLP-1 pathway activator (such as a GLP-1 analogue) but failed to achieve a weight loss of at least 10% of baseline body weight after 24 weeks of treatment with a GLP-1 pathway activator. "Baseline body weight" refers to the subject's body weight immediately prior to or at the start of treatment (e.g., prior to 24 weeks of treatment) against which weight change in response to treatment is determined.

[0015] The data provided herein raises the possibility that patients who do not respond adequately to GLP-1 pathway activators or who cannot tolerate the required doses of GLP-1 pathway activators, such as those who are unable to achieve a 5-10% weight loss goal with treatments that include a GLP-1 pathway activator, may benefit from a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin-selective inhibitor) used in conjunction with a GLP-1 pathway activator. Without being bound by any particular theory, it is contemplated herein that parallel inhibition of the myostatin pathway may enhance the effect of GLP-1 pathway activation, improving treatment outcomes, or may allow treatment outcomes with lower doses or less frequent administration of the GLP-1 pathway activator. Thus, patients who are poorly responsive to GLP-1 activation therapy may benefit from the inclusion of a myostatin pathway inhibitor, either as a combination therapy or as an adjunctive therapy. In the context of the present disclosure, the expression "patients with poor response to GLP-1 activation therapy" refers to patients who fail to achieve their intended weight management goals, such as at least 5% or at least 10% weight loss after treatment (such as 3 months, 6 months, 9 months, and 12 months of treatment) with a GLP-1 pathway activator, or who are unable to tolerate the treatment regimen required to reach their intended weight management goals. In a preferred embodiment, the myostatin pathway inhibitor is a myostatin-selective inhibitor, such as an antibody or antigen-binding fragment that selectively binds to myostatin, for example, apitegromab, GYM329 (also known as RO7204239), trevogrumab, or any variant thereof. In another preferred embodiment, the myostatin pathway inhibitor is an MST1032 variant as disclosed in PCT / JP2015 / 006323. In certain embodiments, the GLP-1 pathway activator is a GLP-1 analogue, such as semaglutide, exenatide ER, liraglutide, lixisenatide, tirzepatide, dulaglutide, XW003, neuiglutide, MEDI0382, and albiglutide. In some embodiments, the GLP-1 pathway activator is an agent that affects the GLP-1 pathway, such as metformin, among other activities.

[0016] In some embodiments, the treatment comprising a GLP-1 pathway activator further comprises a calorie restriction regimen (e.g., a low-calorie diet) and / or an exercise regimen (e.g., increased physical activity) as part of the treatment. In some embodiments, a subject who has undergone or is undergoing treatment with a GLP-1 pathway activator is unable to implement or adhere to a calorie restriction regimen and / or an exercise regimen as part of the GLP-1 pathway activator treatment. A myostatin pathway inhibitor used in conjunction can enhance the effect of the GLP-1 pathway activator even in the absence or incomplete adherence to a calorie restriction regimen and / or an exercise regimen. In a preferred embodiment, the myostatin pathway inhibitor is a myostatin selective inhibitor, such as an antibody or antigen-binding fragment that selectively binds to myostatin, e.g., apitegromab, GYM329, trevogrumab, MST1032, or any variant thereof. In some embodiments, the GLP-1 pathway activator is a GLP-1 analogue, such as semaglutide, exenatide ER, liraglutide, tirzepatide, lixisenatide, dulaglutide, XW003, neuiglutide, MEDI0382 and albiglutide. In some embodiments, the GLP-1 pathway activator is a small molecule GLP-1 receptor agonist. In some embodiments, the GLP-1 pathway activator is a long-acting small molecule GLP-1 receptor agonist.

[0017] The present disclosure also provides an activator of the GLP-1 signaling pathway for use in treating obesity or overweight in a subject as an adjunct therapy, where the subject is being treated with an inhibitor of the myostatin signaling pathway.

[0018] The disclosure further provides an inhibitor of the myostatin signaling pathway and an activator of the GLP-1 signaling pathway as a combination therapy for use in treating obesity or overweight in a subject. The disclosure also provides an inhibitor of the myostatin signaling pathway together with an activator of the GLP-1 signaling pathway for use in treating obesity or overweight in a subject. The myostatin signaling inhibitor and the GLP-1 signaling activator can be formulated in a single pharmaceutical composition or can be formulated in separate pharmaceutical compositions and used in amounts sufficient to treat obesity or overweight for weight management.

[0019] In various embodiments, the use of the myostatin signaling pathway inhibitor and the GLP-1 signaling pathway activator may optionally be combined with calorie restriction (e.g., dieting) and / or moderate exercise to further enhance efficacy. In some embodiments, the calorie restriction and / or physical activity requirements of a GLP-1 agonist approved for the treatment of obesity or overweight (such as semaglutide) may be replaced by a myostatin pathway inhibitor (e.g., a myostatin inhibitor) used in conjunction with the approved GLP-1 agonist. In some embodiments, the degree (amount) of calorie restriction and physical activity requirements of the approved GLP-1 agonist may be reduced with the use of a myostatin pathway inhibitor, resulting in less severe calorie restriction and / or less severe physical activity being required to achieve equivalent weight loss benefits.

[0020] In some embodiments, the use of an inhibitor of the myostatin signaling pathway (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) and an activator of the GLP-1 signaling pathway results in overall weight loss while increasing lean mass during the same duration of use. In some embodiments, lean mass (e.g., % change in lean mass) is measured using suitable techniques such as ultrasound, quantitative nuclear magnetic resonance (qNMR), dual energy X-ray absorptiometry (DXA), liver fat percentage obtained by magnetic resonance imaging (MRI), hydrodensitometry, air displacement plethysmography (ADP), bioelectrical impedance analysis (BIA), bioimpedance spectroscopy (BIS), electrical impedance myography (EIM), three-dimensional bioscanner and multi-compartment models (e.g., three-compartment and four-compartment models). Thus, the use of a myostatin pathway inhibitor in conjunction with a GLP-1 pathway activator can result in overall weight loss while preventing muscle loss.

[0021] In some embodiments, the use of an inhibitor of the myostatin signaling pathway (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) and an activator of the GLP-1 signaling pathway can reduce or delay the increase in body fat mass compared to the latter alone. In some embodiments, the use of an inhibitor of the myostatin signaling pathway (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) can facilitate maintaining weight loss in subjects who have previously achieved weight loss. In some embodiments, body fat mass (e.g., % change in body fat mass) is measured using suitable techniques such as ultrasound, quantitative nuclear magnetic resonance (qNMR), dual energy X-ray absorptiometry (DXA), liver fat percentage obtained by magnetic resonance imaging (MRI), hydrodensitometry, air displacement plethysmography (ADP), bioelectrical impedance analysis (BIA), bioimpedance spectroscopy (BIS), electrical impedance myography (EIM), three-dimensional bioscanners and multi-compartment models (e.g., three-compartment and four-compartment models).

[0022] In some embodiments, the disclosure encompasses the use of a myostatin signaling pathway inhibitor (e.g., a myostatin inhibitor, such as a myostatin selective inhibitor) to maintain weight loss in an obese or overweight subject. Without wishing to be bound by theory, it is contemplated that myostatin inhibition may lead to the reversal of certain metabolic changes associated with weight loss in subjects that are otherwise prone to weight regain. Such metabolic changes include, but are not limited to, reduced energy expenditure, reduced lean muscle mass, reduced mechanical efficiency of skeletal muscle, reduced insulin sensitivity, reduced circulating leptin levels, and / or reduced circulating adiponectin levels compared to levels found in subjects prior to treatment, i.e., in the absence of a myostatin inhibitor. Thus, in certain embodiments, the disclosure encompasses the use of a myostatin signaling pathway inhibitor (e.g., a myostatin inhibitor, such as a myostatin selective inhibitor) to reverse metabolic changes associated with weight loss in obese or overweight subjects. The effects of such use may include increased energy expenditure, increased lean muscle mass, increased overall skeletal muscle function, increased insulin sensitivity, increased circulating leptin levels, and / or increased circulating adiponectin levels compared to baseline, i.e., in the absence of a myostatin inhibitor. In certain embodiments, the disclosure encompasses the use of a myostatin signaling pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) to maintain weight loss in an obese or overweight subject. In certain embodiments, the myostatin signaling pathway inhibitor is a myostatin selective inhibitor, such as an antibody or antigen-binding fragment that selectively binds to myostatin, e.g., apitegromab, GYM-329, trevogrumab, or MST1032 variant. In certain embodiments, the myostatin signaling pathway inhibitor is administered intravenously or subcutaneously. In certain embodiments, the subject has previously been administered at least one dose of a GLP-1 pathway activator. In certain embodiments, the subject has been administered a GLP-1 pathway activator. In certain embodiments, a myostatin signaling pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) is used in conjunction with a GLP-1 pathway activator.

[0023] In some embodiments, the disclosure encompasses the use of apitegromab to maintain weight loss in an obese or overweight subject, the obese or overweight subject optionally having type 2 diabetes. In some embodiments, apitegromab is administered in conjunction with or supplemented with a GLP-1 pathway activator. In some embodiments, the disclosure encompasses the use of GYM-329 to maintain weight loss in an obese or overweight subject, the obese or overweight subject optionally having type 2 diabetes. In some embodiments, GYM-329 is administered in conjunction with or supplemented with a GLP-1 pathway activator. In some embodiments, the disclosure encompasses the use of treboglumab to maintain weight loss in an obese or overweight subject, the obese or overweight subject optionally having type 2 diabetes. In some embodiments, treboglumab is administered in conjunction with a GLP-1 pathway activator. In some embodiments, the disclosure encompasses the use of an MST1032 variant to maintain weight loss in an obese or overweight subject, the obese or overweight subject optionally having type 2 diabetes. In some embodiments, the MST1032 variant is administered in conjunction with a GLP-1 pathway activator.

[0024] In some embodiments, the disclosure encompasses the use of myostatin signaling pathway inhibitors (e.g., myostatin inhibitors, such as myostatin selective inhibitors) to treat certain metabolic phenotypes in subjects with leptin deficiency. Such subjects may exhibit metabolic phenotypes including, but not limited to, lower energy expenditure, lower lean muscle mass, lower skeletal muscle mechanical efficiency, lower insulin sensitivity, lower circulating leptin levels, and / or lower circulating adiponectin levels compared to individuals with normal leptin levels (Rosenbaum et al. J Clin Invest. 2005 Dec;115(12):3579-86). Thus, in certain embodiments, the disclosure encompasses the use of myostatin signaling pathway inhibitors (e.g., myostatin inhibitors, such as myostatin selective inhibitors) to reverse metabolic changes associated with leptin deficiency in a subject. Effects of such use may include increased energy expenditure, increased lean muscle mass, increased overall skeletal muscle function, increased insulin sensitivity, and / or increased circulating adiponectin levels compared to baseline. In certain embodiments, the leptin-deficient subject is obese or overweight. In certain embodiments, the disclosure encompasses the use of a myostatin signaling pathway inhibitor (e.g., a myostatin inhibitor, such as a myostatin selective inhibitor) for weight loss in an obese or overweight subject with leptin deficiency. In certain embodiments, the use of a myostatin signaling pathway inhibitor maintains weight loss in a leptin-deficient subject. In certain embodiments, the subject has previously been administered at least one dose of a GLP-1 pathway activator. In certain embodiments, the subject has been administered a GLP-1 pathway activator. In certain embodiments, the myostatin signaling pathway inhibitor, a myostatin signaling pathway inhibitor (e.g., a myostatin inhibitor, such as a myostatin selective inhibitor) is used in conjunction with a GLP-1 pathway activator.

[0025] According to the present disclosure, inhibitors of the myostatin signaling pathway are used as monotherapy or in conjunction with activators of the GLP-1 signaling pathway to treat obesity (e.g., excess adiposity, overweight) in a subject. Inhibitors of the myostatin signaling pathway can be any agent capable of suppressing myostatin signaling. In some embodiments, inhibitors of the myostatin signaling pathway useful in practicing the present invention can be myostatin-selective inhibitors. In some embodiments, inhibitors of the myostatin signaling pathway useful in practicing the present invention can be myostatin-nonselective inhibitors. In various embodiments, inhibitors of the myostatin signaling pathway useful in practicing the present invention can include low molecular weight compounds (i.e., small molecules) that inhibit the pathway, as well as biologics such as antibodies or antigen-binding fragments thereof, and engineered protein constructs that contain ligand-binding domains (such as soluble receptor ligand traps, follistatin-based engineered constructs, and adnectins). In some embodiments, the myostatin pathway inhibitor is an antibody (e.g., an antigen-binding fragment thereof or an engineered construct comprising such a fragment) that binds to mature myostatin (also known as GDF-8 or GDF8). Such an antibody is typically referred to as a neutralizing antibody because it blocks its ability to bind to endogenous receptors to bind growth factors and thereby activate signaling pathways. In some embodiments, the myostatin pathway inhibitor binds to the myostatin receptor, thereby interfering with ligand binding. These include antibodies that bind to one or more extracellular portions of the receptor. A non-limiting example of such an antibody is bimagrumab (BYM338), which binds to ActRIIB. In preferred embodiments, the inhibitor of the myostatin signaling pathway useful in practicing the present invention is a myostatin selective inhibitor. In some embodiments, the myostatin selective inhibitor is a neutralizing antibody that selectively binds to mature myostatin (but not to other growth factors such as GDF11 or activin A). One example of such a myostatin-selective antibody is trevoglumab, also known as REGN1033.In some embodiments, the myostatin selective inhibitor is an antibody or antigen-binding fragment thereof that binds to the pro / latent myostatin complex, thereby inhibiting activation (e.g., release) of mature myostatin. Such an antibody or antigen-binding fragment is referred to as an "activation inhibitor" of myostatin. Non-limiting examples of myostatin selective activation inhibitors include apitegromab (also known as SRK-015) and variants thereof, GYM329 and variants thereof, or MST1032 variants as described in PCT / JP2015 / 006323.

[0026] According to the present disclosure, an activator of the GLP-1 signaling pathway is used in combination with an inhibitor of the myostatin signaling pathway to treat obesity (e.g., excess fat accumulation, overweight) in a subject. An activator of the GLP-1 signaling pathway can include any agent capable of enhancing GLP-1 signaling. In some embodiments, an activator of the GLP-1 signaling pathway useful in carrying out the present invention is a GLP-1 receptor agonist, such as a GLP-1 analog. Endogenous GLP-1 is easily degraded by peptidases and has a short half-life. Therefore, certain modifications can be introduced into peptide hormones to create GLP-1 analogs, with the goal of extending their active time or stabilizing their activity. In some embodiments, the GLP-1 analog can include the amino acid sequence EGTFTSD (SEQ ID NO: 116). In some embodiments, the GLP-1 analog includes the amino acid sequence HXXGXFTXD (SEQ ID NO: 117), where X is any amino acid residue. Non-limiting examples of GLP-1 analogues include semaglutide, exenatide ER, liraglutide, lixisenatide, tirzepatide, dulaglutide, XW003, neuiglutide, MEDI0382 and albiglutide. In some embodiments, activators of the GLP-1 signaling pathway useful in carrying out the present invention are inhibitors of peptidases involved in the degradation of GLP-1. In some embodiments, the peptidase inhibitor inhibits dipeptidyl peptidase 4 (DPP-4) and / or neutral endopeptidase (NEP). In some embodiments, activators of the GLP-1 signaling pathway useful in practicing the present invention are agents capable of modulating GLP-1 receptor transport (e.g., agents that cause a slowdown in receptor internalization); agents that increase cell surface GLP-1 receptor expression; agents that enhance ligand-induced GLP-1 receptor activation; agents that enhance pathways downstream of the GLP-1 receptor, such as cAMP signaling, and / or agents that antagonize phosphatases involved in the dephosphorylation of the GLP-1 receptor.

[0027] The therapeutic methods disclosed herein aim to provide improved weight management regimens. Thus, inhibitors of the myostatin signaling pathway are used as monotherapy or in combination with activators of the GLP-1 signaling pathway to treat obesity or overweight in subjects in need thereof for weight management, e.g., chronic weight management.

[0028] In some embodiments, the determination of obesity and overweight is based on the body mass index (BMI), which is the most commonly used tool for predicting and screening for overweight and obesity in adults and children. In some embodiments, the BMI of an adult subject treated with a combination or adjunctive therapy according to the present disclosure is 25 kg / m 2 In some embodiments, the adult subject is between 25 and 29.9 kg / m 2 In some embodiments, an adult subject treated with a combination or adjunctive therapy of the present disclosure has a BMI of 30 kg / m 2 or higher (obese). In some embodiments, the BMI of an adult subject treated with a combination or adjunctive therapy of the present disclosure is 30-40. In some embodiments, the BMI of an adult subject treated with a combination or adjunctive therapy of the present disclosure is 40 or higher (extremely obese). In some embodiments, the BMI of a child or adolescent subject aged 2-19 years treated with a combination or adjunctive therapy of the present disclosure is equal to or higher than the 85th percentile of a child or young adult of the same sex and age (e.g., a BMI equal to or higher than the 85th percentile on the CDC growth curve). In some embodiments, the BMI of a subject aged 2-19 years treated with a combination or adjunctive therapy of the present disclosure is equal to or higher than the 95th percentile of a child or young adult of the same sex and age (e.g., a BMI equal to or higher than the 95th percentile on the CDC growth curve). In some embodiments, a subject between 2 and 19 years of age treated with a combination or adjunctive therapy disclosed herein has a BMI that is greater than or equal to 120 percent of the 95th percentile for children or young adults of the same sex and age (e.g., a BMI greater than or equal to 120 percent of the 95th percentile on the CDC growth curve).

[0029] In some embodiments, the subject also suffers from or is at risk of developing one or more weight-related conditions, such as high blood pressure, type 2 diabetes, heart disease, stroke, fatty liver disease, kidney disease, or high cholesterol. In some embodiments, the subject is an adult subject. In some embodiments, the subject is a child or adolescent subject between the ages of 2 and 19. In some embodiments, the adult subject is over 27 kg / m2 with at least one weight-related condition. 2 or for adult subjects, a body mass index (BMI) of 30 kg / m 2 In some embodiments, the adult subject has a BMI of 28 to 40 kg / m 2 and optionally the subject has an HbA1c of 6.5-10%. In some embodiments, the subject is 2-19 years old and has a BMI that is equal to or greater than the 85th percentile of a child or young adult of the same sex and age (e.g., a BMI equal to or greater than the 85th percentile on the CDC growth curve). In some embodiments, the subject is 2-19 years old and has a BMI that is equal to or greater than the 95th percentile of a child or young adult of the same sex and age (e.g., a BMI equal to or greater than the 95th percentile on the CDC growth curve). In some embodiments, the subject is 2-19 years old and has a BMI that is equal to or greater than the 120th percentile of a child or young adult of the same sex and age (e.g., a BMI equal to or greater than the 95th percentile on the CDC growth curve). In some embodiments, the subject is on a diet (e.g., a calorie restriction). In some embodiments, the subject is on an exercise regimen.

[0030] Thus, the therapeutic methods disclosed herein may be useful for chronic weight management with the goal of treating excess adiposity and metabolic derangements in obese subjects, optionally having or at risk of developing obesity-related type 2 diabetes, while maintaining or improving the subject's body composition (e.g., lean mass to fat mass ratio).

[0031] The present disclosure also encompasses various embodiments of therapeutic uses in which an inhibitor of the myostatin signaling pathway is administered as monotherapy or in conjunction with an activator of the GLP-1 signaling pathway to improve body composition in a subject. In some embodiments, the subject may be obese or overweight. In some embodiments, the subject may have or be at risk of developing one or more metabolic conditions, such as type 2 diabetes or metabolic syndrome. In some embodiments, the therapeutic uses disclosed herein can improve body composition in a subject even if the subject does not experience overall weight loss (e.g., neutral effect on body weight) or even if the subject experiences overall weight gain. In some embodiments, the therapeutic uses increase lean mass. In some embodiments, the therapeutic uses decrease fat mass. In some embodiments, the therapeutic uses increase the lean mass to fat mass ratio. In some embodiments, the therapeutic uses have a neutral effect on body weight. In some embodiments, the therapeutic uses achieve overall weight loss in the subject. In some embodiments, the therapeutic uses maintain weight loss in subjects who previously achieved weight loss. Body composition may be determined or measured using suitable techniques such as qNMR, dual energy x-ray absorptiometry (DXA), liver fat percentage obtained by magnetic resonance imaging (MRI), hydrodensitometry, air displacement plethysmography (ADP), bioelectrical impedance analysis (BIA), bioimpedance spectroscopy (BIS), electrical impedance myography (EIM), three-dimensional bioscanners and multi-compartment models (e.g., three-compartment and four-compartment models).

[0032] Advantageously, preferred embodiments utilize selective inhibitors of myostatin that offer an improved safety profile compared to non-selective inhibitors of myostatin. [Brief description of the drawings]

[0033] [Figure 1A]1 shows overall (whole body) weight loss in groups treated with either the myostatin selective inhibitor Ab2 or a control antibody during 20% ​​or 30% calorie restriction (CR). The left panel shows the animal weights over the course of the study, and the right panel shows the percent change in weight. [Figure 1B] 1 shows the percent change in lean muscle mass in groups treated with either the myostatin selective inhibitor Ab2 or a control antibody during 20% ​​or 30% caloric restriction (CR) as measured by qNMR. [Figure 1C] A comparison of the percent change in lean mass during the study measured on day 44 compared to baseline is shown. [Figure 1D] The mean weight in milligrams of the left and right gastrocnemius muscle samples taken at the end of the study are shown. [Figure 1E] The percentage difference in weight from the gastrocnemius muscle shown in FIG. 1D compared to the gastrocnemius muscle weight of control mice fed a high-fat diet ad libitum is shown. [Figure 1F] The percent fat mass loss is shown compared to the percent fat mass loss achieved by control mice fed a high fat diet ad libitum. [Figure 1G] 1 shows that Ab2 significantly increased the rate of fat mass loss in 20% calorie restricted animals. [Figure 1H] Inguinal fat pad weights (average of both left and right fat pads) collected at the end of the study are shown. Ab2 significantly reduced inguinal fat pad weight in 20% calorie restricted animals. [Figure 1I] (Left panel) Serum leptin levels in mice on a 20% calorie restriction compared to mice fed a high fat diet ad libitum. (Right panel) Leptin levels normalized by percentage fat. [Figure 2A] 1 shows the results of an initial dose-ranging study for liraglutide in the C57BL / 6NTac DIO mouse model. [Figure 2B]Percent body weight change from baseline at the end of the 30-day study in mice receiving control IgG1 alone, Ab2 alone, or IgG1 or Ab2 in combination with liraglutide at 0.03, 0.06, or 0.10 mg / kg. Liraglutide induced a dose-proportional loss in body weight, with Ab2 enhancing weight loss in the 0.1 mg / kg liraglutide group compared to the IgG control. [Figure 2C] 4 shows the percent change in lean mass compared to baseline as measured by qNMR. [Figure 2D] Percent change in gastrocnemius muscle mass is shown. [Figure 2E] 1 shows whole body percent change in fat mass compared to baseline as measured by qNMR. [Figure 2F] The percent change in inguinal fat pad weight is shown. [Figure 2G] Shown are total lipid counts in livers from mice at the end of the study as measured by histology. [Figure 3A] Figure 2 shows that myostatin-blocking antibody Ab2 increased VO2 in 30% calorie-restricted animals. [Figure 3B] 1 shows that myostatin-blocking antibody Ab2 increased VO2 in 30% calorie-restricted animals when normalized for reduced body weight. [Figure 3C] These results show that when normalized for lean mass, myostatin-blocking antibody Ab2 had no effect on VO2 in 30% calorie-restricted animals. [Figure 3D] 1 shows that myostatin-blocking antibody Ab2 increased energy expenditure in 30% calorie-restricted animals. [Figure 3E] 1 shows that the myostatin-blocking antibody Ab2 had no effect on respiratory exchange ratio (RER) in 30% calorie-restricted animals. [Figure 4] 1 shows body weight over time in mice undergoing 20% ​​caloric restriction treated with antibody Ab2. The left panel shows total body weight over the 44 day study, and the right panel shows percent change. [Figure 5A] 1 shows that myostatin-inhibiting antibody Ab2 preserved lean body mass in 20% calorie-restricted animals. [Figure 5B] Animals treated with anti-myostatin antibody Ab2 showed significantly less loss of lean body mass compared to animals treated with the control antibody. [Figure 5C] 1 shows that the myostatin inhibitor Ab2 increased gastrocnemius muscle weight in 20% calorie restricted animals. [Figure 5D] Calorie restricted animals treated with control antibody lost gastrocnemius muscle mass, whereas calorie restricted animals treated with Ab2 showed a significant increase in gastrocnemius muscle mass. [Figure 5E] 1 shows that myostatin inhibitor Ab2 reduced both O2 consumption and CO2 output in 20% calorie restricted animals compared to control antibody treated mice. [Figure 5F] Shown is the average respiratory exchange ratio as a function of time over the entire 72-hour period that mice spent in metabolic cages. [Figure 5G] Physical activity levels in mice subjected to 20% calorie restriction and treated with antibody Ab2. [Figure 6] 1 shows the reduction in fat mass in mice treated with Ab2 and / or 20% calorie restriction. [Figure 7] Oxygen and energy consumption in mice treated with Ab2 and / or 20% caloric restriction are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The present invention is based, at least in part, on the discovery that administration of a myostatin inhibitor, such as a myostatin pathway inhibitor, e.g., a myostatin selective inhibitor, in a subject having a metabolic disease, e.g., obesity and / or type 2 diabetes mellitus (T2DM), significantly improves both the physiological and functional characteristics of the affected subject (e.g., a mammalian subject) (e.g., improved body composition, glucose tolerance, etc.). In particular, the inventors have surprisingly discovered that administration of a combination of a myostatin pathway inhibitor and a GLP-1 pathway activator can promote the preservation of lean mass in such subjects, prevent or reduce the increase in body fat mass, improve body composition by increasing muscle-to-fat ratio, and / or provide additional benefits in conjunction with or without the need for caloric restriction and / or increased exercise.

[0035] definition The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical referent of the article. By way of example, "an element" means one element or more than one element.

[0036] Except in the examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." The term "about," when used in connection with percentages, can mean ±1%. Additionally, the term "about" can mean within ±1% of a value.

[0037] GLP-1 signaling pathway activator: The terms "GLP-1 signaling pathway activator" and "GLP-1 pathway activator" are used interchangeably herein and encompass any agent that increases or enhances the activity of the GLP-1 signaling pathway, regardless of mechanism of action. Increased or enhanced activity of the GLP-1 signaling pathway can be the result of, for example, greater activity, longer duration of activity, increased availability of one or more components of the signaling pathway, etc.

[0038] Adjunctive Therapy: The terms "adjunctive therapy" and "add-on" therapy are used interchangeably herein and are intended to refer to a treatment regimen in which a second agent (used as adjunctive therapy) is administered to a subject who is taking, has been administered, or is being treated with a first agent (e.g., background therapy). The terms "in conjunction with" and "complementary to" are used interchangeably herein and are intended to refer to therapies used together, whether parallel in time or partially overlapping.

[0039] Administer / Administration: The terms "administer," "administering," or "administration" include any method or act of delivery of a pharmacological agent (e.g., a pharmaceutical agent) to an intended subject, e.g., a patient. A pharmacological agent can be a biologic, such as an antibody or antigen-binding fragment thereof (e.g., a pharmaceutical composition comprising such an antibody or antigen-binding fragment, a peptide drug such as a hormone and modified or synthetic analogues thereof, or a low molecular weight drug (i.e., a structurally defined small molecule or chemical entity), either to the subject's entire body or to a specific area in or on the subject's body (systemic administration and local administration, respectively).

[0040] Adult: As used herein, in the context of weight management, an "adult" patient refers to an individual who is greater than 19 years of age (ie, 20 years of age or older) unless otherwise specified.

[0041] Antibody: As used herein, the term "antibody" includes full-length immunoglobulins, antigen-binding portion(s) / fragment(s) thereof, and any variants thereof that retain the ability to bind to a target antigen. Antibodies include human antibodies and humanized antibodies.

[0042] Background Therapy: In relation to add-on or adjunctive therapy, the term "background therapy" refers to an approved treatment that a patient or patient population has received or is receiving before a second therapy (e.g., before adjunctive administration of a myostatin pathway inhibitor) that is to be added as an adjunctive therapy for the same indication that is available to the patient or patient population for a particular indication or condition related to that indication. Typically, background therapy is the standard of care for the condition. For example, a type 2 diabetes patient may be taking a biguanide as background therapy, such as metformin (e.g., Fortamet, Glucophage, Glucophage XR, Glumetza, Riomet, Obimet, Gluformin, Dianben, Diabex, Diaformin, Metsol, Siofor, Metforgamma, and Glifor) or a metformin-containing medication that includes one or more additional active agents (e.g., thiazolidinediones (glitazones) and rosiglitazone). For example, a type 2 diabetes patient may be taking a sulfonylurea (e.g., glimepiride, glipizide, glyburide, glyparamide, or chlorpropamide. By way of example, a type 2 diabetes patient may also be taking a thiazolidinedione, such as pioglitazone.

[0043] Baseline: The terms "baseline" and "baseline measurement" refer to a measurement of a particular metric in a subject or population of subjects before an action is taken to alter that metric, e.g., at the beginning of a study (before a dose is administered). For example, in studying the treatment of a metabolic disease or disorder, baseline or baseline measurement can refer to, among other measurements, leptin levels, ghrelin levels, adiponectin levels, body weight, fat mass (e.g., total fat mass or visceral fat mass), lean mass (e.g., muscle mass), insulin levels (fasting or non-fasting), insulin sensitivity, glucose levels (fasting or non-fasting), gastric emptying rate, metabolic rate (e.g., measured by oxygen consumed and carbon dioxide excreted in a defined period of time), HbA1c levels, blood pressure and pulse, lipoprotein lipid measurements, and / or waist circumference measurements.

[0044] Body composition: The term "body composition" refers to the relative constituents that make up the body, including fat mass, muscle (lean) mass, bone, and water. In particular, in the context of weight management, body composition refers to the ratio of lean to fat mass of the body. Unless expressly stated, the term as used herein refers to total body (whole body) body composition. Body composition can be measured by a variety of suitable methods and techniques, including body density, dual energy x-ray absorptiometry (DEXA), air displacement plethysmography (ADP), bioelectrical impedance analysis (BIA), body volume index (BVI), subcutaneous fat (with measuring calipers), ultrasound, quantitative magnetic resonance (QMR), circumference (e.g., measured by waist circumference), and other measurements.

[0045] Body Mass Index (BMI): The term "Body Mass Index" or "BMI" is a number derived from a person's mass and height and is defined as weight in kilograms divided by height in meters squared (kg / m 2 BMI is expressed in units of 100. BMI provides a general weight-height relationship that can be used to classify people as underweight, normal weight, overweight, obese, or extremely obese based on tissue mass (muscle, fat, and bone) relative to their height.

[0046] Calorie restriction / caloric restriction: This term refers to a form of dieting that requires a reduction in overall calorie intake.

[0047] Combination therapy: As used herein, "combination therapy" refers to a treatment regimen involving the administration of two or more active agents (e.g., two or more pharmacological agents) intended to treat a given indication and / or its associated pathology. The two or more agents may be formulated as separate compositions (e.g., formulations) or may be formulated as a single composition (formulation). "Combination therapy" encompasses therapies used in conjunction with and as complements of each other.

[0048] The term "reduce" or "reducing" as used herein refers to a statistically significant decrease in such levels in relation to disease symptoms. The decrease can be, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% below the detection level of the detection method. The decrease can also be, for example, about 1-10%, 10-20%, 1-30%, 20-50%, 30-60%, 40-70%, 50-80% or 60-90%. In certain embodiments, the reduction in an individual with a disorder can achieve a level that is accepted as being within the normal range for an individual without such disorder.

[0049] Diet / Dietary Regimen: In the context of the present disclosure, certain diets may be adopted as part of weight management, e.g., obesity treatment, including pharmacological intervention. Diets may include caloric restriction / calorie restriction (i.e., reduced caloric intake or reduced absorption of calories) as well as changes in the choice regarding the type of food consumed (e.g., high protein, low fat or low carbohydrate regimens). Thus, a patient is "on a diet or low calorie regimen" when he or she adopts or is instructed by a physician or equivalent to adopt a diet as part of an overall treatment regimen, e.g., weight management.

[0050] Effective amount: As used herein, the terms "effective amount" and "effective dose" refer to any quantity or dose of a compound or composition sufficient to achieve its intended purpose or purposes, i.e., a desired biological or medical response, in a tissue or subject at an acceptable benefit-to-risk ratio. For example, in certain embodiments of the invention, the intended purpose may be to inhibit activation of myostatin in vivo and achieve a clinically meaningful outcome associated with myostatin inhibition.

[0051] In some embodiments, an effective amount is an amount that, when administered according to a particular regimen, results in a positive clinical outcome with a reasonably acceptable level of adverse effects (e.g., toxicity), such that the adverse effects, if any, are sufficiently tolerable for the patient to continue the treatment regimen, and the benefits of the therapy outweigh the risks of toxicity. One of skill in the art will appreciate that in some embodiments of the invention, a unit dosage amount may be considered to contain an effective amount if it contains an amount appropriate for administration in conjunction with a dosage regimen that correlates with a positive outcome.

[0052] The therapeutically effective amount is often administered in a dosage regimen that may include multiple unit doses. For any particular pharmaceutical agent, the therapeutically effective amount (and / or the appropriate unit dose within the effective dosage regimen) may vary, for example, depending on the route of administration, combination with other pharmaceutical agents. In some embodiments, the specific therapeutically effective amount (and / or unit dose) for any particular patient may depend on a variety of factors, including the disorder under treatment and the severity of the disorder; the activity of the specific pharmaceutical agent used; the specific composition used; the age, weight, general health, sex and diet of the patient; the timing, route of administration and / or excretion or metabolic rate of the specific pharmaceutical agent used; duration of treatment; and similar factors as are well known in the medical arts.

[0053] As used herein, "effective amount" can also refer to the amount of each active agent required to confer a therapeutic effect on a subject, either alone or in combination with one or more other active agents. For example, an effective amount refers to an amount of a myostatin inhibitor, such as an antibody or antigen-binding fragment thereof, disclosed herein that is sufficient to achieve a biological effect, such as an increase in muscle mass or muscle fiber diameter, a conversion of muscle fiber type, an increase in the amount of force generated by a muscle, an increase in the amount and / or function of muscle tissue in a subject; an increase in metabolic rate in a subject; an increase in insulin sensitivity in a subject; an increase in the level of brown adipose tissue in a subject; an increase in the level of beige adipose tissue in a subject; a decrease in the level of white adipose tissue in a subject; a decrease in the level of visceral adipose tissue in a subject; a decrease in the ratio of fat to muscle tissue in a subject; an increase in glucose uptake by brown adipose tissue, beige adipose tissue, or muscle tissue in a subject; a decrease in glucose uptake by white adipose tissue or hepatic tissue; a decrease in muscle protein catabolism and / or muscle amino acid release in a subject; an increase in insulin-dependent glycemic control in a subject; a decrease in intramuscular fat infiltration in a subject; or a clinically important outcome in a subject, such as partial or complete reversal of insulin resistance, overweight, or glycemic control or prevention of muscle loss or muscle atrophy; and / or prevention of the onset of a metabolic disease in a subject.

[0054] Effective amounts, as will be appreciated by those of skill in the art, will vary depending on the precise condition under treatment, the severity of the condition, individual patient parameters including age, health, size, sex and weight, duration of treatment, the nature of concurrent therapy (if any), the particular route of administration, and similar factors within the knowledge and expertise of the health practitioner, which are well known to those of skill in the art and can be addressed with no more than routine experimentation.

[0055] Efficacy: The term "efficacy" refers to a measurable biological or medical response in a subject as a result of administering a therapy in an amount sufficient to achieve its intended purpose or purposes. For example, in certain embodiments of the invention, the efficacy of a myostatin pathway inhibitor may refer to any desired, clinically meaningful outcome associated with the use of the inhibitor. In some embodiments, the efficacy of a GLP-1 pathway activator may refer to any desired, clinically meaningful outcome associated with the use of a GLP-1 pathway activator. In some embodiments, the efficacy of a myostatin pathway inhibitor used in conjunction with a GLP-1 pathway activator is measured in a patient or patient population. In some embodiments, the efficacy of a combination therapy comprising a myostatin pathway inhibitor and a GLP-1 pathway activator is measured in a patient or patient population treated with the combination. In some embodiments, the efficacy of an adjunctive therapy comprising a myostatin pathway inhibitor is measured in a patient or patient population treated with a GLP-1 pathway activator. In some embodiments, the efficacy of an adjunctive therapy comprising a GLP-1 pathway activator is measured in a patient or patient population treated with a myostatin pathway inhibitor. In combination or adjunctive therapy, the efficacy measured is preferably higher (more clinically meaningful) than either monotherapy. In some embodiments, therapeutic efficacy refers to a clinically meaningful outcome for a subject (e.g., a mammalian subject) with a metabolic disorder, such as alleviation, reduction, delay, or prevention of one or more symptoms of the metabolic disorder in a subject receiving a myostatin pathway inhibitor in combination with a GLP-1 pathway activator. In some embodiments, such efficacy is assessed by measuring changes in body composition (e.g., changes in muscle-to-fat ratio) at the end of a study or treatment regimen, for example, by DXA, qNMR, etc. In some embodiments, therapeutic efficacy is determined by measuring glycemic control / insulin sensitivity (e.g., HbA1c measurements, fasting glucose and insulin, etc.), anthropometric measurements (weight, BMI, waist circumference, weight-to-hip ratio, fat or muscle loss, stability, or gain), each compared to baseline. In some embodiments, measurements of PK / PD markers, such as serum latent myostatin, can be used as efficacy measurements.In some embodiments, a therapeutically effective outcome of treatment with a myostatin pathway inhibitor and a GLP-1 pathway activator is an increase in muscle-to-fat ratio, regardless of changes in other characteristics, such as body weight, relative to baseline. In some embodiments, a therapeutically effective outcome includes a reduction in body weight, a reduction or stabilization of body fat mass, and / or an increase in muscle mass compared to baseline.

[0056] Exercise / exercise regimen: As used herein, the term "exercise" includes any physical activity. A patient is on an "exercise regimen" when he or she has incorporated, or is instructed by a physician or equivalent to incorporate, increased physical activity into an overall treatment regimen, e.g., as part of weight management.

[0057] Fc variant: The term "Fc variant" refers to an antibody (immunoglobulin) that contains one or more mutations in its Fc region, typically with the same CDR sequences as the parent (reference) antibody from which it is derived. Fc variants can be engineered to have altered (e.g., increased) affinity for an FcR, such as FcRn. In some embodiments, the altered affinity for FcRn results in a changed in vivo biological activity, such as a longer serum half-life, of the Fc variant compared to its parent antibody lacking one or more Fc mutations.

[0058] The term "gastric emptying" refers to the process by which the contents of the stomach enter the small intestine (i.e., the duodenum). The term "postprandial gastric emptying" refers to the rate at which gastric emptying occurs after a meal. Gastric emptying can be measured by any method known in the art, such as scintigraphy, magnetic resonance imaging (MRI), and real-time ultrasound.

[0059] GLP-1 analogues: As used herein, the term "GLP-1 analogues" refers to peptides or modified peptides that have structural and functional similarity to naturally occurring GLP-1 and have the ability to bind and activate GLP-1 receptors. Non-limiting examples of GLP-1 analogues include semaglutide, exenatide ER, liraglutide, lixisenatide, tirzepatide, dulaglutide, XW003, neuiglutide, MEDI0382 and albiglutide. GLP-1 analogues are intended to include peptides or modified peptides that include the amino acid sequence EGTFTSD (SEQ ID NO: 116). GLP-1 analogues are intended to include peptides or modified peptides that include the amino acid sequence HXXGXFTXD (SEQ ID NO: 117), where X is any amino acid residue.

[0060] GLP-1 receptor agonist: The term "GLP-1 receptor agonist" refers to an agent capable of binding to and activating the GLP-1 receptor. GLP-1 is a naturally occurring agonist of the GLP-1 receptor. GLP-1 receptor agonists include GLP-1 analogs. The GLP-1 receptor agonist can be a small molecule GLP-1 receptor agonist. In some embodiments, the GLP-1 receptor agonist is a long-acting small molecule GLP-1 receptor agonist.

[0061] GLP-1 Pathway Activators: The terms "GLP-1 pathway activator" and "activator of the GLP-1 signaling pathway" are used interchangeably herein and encompass any agent that increases or enhances the activity of the GLP-1 signaling pathway, regardless of mechanism of action. Increased or enhanced activity of the GLP-1 signaling pathway can be the result of, for example, a greater degree of activity, a longer duration of activity, increased availability of one or more components of the signaling pathway, etc. In some embodiments, GLP-1 pathway activators include agents that modulate upstream regulators of GLP-1 (e.g., dipeptidyl peptidase (DPP-IV) inhibitors). In some embodiments, GLP-1 pathway activators include agents that control the amount or activity of GLP-1 (e.g., agents that increase production or secretion of GLP-1; GLP-1 stabilizers). In some embodiments, GLP-1 pathway activators include agents that increase activation of the GLP-1 receptor (GLP-1R). In some embodiments, agents that increase activation of GLP-1R include GLP-1 agonists, including GLP-1 analogs. In some embodiments, GLP-1 pathway activators include agents that activate downstream signaling of GLP-1R (e.g., activators of PI3K, PKC, cAMP, etc.). In some embodiments, GLP-1 pathway activators can include agents that modulate receptor GLP-1R expression and / or trafficking (e.g., inhibitors of GLP-1R internalization; see, e.g., Jones et al., Nat. Comm. (2018) 9:1602). GLP-1 pathway activators include activators of GLP-1R. In a preferred embodiment, the GLP-1 pathway activator is a GLP-1 receptor agonist. GLP-1 pathway activators include, but are not limited to, antibodies and antigen-binding fragments thereof, engineered protein constructs (such as Fc conjugates and multifunctional molecules that include GLP-1 analogs), peptides, and small molecules. Treatment regimens that include a GLP-1 pathway activator (such as a GLP-1 analogue) may further include diet (e.g., a low-calorie diet or calorie restriction) and / or exercise (e.g., increased physical activity) as part of the treatment.In some embodiments, the patient is provided with diet and / or exercise counseling.

[0062] Human / humanized antibodies: The term "human antibodies" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences and fragments thereof. Human antibodies of the present disclosure may include, for example, in the CDRs and particularly CDR3, amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The term "humanized antibodies" as used herein refers to antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibodies produced in humans. "Humanized antibodies" may refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0063] The term "increase" refers to a statistically significant increase in such levels, for example in association with a disease where a disease-related loss or amount of function, such as loss of muscle mass, is a symptom. The increase can be, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or above the detection level of the detection method. The increase can also be, for example, about 1-10%, 10-20%, 1-30%, 20-50%, 30-60%, 40-70%, 50-80% or 60-90%. In certain embodiments, the increase is up to a level that is accepted as being within the normal range for individuals without such disorder, which can be referred to as normalization of the level. In certain embodiments, the increase is a normalization of the level of a sign or symptom of the disease, an increase in the difference between the subject level of the sign of the disease and the normal level of the sign of the disease. In certain embodiments, the method comprises an increase in muscle tissue mass and / or function following treatment of a subject with an antibody that specifically binds pro / latent myostatin. In certain embodiments, the method comprises an increase in promyostatin levels in a target muscle compared to control promyostatin levels.

[0064] Inhibitors of myostatin signaling pathway: The terms "inhibitors of the myostatin signaling pathway" and "myostatin pathway inhibitors" are used interchangeably herein and encompass any agent that decreases (reduces) or suppresses the activity of the myostatin signaling pathway, regardless of mechanism of action. A reduction or suppression of myostatin signaling pathway activity can be the result, for example, of a reduced magnitude of activity, a reduced duration of activity, a reduced availability of one or more components of the signaling pathway, etc.

[0065] Insulin sensitivity; insulin resistance: The term "insulin sensitivity" refers to the metabolic action of insulin to promote glucose disposal in the body of a subject. A subject is said to exhibit increased insulin sensitivity when the subject requires less insulin to lower blood glucose levels compared to the average in the human population. In contrast, a subject is said to exhibit decreased insulin sensitivity when the subject requires more insulin to lower blood glucose levels. A subject is said to exhibit "insulin resistance" when the amount of exogenous or endogenous insulin required to increase glucose uptake and utilization of the subject is significantly higher than that of a healthy subject. For example, a subject is said to be "insulin resistant" when the amount of exogenous or endogenous insulin required to increase glucose uptake and utilization of the subject is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to a healthy subject.

[0066] Lean / Lean Mass: As used herein, "lean" mass or tissue generally corresponds to muscle mass or tissue, as opposed to fat mass or tissue (e.g., fat). Typically, a greater ratio of lean mass compared to fat mass in the body indicates a more desirable body composition.

[0067] Ligand trap: As used herein, the term "ligand trap" refers to a molecule or class of molecules that contain a ligand-binding fragment / moiety that acts as a "trap" to sequester a ligand (such as a growth factor) and thereby prevent the ligand from binding to and activating an endogenous receptor. A ligand trap can be derived by importing the ligand-binding moiety from the ligand's naturally occurring receptor. Examples of myostatin ligand traps include, without limitation, soluble receptor-based ligand traps, follistatin-based ligand traps, and the like. A myostatin ligand trap may not be selective for myostatin and may also bind to and inhibit additional ligands, such as GDF11 and activin A.

[0068] Mature myostatin: The term "mature myostatin" refers to the dimeric growth factor, also known as GDF8, which is released from the latent myostatin complex. Mature myostatin is a soluble, biologically active ligand that has the ability to bind to and activate its receptor. Unless otherwise specified, the term "mature myostatin" refers to the fully processed, biologically active form of myostatin. The wild-type sequence of mature myostatin is provided as SEQ ID NO: 56. In some cases, mature myostatin may contain one or more mutations, which may exhibit changes in structure / function or stability.

[0069] SEQ ID NO:56 DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS

[0070] Metabolic Disorder: The term "metabolic disorder" may also be referred to as metabolic disease or metabolic condition and includes any condition involving dysregulation of the body's metabolic functions resulting in a disturbance in the normal physiological state of homeostasis due to altered metabolism (anabolic and / or catabolic). Metabolic disorders may be inherited or acquired. Non-limiting examples of metabolic disorders include obesity / overweight, type 2 diabetes mellitus, obesity-associated type 2 diabetes mellitus, and metabolic syndrome.

[0071] Metabolic rate: The term "metabolic rate" refers to the amount of energy expended during a particular period of time. It is typically measured in calories, kilocalories, or joules. Metabolic rate can be expressed as oxygen consumed or carbon dioxide produced per unit of time.

[0072] Metabolism: The term "metabolism" refers to the processes involved in the synthesis and breakdown of components of the body, such as fat (e.g., adipose tissue), muscle, and bone. Thus, "fat metabolism" refers to the process of fat synthesis and breakdown.

[0073] Myostatin: In the context of this disclosure, unless expressly defined otherwise, the term "myostatin" can refer to any form of myostatin protein, including promyostatin, latent myostatin, and mature myostatin, each of which exists as a dimer in vivo.

[0074] Myostatin inhibitor: As used herein, the term "myostatin inhibitor" refers to any agent that inhibits one or more forms of myostatin (e.g., promyostatin, latent myostatin, and / or mature myostatin) and includes both selective inhibitors of myostatin and non-selective inhibitors of myostatin. A selective inhibitor of myostatin (i.e., a myostatin selective inhibitor) is substantially specific for and potent toward myostatin compared to other structurally related members of the TGFβ superfamily at a given concentration. In some embodiments, a myostatin inhibitor is selective if it binds to myostatin with 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold or more affinity compared to another member of the TGFβ superfamily (e.g., GDF11 or activin A). Examples of myostatin selective inhibitors known in the art include apitegromab, trevogrumab and GYM329. In contrast, non-selective inhibitors of myostatin inhibit myostatin and one or more additional members of the TGFβ superfamily, such as GDF11, activin A, activin B, BMP, etc., at a given concentration. Most myostatin inhibitors in the literature fall into the non-selective category. The term myostatin inhibitor encompasses any molecular modality, including macromolecules (biologicals, such as antibodies and engineered protein constructs) and small molecules (such as structurally defined low molecular weight chemical entities). Myostatin inhibitors can be anti-myostatin antibodies or antigen-binding fragments thereof that bind to pro- and / or latent myostatin and / or mature myostatin. In some embodiments, the myostatin inhibitor can be an anti-pro / latent myostatin antibody or an antigen-binding fragment thereof that preferentially (e.g., selectively) binds to pro- and / or latent myostatin relative to mature myostatin. In various embodiments, the myostatin inhibitor can be an antibody (such as a neutralizing antibody), an activation inhibitor (e.g., an antibody that inhibits activation of pro- and / or latent myostatin), an adnectin, a peptibody, a receptor trap, or a ligand trap. In some embodiments, the myostatin inhibitor is a small molecule inhibitor.In another embodiment, myostatin inhibitor refers to gene therapy.

[0075] Myostatin pathway inhibitors: The terms "myostatin pathway inhibitor" and "inhibitor of the myostatin signaling pathway" are used interchangeably herein and encompass any agent that reduces (reduces) or inhibits the activity of the myostatin signaling pathway, regardless of mechanism of action. Reducing or inhibiting the activity of the myostatin signaling pathway can be the result of, for example, reducing the magnitude of activity, shortening the duration of activity, reducing the availability of one or more components of the signaling pathway, and the like. Such inhibition can be selective for myostatin itself, or can be non-selective such that it also inhibits at least one additional growth factor, such as GDF11 and activin A. Such agents can reduce signaling associated with myostatin by acting upstream, on or downstream of myostatin via members of the myostatin-induced signaling cascade to alter the signaling function of the myostatin pathway. In some embodiments, myostatin pathway inhibitors include inhibitors that act on molecules upstream of myostatin to prevent activation of proteases that cleave pro- and / or latent myostatin to their active forms, for example. In some embodiments, myostatin pathway inhibitors include myostatin inhibitors that act on myostatin itself to prevent its activation or its interaction with the receptor, including myostatin selective inhibitors. In some embodiments, myostatin pathway inhibitors include antibodies and other inhibitors that act on the myostatin receptor or downstream to prevent one or more effects of the myostatin signaling cascade. In some embodiments, myostatin pathway inhibitors include, but are not limited to, antibodies or antigen-binding fragments thereof, small molecules, receptor traps, adnectins, affibodies, DARPins, anticalins, avimers, versabodies, receptor inhibitors (such as receptor antibodies or receptor kinase inhibitors), or gene therapy.

[0076] Myostatin selective inhibitor: The term "myostatin selective inhibitor" refers to an inhibitor of the myostatin signaling pathway (i.e., a myostatin pathway inhibitor) that is selective for myostatin but not for GDF11, activin A, or other TGFβ family members. In some embodiments, a myostatin inhibitor is selective if it binds to myostatin with 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold or more affinity compared to another member of the TGFβ superfamily (e.g., GDF11 or activin A). In preferred embodiments, a selective myostatin inhibitor does not exhibit detectable binding or potency for other TGFβ family members. In some embodiments, a myostatin selective inhibitor is a neutralizing antibody that binds to mature myostatin and thereby inhibits its activity. In some embodiments, the myostatin selective inhibitor is an antibody that binds to pro- and / or latent myostatin, thereby inhibiting the activation process of myostatin, hi some embodiments, the myostatin selective inhibitor is Ab2, apitegromab, trevoglumab, GYM329, or a variant of any one of the foregoing.

[0077] Overweight / Obesity: A person who weighs more than what is considered normal weight adjusted for height is described as being overweight or having obesity. Using a BMI-based classification, for human adults (age 20 and over), a BMI of 18.5-24.9 is considered normal weight; a BMI of 25-29.9 is considered overweight; a BMI of 30 or more is considered obese (including extreme obesity); and a BMI of 40 or more is considered extremely obese. For children and adolescents (age 2-19), a BMI at or above the 85th percentile on the CDC growth chart is considered overweight or obese; a BMI at or above the 95th percentile on the CDC growth chart is considered obese (including extreme obesity); and a BMI at or above 120 percent of the 95th percentile on the CDC growth chart is considered extremely obese.

[0078] Pro / Latent Myostatin: As used herein, the term "pro / latent myostatin" refers to promyostatin, latent myostatin, or both (i.e., the pro-form or precursor of myostatin), but excludes mature myostatin. The pro and latent forms of myostatin remain associated with the prodomain (e.g., LAP) and are inactive in that they are unable to bind to the cellular myostatin receptor.

[0079] "Specific" and "specificity" refer to the selective reactivity of an interaction between members of a specific binding pair (e.g., a ligand and a binding site, an antibody and an antigen, biotin and avidin). The phrase "specifically binds to" and similar phrases, in the context of an antibody, refer to the ability of the antibody (or an antigenically reactive fragment thereof) to specifically bind to an antigen (or a fragment thereof) and not to other entities. Specific binding is understood as a preference for binding to a given antigen, epitope, receptor ligand or binding partner, for example 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 refers to the ability of an antibody to bind to a given antigen, epitope, receptor ligand or binding partner, for example 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. on , K off and K D For example, a ligand may have a binding affinity of 10 as measured by, for example, ELISA and Octet. -2 sec -1 Below, 10 -3 sec -1 Below, 10 -4 sec -1 Below, 10 -5 sec -1 Less than or equal to 10 -6 sec -1 The following K off and / or 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, or 10 -11 M or less, or 10 -12 K below M DIt can be understood that an antibody specifically binds to its target site if it has a binding affinity of 0.01 to 0.01%. It is understood that various proteins can share common epitopes or other binding sites (e.g., kinase reaction sites). In certain embodiments, a binding site can bind to more than one ligand, but still be considered specific based on binding preference compared to non-specific antigens and / or having certain binding kinetic parameters. How to select an appropriate non-specific control is within the capabilities of one of ordinary skill in the art. Binding assays are typically performed under physiological conditions. In some embodiments, an antibody can also "selectively" (i.e., "preferentially") bind to a target antigen if it binds to its target with a strength relatively greater than the strength of binding shown to other antigens, for example, binding to a target antigen (e.g., pro / latent myostatin) with 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or more affinity than to a non-target antigen (e.g., GDF11, activin A, or other TGFβ superfamily members). In a preferred embodiment, the selective myostatin inhibitor exhibits no detectable binding or potency against other TGFβ family members.

[0080] Subject: As used herein, the term "subject" is a target to which one or more of the therapies described herein may be administered. In the clinical context, the terms "subject" (e.g., human subject) and "patient" may be used interchangeably. In some embodiments, the subject is a vertebrate, particularly a mammal, in need of treatment, such as companion animals (e.g., dogs, cats, etc.), agricultural animals (e.g., cows, pigs, horses, sheep, goats, poultry, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). In some embodiments, the subject is a human who would benefit from or require treatment. In some embodiments, the subject is a human subject. In some embodiments, the subject is a pediatric or adolescent subject between the ages of 2 and 19. In some embodiments, the subject is a subject between the ages of 12 and 17. In some embodiments, the subject is an adult subject between the ages of 20 and 17. In some embodiments, the subject is an overweight, obese, or extremely obese human. In some embodiments, the subject has metabolic syndrome, type 2 diabetes or obesity-associated type 2 diabetes.

[0081] The term "total fat mass" refers to the cumulative fat content in a subject's body. Total fat mass includes fat composed of various types of adipocytes, such as white fat, brown fat, and beige fat, and includes fat stores in various body compartments, such as essential fat, subcutaneous fat, and visceral fat. Total fat mass can be measured or estimated by any method known in the art, including by skinfold thickness measurement using a skinfold gauge, measuring the circumference of certain body parts, dual energy X-ray absorptiometry (DXA), hydrostatic weighing, air displacement plethysmography, bioelectrical impedance analysis, bioimpedance spectroscopy, electrical impedance myography, three-dimensional body scanner, multicompartment model, or magnetic resonance imaging. The term "body fat mass gain" or "body fat mass loss" refers to the change in the magnitude of measured body fat mass compared to baseline measurement. For example, a subject with a metabolic disorder may exhibit body fat mass loss or visceral fat mass loss following treatment for the metabolic disorder (e.g., treatment with a myostatin pathway inhibitor, e.g., in conjunction with a GLP-1 pathway activator). The term "visceral fat" refers to the fat content found primarily in the abdominal area of ​​a subject's body and surrounding the subject's major organs, such as the liver, kidneys, pancreas, intestines, and heart, which is predominantly composed of white adipocytes.

[0082] Treat / Treating / Treatment: Treating a condition or disease in a subject refers to the act of providing a therapeutic regimen with the goal of reducing, preventing, or ameliorating a medical condition. Thus, the term treating or treatment does not necessarily require complete cure or prevention of a disease or disorder. In one embodiment, the symptoms of a 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%.

[0083] Weight loss: Weight loss refers to a reduction in body weight, regardless of whether one or more specific tissues are lost. For example, weight loss itself does not distinguish between loss of fat mass and loss of muscle mass. An overall loss of total body weight does not necessarily reflect an improvement in body composition.

[0084] Weight management: As used herein, the term "weight management" includes measures taken to lose weight, maintain weight, and reduce body fat tissue, increase lean mass, or otherwise improve or sustain body composition. Clinically meaningful weight management success may or may not involve overall weight loss. Thus, weight management may include diet (e.g., calorie-restricted diet, e.g., reducing calorie intake or reducing calorie absorption), exercise regimen, and / or medication (e.g., myostatin pathway inhibitor and / or GLP-1 pathway activator) to reduce the subject's total body weight, reduce the total fat mass, reduce the visceral fat mass, increase metabolic rate, increase the lean mass, and / or increase muscle-to-fat ratio, or otherwise improve body composition.

[0085] Weight-related condition: The term "weight-related condition" or "weight-related problem" as used herein refers to one or more medical conditions related to excess body fat mass (i.e., in addition to being overweight or obese), in which the subject's excess body fat mass is a contributing factor.Non-limiting examples of weight-related conditions include type 2 diabetes mellitus, high blood pressure, high triglyceride or cholesterol levels, heart disease, stroke, kidney disease, fatty liver (e.g., NAFLD and NASH) and sleep apnea.

[0086] Glucagon-like peptide 1 receptor (GLP-1R) In various embodiments, agents useful for carrying out the present invention include agents that enhance the GLP-1 signaling pathway. GLP-1 signals through its endogenous receptor, the GLP-1 receptor (GLP-1R). Human GLP-1R (UniProt Accession No. P43220) is a 463 amino acid G protein-coupled receptor formed by eight hydrophobic domains and is expressed primarily in pancreatic islets. This receptor, which functions on the cell surface, becomes internalized upon binding by the peptide hormone glucagon-like peptide 1 (GLP-1) and GLP-1 analogs, which play a key role in the signaling cascade leading to insulin secretion.

[0087] GLP-1R is particularly abundant in pancreatic β-cells, where it drives glucose-dependent insulin secretion, but is also present in pancreatic α-cells, where it mediates inhibition of glucagon secretion, and is also present in the intestine, lungs, kidneys, heart, blood vessels, lymphocytes, and the peripheral and central nervous systems. In the cardiovascular system, GLP-1R is abundant in the vasculature, primarily in arteries and arterioles, where GLP-1 exerts vasodilatory effects, while in the heart, GLP-1R expression is approximately six-fold higher in atrial myocytes compared to ventricular myocytes.

[0088] Wild-type glucagon-like peptide-1 (7-36) amide (GLP-1) is a secreted peptide that serves as a key determinant of blood glucose homeostasis due to its ability to delay gastric emptying, enhance pancreatic insulin secretion, and inhibit pancreatic glucagon secretion (see, e.g., Prog Mol Biol Transl Sci. 2014;121:23-65.). GLP-1 is secreted from L-cells of the gastrointestinal mucosa in response to food intake and plays a role in lowering blood glucose. Wild-type GLP-1 is rapidly inactivated due to its enzymatic degradation by dipeptidyl peptidase-IV (DPP-IV). Released GLP-1 activates enteric and autonomic reflexes while also circulating as an incretin hormone to control pancreatic endocrine function. Upon binding of the ligand (GLP-1), GLP-1R initiates a cascade involving the activation of membrane-bound adenyl cyclase and the resulting production of cyclic adenosine monophosphate (cAMP). Downstream of cAMP formation, several signaling pathways can be initiated, but they generally require activation of either or both of the cellular cAMP effectors protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC). The downstream signaling cascade can induce inhibition of glucose-stimulated insulin secretion (GSIS) in β cells and α cell glucagon release. In addition, binding of GLP-1R by GLP-1 and its analogs also initiates a variety of antidiabetic effects, including but not limited to reduced gastric emptying, increased satiety and inhibition of food-motivated behavior, replenishing insulin stores, and cytoprotective and anti-inflammatory effects on β cells.

[0089] GLP-1 pathway activators The present disclosure provides the use of agents that activate or enhance the GLP-1 signaling pathway in conjunction with myostatin pathway inhibitors for weight management and improved body composition. The combined use may involve combination therapy or adjunctive therapy. The GLP-1 signaling pathway is an attractive therapeutic target for the development of treatments for metabolic disorders, including, but not limited to, treatments that include activators of GLP-1 / GLP-1R and related signaling pathways, mainly due to its potential beneficial effect on glucose homeostasis through its regulation of β-cell mass, function and viability. For example, the use of GLP-1 pathway activators can provide a number of benefits to subjects (e.g., mammals) with type 2 diabetes mellitus (T2DM), such as enhancing proper pancreatic β-cell (insulin and amylin) secretion, pancreatic α-cell (glucagon) suppression, reducing hepatic glucose production, increasing satiety through the central nervous system, slowing down gastric emptying time and increasing insulin uptake in peripheral tissues via weight loss, with limitations including the need for a parallel diet and increased physical activity, as well as safety concerns. The novel weight management approach disclosed herein combines the benefits of this GLP-1 pathway activator with a myostatin pathway inhibitor to enhance clinical benefit. Without wishing to be bound by theory, it is contemplated that at least a portion of the weight loss effects of GLP1R agonists are mediated by a direct effect on CNS GLP1R or as a result of downstream activation of afferent neuron GLP1R.

[0090] According to the present disclosure, "activators of the GLP-1 pathway" or "GLP-1 pathway activators" include agents that act on multiple pathways, including but not limited to insulin production pathway activators (e.g., activators of the cAMP, EPAC, CREB, or EGFR signaling pathways) and metformin, including effects on insulin production. Also included are GLP-1 promoters (e.g., agents that increase GLP-1 production or secretion, GLP-1 stabilizers); modulators of GLP-1R transport (e.g., promoters of receptor membrane localization, inhibitors of receptor internalization), GLP-1 degradation inhibitors (e.g., DPP-IV inhibitors); GLP-1 secretagogues and GLP-1 receptor agonists.

[0091] "Insulin secretion" refers to the level of insulin released by the pancreas. A subject's insulin secretion level may be estimated using measurements of fasting and / or non-fasting blood glucose concentrations as a proxy. For example, normal fasting blood glucose concentrations in humans are 80 mg / 100 mL to 90 mg / 100 mL, which is associated with very low insulin secretion levels. Serum insulin levels may be measured using any method known in the art, such as by enzyme-linked immunosorbent assay (ELISA). Agents that enhance the GLP-1 signaling pathway may promote / stimulate insulin secretion, including GLP-1 (and GLP-1 analogs) and gastric inhibitory intestinal polypeptide (GIP).

[0092] In some embodiments, useful activators of the GLP-1 signaling pathway include GLP-1 receptor agonists, such as GLP-1 analogs. GLP-1 analogs include modified peptides that share certain structural similarities with the GLP-1 peptide and retain the ability to bind and activate the GLP-1 receptor. In some embodiments, the GLP-1 analog comprises the amino acid sequence EGTFTSD (SEQ ID NO: 116). In some embodiments, the GLP-1 analog comprises the amino acid sequence HXXGXFTXD, where X is any amino acid residue (SEQ ID NO: 117). According to the present disclosure, the GLP-1 signaling pathway activator may be used in conjunction with a myostatin pathway inhibitor. In some embodiments, the GLP-1 pathway activator and the myostatin pathway inhibitor may be used as a combination therapy or an adjunctive (or add-on) therapy in the treatment of a metabolic disorder, such as obesity, in a subject. In some embodiments, the GLP-1 pathway activator and the myostatin pathway inhibitor may be used as, in conjunction with, or supplemental combination or adjunctive (or add-on) therapy in a subject's weight management regimen. In some embodiments, the GLP-1 pathway activator and the myostatin pathway inhibitor may be used as, in conjunction with, or supplemental combination or adjunctive (or add-on) therapy in the treatment of a metabolic disorder to improve the body composition of a subject.

[0093] Non-limiting examples of GLP-1 analogues include albiglutide, taspoglutide, semaglutide, exenatide, BPI-3016, GW002, glutazumab, exendin-4, exenatide, GLP-1(7-36)NH2, everestumab, liraglutide, lixisenatide, tirzepatide, XW003, neuiglutide, MEDI0382 and dulaglutide. In one embodiment, the GLP-1 analogue is liraglutide, which is currently approved for type 2 diabetes and obesity.

[0094] Liraglutide (Victoza®, Saxenda®, CAS number 204656-20-2) is an acylated GLP-1 agonist derived from GLP-1(7-37). While wild-type GLP-1 has a plasma half-life of 1.5-2 minutes due to degradation by the enzymes dipeptidyl peptidase-4 (DPP-IV) and / or neutral endopeptidase, liraglutide is an acylated form of GLP-1 engineered to bind to albumin upon administration to a subject, thus significantly extending its half-life compared to GLP-1(7-37), with a plasma half-life of 13 hours. Liraglutide is also available in combination with insulin degludec under the trade name Xultophy®. Semaglutide (Ozempic®, Rybelsus®, Wegovy®, CAS number 910463-68-2) is a modified GLP-1 peptide containing two amino acid substitutions: 2-aminoisobutyric acid replaces the alanine moiety at position 8, and arginine replaces the lysine moiety at position 34. The substitution at position 8 prevents degradation by the enzyme DPP-IV. Semaglutide was approved by the US FDA in 2017. See, e.g., Novo Nordisk A / S. Ozempic (semaglutide) injection, for subcutaneous use; prescribing information; 2020.

[0095] Dulaglutide (Trulicity®, CAS number 923950-08-7) contains GLP-1(7-37) covalently linked to the Fc fragment of human IgG4. Dulaglutide is approved (in combination with diet and exercise) for adults with type 2 diabetes mellitus. See, e.g., Meece J., Adv Ther. 2017;34:638-657.

[0096] Exenatide (Byetta®, Bydureon®, CAS number 141758-74-9) is a synthetic form of the GLP-1 mimetic exendin-4 (a Gila monster salivary hormone) approved in 2005 by the U.S. FDA for the treatment of diabetes mellitus type 2 in combination with diet and exercise.

[0097] Glutazumab is an antibody fusion protein engineered by linking a human GLP-1 derivative to a humanized GLP-1R antibody via a peptide linker. Li et al.,Biochem Pharmacol.2018 Apr;150:46-53.

[0098] Lixisenatide (Lyxumia®, Adlyxin®, CAS number 320367-13-3) is a GLP-1 mimetic approved for the treatment of diabetes mellitus type 2 in conjunction with diet and exercise. See, e.g., Sanofi-Aventis US. Adlyxin (lixisenatide), prescribing information, 2016. Lixisenatide is also available in combination with insulin glargine under the trade names Soliqua® / Suliqua®.

[0099] Everestumab is a long-acting GLP-1 / anti-GLP1R fusion protein containing a mutant GLP-1 (A8G) fused to a tandem bispecific humanized GLP-1R targeting and albumin-binding nanobody designed for the treatment of diabetes mellitus type 2. Pan et al., ARTIFICIAL CELLS, NANOMEDICINE AND BIOTECHNOLOGY 2020, VOL. 48, NO. 1, 854-866.

[0100] Albiglutide (Eperzan®, Tanzeum®, CAS number 782500-75-8) is a GLP-1R agonist approved for type 2 diabetes mellitus. Its production was discontinued in 2018 due to lack of sales. See, e.g., GlaxoSmithKline LLC. Tanzeum® (albiglutide) for injection, for subcutaneous use prescribing information; 2017.

[0101] Taspoglutide is a GLP-1R agonist that was previously in clinical trials for the treatment of type 2 diabetes mellitus.

[0102] Tirzepatide (LY3298176) is a dual glucose-dependent insulinotropic polypeptide, GIP and GLP-1 receptor agonist in clinical trials for the treatment of type 2 diabetes. It was created by engineering GLP-1 activity into the GIP sequence and has been shown to improve glycemic control and reduce body weight (Rosenstock et al., Lancet (2020) 398:143-155).

[0103] BP3016 is a long-acting hGLP-1 analogue that contains human GLP-1(7-37) engineered for resistance to DPP-IV cleavage. See, e.g., Pharmacol Res. 2017 Aug;122:130-139.

[0104] XW003 is an acylated human glucagon-like peptide-1 (GLP-1) analogue that has been shown to induce dose-dependent weight loss in early clinical trials. The drug is currently in phase 2 development for the treatment of obesity. See, e.g., ClinicalTrials.gov identifier NCT05111912.

[0105] Neuiglutide is a 40-mer exenatide analogue that acts as a glucagon-like peptide-1 (GLP-1) agonist. The drug is being developed by Jiangsu Hansoh Pharmaceutical for the treatment of obesity. It is currently in phase 2 clinical development. See, e.g., Cabri et al. Front Mol Biosci. 2021;8:697586.

[0106] MEDI0382 is an oxyntomodulin-like peptide with targeted GLP-1 and glucagon receptor activity. It is currently in phase 2 clinical development for the treatment of obesity. See, e.g., Ambery et al. Lancet. 2018 Jun 30;391(10140):2607-2618.

[0107] According to the present disclosure, GLP-1 pathway activators include GLP-1 receptor agonist / GIP receptor antagonist combinations such as danugliplon (Pfizer), AMG 133 (Amgen), mas receptor activators, e.g., GLP-1 / GIP combinations such as Sarconeos, tirzepatide (Eli Lilly), amylin / GLP-1 combinations such as caglilintide / semaglutide combinations (Novo Nordisk), GLP-1 / glucagon combinations such as DD01 (Neuraly), GLP-1 / glucagon combinations such as ALT-801 (Altimmune), GLP-1 / GIP such as CT-388 (Carmot), IBI362 (mazdutide), LY-330567 (Innovent / Eli Lilly), GLP-1 / GIP / Glucagon triple receptor agonists such as danugliplon (PF-06882961) (Pfizer), setomelanotide (Rhythm), LY3437943 (Eli Lilly), and dual GIP / GLP-1 receptor agonists such as LY3298176 (Eli Lilly).

[0108] GLP-1 pathway activators further include agents that act on multiple pathways, including the GLP-1 pathway, such as metformin and its derivatives. Rena et al., Diabetologia 60:1577-85 (2017). Non-limiting examples of metformin include Fortamet, Glucophage, Glucophage XR, Glumetza, Riomet, Obimet, Gluformin, Dianben, Diabex, Diaformin, Metsol, Siofor, Metforgamma, and Glifor. GLP-1 pathway activators also include metformin-containing medications that include additional active agents. Examples include, but are not limited to, thiazolidinediones (glitazones) and rosiglitazone. In some embodiments, the GLP-1 pathway activator is FGF-2 or a peptide derived therefrom.

[0109] Myostatin Myostatin, also known as GDF8 (or GDF-8), is a member of the TGFβ superfamily and belongs to a subfamily that contains two members: myostatin and GDF11. Like other members of the TGFβ superfamily, myostatin and GDF11 are both initially expressed as inactive precursor polypeptides (termed promyostatin and proGDF11, respectively). In the overall structure of promyostatin, the mature growth factor is held locked in a cage containing two α-helices connected by a loop termed the "latency lasso." Human myostatin corresponds to UniProt accession number O14793; mouse myostatin corresponds to UniProt accession number O08689.

[0110] Myostatin is a well-characterized negative regulator of skeletal muscle mass that is released by two distinct protease cleavage steps from an autoinhibitory N-terminal prodomain. These cleavage events, for example within the myofiber microenvironment, can be referred to as supracellular activation. After activation, mature myostatin signals by binding to a complex of type I and type II cell surface receptors (Alk4 / 5 and ActRIIB), whose downstream signaling induces muscle destruction and atrophy. Myostatin has long been of interest as a potential target for the treatment of muscle wasting. However, the failure and discontinuation of nearly all clinical programs to date has cast doubts on its potential. Indeed, some reports suggest that myostatin expression levels appear to be decreased in a number of muscle disorders, leading to increased skepticism of myostatin as a therapeutic target (see, e.g., Mariot et al. (2017) Nat. Com. 8:1859; Burch et al. (2017) J. Neurol). Moreover, Latres et al. suggested in a 2017 paper that activin A, not myostatin, appears to be the predominant regulator of skeletal muscle growth. Notwithstanding the foregoing, the data presented herein support the idea that myostatin inhibition may be an effective approach for weight management and that its effects may be enhanced when the GLP-1 signaling pathway is activated in parallel. Thus, the present disclosure includes the use of any agent that inhibits or suppresses the myostatin signaling pathway in conjunction with an activator of the GLP-1 signaling pathway as described herein.

[0111] Activation and release of mature myostatin is achieved by several distinct protease cleavage events. The first cleavage step of promyostatin and proGDF11 is performed by a proprotein convertase, which cleaves at a conserved RXXR site between the prodomain and the mature growth factor. This cleavage produces "latent myostatin," in which mature myostatin is shielded from binding to its receptor by the prodomain. Activation and release of mature active myostatin growth factor is achieved following cleavage of latent myostatin by additional proteases from the BMP / Tolloid family, such as mTLL-2. As used herein, the term "mature myostatin" can refer to both full-length mature myostatin as well as fragments of full-length mature myostatin that retain biological activity.

[0112] The term "promyostatin," also known as "proGDF8," refers to an inactive precursor of mature myostatin that comprises a disulfide-linked homodimer, each molecule of which comprises an amino-terminal prodomain covalently linked to a carboxyl-terminal mature myostatin domain. In one embodiment, "promyostatin" has not yet been cleaved by either a proprotein convertase or a protease from the BMP / thoroid family. Exemplary promyostatin sequences, variants thereof, and methods for producing promyostatin are well known in the art and are described in further detail herein.

[0113] The term "latent myostatin" as used herein refers to an inactive precursor of mature myostatin that comprises a disulfide-bonded homodimer, each molecule of which comprises an amino-terminal prodomain non-covalently bound to a carboxyl-terminal mature myostatin domain. In one embodiment, "latent myostatin" is generated from promyostatin that has been cleaved by a proprotein convertase but has not yet been cleaved by a protease from the BMP / Tolloid family. In another embodiment, "latent myostatin" can be generated by combining the prodomain and the carboxy-terminal mature myostatin domain in vitro and allowing them to fold properly. See, e.g., Sengle et al., J. Biol. Chem., 286(7):5087-5099, 2011. Exemplary latent myostatin sequences, variants thereof, and methods for generating latent myostatin are known in the art and are described in further detail herein.

[0114] Pro-GDF8 (human): [ka]

[0115] Pro-GDF8 (rat): [ka]

[0116] Pro-GDF8 (mouse): [ka]

[0117] Pro-GDF8 (Cynomolgus monkey): [ka]

[0118] Exemplary pro-GDF8 sequences for human, rat, mouse, and cynomolgus monkey are provided above. In these pro-GDF8 sequences, the proprotein convertase cleavage site is indicated in bold and the toroid protease site is underlined. In some embodiments, the proprotein convertase cleavage site comprises amino acid residues 240-243 of SEQ ID NOs: 52-55. In some embodiments, the toroid protease site comprises amino acid residues 74-75 of SEQ ID NOs: 52-55. The exemplary pro-GDF8 sequences provided herein are not intended to be limiting, and it should be understood that additional pro-GDF8 sequences from other species, including any isoforms thereof, are within the scope of the present disclosure.

[0119] The prodomain of the myostatin polypeptide comprises several structural domains as previously described (WO 2014 / 182676), including, for example, the Straight Jacket region, the zipper region, the arm region, finger region 1, finger region 2, the cryptic loop, the alpha-1 helix region, and the bowtie region. In some embodiments, the preferred antibody or fragment thereof binds to an epitope within the arm region of the myostatin prodomain. In some embodiments, the epitope comprises at least one amino acid residue from the "KALDEN" (SEQ ID NO: 118) polypeptide stretch within the arm region of the prodomain. In some embodiments, the amino acid residue within the arm region of the prodomain that makes contact with the antibody upon binding to the antigen is a residue that is not conserved between myostatin and GDF11. In some embodiments, such one or more residues are K, E, and / or N of this polypeptide stretch (shown in bold above). In some embodiments, the epitope comprises at least one amino acid residue from the "FVQILRLIKPMKDGTRYTGIRSLK" (SEQ ID NO:57) polypeptide stretch within the arm region of the prodomain. In some embodiments, such one or more residues are F, Q, L, Y, R, S and / or K of this polypeptide stretch (shown in bold above). See Dagbay et al. (J. Biol. Chem. (2020) 295(16):5404-5418), the contents of which are incorporated herein in their entirety. In some embodiments, the antibody or antigen-binding fragment thereof that binds to such an epitope is apitegromab or a variant thereof, and optionally, the variant is an Fc variant. In other embodiments, the antibody or antigen-binding fragment thereof that binds to such an epitope is not apitegromab or a variant thereof.

[0120] Myostatin Pathway Inhibitors There are several myostatin pathway inhibitors in various stages of clinical development for the treatment of muscle-related pathologies, including small molecules, antibodies or their antigen-binding portions, and gene therapy. Such inhibitors of the signaling pathway target either the mature growth factor or its type II receptor. Notably, many of these antagonists are not myostatin specific, and therefore they antagonize the signaling of multiple TGFβ family members. For example, several current clinical candidates block additional growth factors, such as activin A, GDF11, and BMP9 and 10, which are regulators of reproductive biology, wound healing, erythropoiesis, and angiogenesis, respectively.

[0121] In some embodiments, as disclosed herein, myostatin pathway inhibitors, such as anti-myostatin antibodies or antigen-binding fragments thereof, bind, e.g., selectively bind, promyostatin and / or latent myostatin, thereby inhibiting myostatin activation. In some embodiments, given the prevalence of latent complexes in the circulation, provided herein are therapies that specifically target the more abundant and longer-lived myostatin precursors, e.g., promyostatin and latent myostatin, rather than the mature growth factor. In some embodiments, myostatin inhibitors provided herein, such as antibodies or antigen-binding fragments thereof, may prevent promyostatin and / or latent myostatin from proteolytically activating to mature myostatin, which is considered the "active" form of myostatin and has the ability to activate the myostatin pathway, e.g., by binding to type I (ALK4 / 5) and type II (ACTRIIA / B) receptors.

[0122] In some embodiments, the myostatin pathway inhibitor is a myostatin inhibitor. In some embodiments, the myostatin inhibitor used herein can be an antibody (e.g., including fragments thereof, such as domain antibodies (dAbs) as described in U.S. Pat. Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; and 6,696,245), a small molecule inhibitor, an adnectin, an affibody, a DARPin, an anticalin, an avimer, a versabody, or a gene therapy.

[0123] In some embodiments, the myostatin pathway inhibitor is a non-selective myostatin inhibitor, such as an inhibitor that also inhibits activin A and / or GDF11. In some embodiments, the non-selective myostatin inhibitor is a ligand trap (e.g., ACE-031, ACE-083, and BIIB-110 / ALG-801); an anti-ActRIIb antibody (e.g., bimagrumab); a neutralizing antibody that binds to mature myostatin (e.g., stamulumab (MYO-029), domagrozumab (PF-06252616), landgrozumab (LY2495655), AMG-745 / PINTA-745 (myostatin peptibody), RG6206 (anti-myostatin Adnectin that is a single-chain fusion protein containing domain of fibronectin), BMS-986089 (anti-myostatin Adnectin also known as taldef globep alfa). In some embodiments, the myostatin pathway inhibitor is a receptor antagonist (e.g., Alk4 / 5 inhibitor).

[0124] In various embodiments, the myostatin pathway inhibitor comprises stamulumab, trevogrumab, LY2495655, AMG 745, bimagrumab, BIIB-110, domagurozumab (PF-06252616), apitegromab, GYM329, taldef globep alfa (also known as BMS-986089), or efumitermant alfa. In some embodiments, the myostatin pathway inhibitor comprises a pH-dependent anti-latent myostatin antibody, such as GYM329 or another MST1032 variant as disclosed in WO 2016 / 098357, which is incorporated herein by reference. In some embodiments, the myostatin pathway inhibitor comprises an MST1032 variant as disclosed in International Patent Application PCT / JP2015 / 006323, the contents of which are incorporated herein in their entirety. In some embodiments, the myostatin pathway inhibitor comprises an antibody or antigen-binding fragment that comprises additional sequences found in, for example, WO 2018 / 129395; WO 2017 / 218592; WO 2017 / 120523; WO 2017 / 049011; WO 2016073853; or WO 2014182676 (the contents of which are incorporated by reference in their entireties).

[0125] "MST1032 variant," as used herein, refers to an antibody or antigen-binding fragment comprising any of the sequences disclosed in PCT / JP2015 / 006323, including but not limited to the sequences of MS1032LO01-SG1, MS1032LO06-SG1, MS1032LO11-SG1, MS1032LO18-SG1, MS1032LO19-SG1, MS1032LO21-SG1, MS1032LO25-SG1, as well as the sequences provided in Table 2a, Table 11a, Table 11b, or Table 13 of PCT / JP2015 / 006323. In a particular embodiment, the MST1032 variant comprises an antibody or antigen-binding fragment comprising a heavy chain variable domain comprising three CDR sequences, HCDR1, HCDR2 and HCDR3, and a light chain variable domain comprising three CDR sequences, LCDR1, LCDR2 and LCDR3, wherein the heavy chain CDRs comprise the amino acid sequences of X1X2DIS (HCDR1; SEQ ID NO: 17); IISYAGSTYYASWAKG (HCDR2; SEQ ID NO: 18); GVPAYSX3GGDL (HCDR3; SEQ ID NO: 19), respectively; and the light chain CDRs comprise the amino acid sequences of X4X5SQSVYX6X7NWLS (LCDR1; SEQ ID NO: 20); WASTLAX8 (LCDR2; SEQ ID NO: 21); and AGGYGGGX9YA (LCDR3; SEQ ID NO: 22), respectively (wherein each of X1 to X9 is any amino acid residue). In a particular embodiment, X1 is S or H; X2 is Y, T or D; X3 is T or H; X4 is Q or T; X5 is S or T; X6 is D or H; X7 is N or E; X8 is S or Y; and X9 is L or R. In a particular embodiment, the MST1032 variant comprises six CDR sequences of SEQ ID NOs: 18, 66, 67, 68-70; SEQ ID NOs: 119, 18, 120, 68, 121, 122; or SEQ ID NOs: 123, 18, 120, 124, 121, 122. In a particular embodiment, the MST1032 variant comprises a heavy chain variable domain comprising any one of the amino acid sequences of SEQ ID NOs: 125-128. In a particular embodiment, the MST1032 variant comprises a light chain variable domain comprising any one of the amino acid sequences of SEQ ID NOs: 129, 126, 127 and 130.In certain embodiments, the MST1032 variant comprises a set of six CDRs (e.g., from the same MST1032 variant antibody) or paired VH / VL (e.g., from the same MST1032 variant antibody) from those listed in the table below.

[0126] [Table 1]

[0127] [Table 2]

[0128] [Table 3]

[0129] [Table 4]

[0130] [Table 5]

[0131] [Table 6]

[0132] In some embodiments, the myostatin antibody or antigen-binding fragment thereof may include or be engineered to include a mutation or modification that results in an increased half-life of the antibody. In some embodiments, such a mutation or modification may be within the Fc domain of the antibody (e.g., an Fc-modified antibody), for example, to enhance circulating half-life or other PK properties. In some embodiments, the mutation is a YTE mutation (see, e.g., Saunders, KO (2019) "Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life," Frontiers in Immunology 10:1296 and U.S. Patent No. 7,083,784, each of which is incorporated by reference herein).

[0133] In some embodiments, the anti-pro / latent myostatin antibody or antigen-binding fragment thereof specifically binds to latent myostatin. In some embodiments, the anti-pro / latent myostatin antibody or antigen-binding fragment thereof specifically binds to both latent myostatin and promyostatin. In preferred embodiments, the anti-pro / latent myostatin antibody or antigen-binding fragment thereof that specifically binds to promyostatin and / or latent myostatin does not bind to mature myostatin. In preferred embodiments, the anti-pro / latent myostatin antibody or antigen-binding fragment thereof that specifically binds to promyostatin and / or latent myostatin does not bind to pro / latent GDF11 or mature GDF11.

[0134] Anti-pro / latent myostatin antibodies and their antigen-binding fragments In some embodiments, the antibodies or antigen-binding fragments thereof described herein have the ability to bind to pro / latent myostatin, thereby inhibiting the proteolytic activity of pro / latent myostatin to mature myostatin. In some examples, the antibodies or antigen-binding fragments thereof described herein can inhibit the proteolytic activity of pro / latent myostatin by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some examples, the antibodies described herein can inhibit the proteolytic cleavage of promyostatin by proprotein convertases (e.g., furin) by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some examples, the antibodies or antigen-binding fragments thereof described herein can inhibit proteolytic cleavage of promyostatin or latent myostatin by toroidal protease (e.g., mTLL2) by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.

[0135] In some embodiments, the antibodies or antigen-binding fragments thereof described herein have the ability to bind to pro / latent myostatin, thereby inhibiting myostatin activity. In some examples, the antibodies or antigen-binding fragments thereof described herein can inhibit myostatin signaling by at least 20%, such as 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some embodiments, inhibition of myostatin signaling can be measured by routine methods, for example, using a myostatin activation assay as described in Example 1 disclosed in WO2016 / 073853, the contents of which are expressly incorporated herein by reference in their entirety. However, it should be understood that additional methods can be used to measure myostatin signaling activity.

[0136] It should be understood that the extent of proteolytic cleavage of myostatin, for example by proprotein convertases and / or thrombolytic proteases, may be measured and / or quantified using any suitable method. In some embodiments, the extent of proteolytic cleavage of myostatin is measured and / or quantified using an enzyme-linked immunosorbent assay (ELISA). For example, an ELISA may be used to measure the level of released growth factors (e.g., mature myostatin). As another example, an antibody or antigen-binding fragment thereof that specifically binds to promyostatin, latent myostatin and / or mature myostatin may be used in an ELISA to measure the level of a specific form of myostatin (e.g., pro / latent / mature myostatin) and quantify the extent of proteolytic cleavage of myostatin. In some embodiments, the extent of proteolytic cleavage of myostatin is measured and / or quantified using immunoprecipitation followed by SDS-PAGE or mass spectrometry of tryptic peptides, techniques based on fluorescence anisotropy measurements, FRET assays, hydrogen deuterium exchange mass spectrometry and / or NMR spectroscopy.

[0137] Anti-pro / latent myostatin antibodies or antigen-binding fragments thereof suitable for use in the methods of the invention include those described in International Patent Application Nos. PCT / US15 / 59468 and PCT / US16 / 52014, the entire contents of each of the foregoing applications being incorporated herein by reference in their entirety.

[0138] In accordance with the present disclosure, antibodies or antigen-binding fragments that bind to human pro / latent myostatin may be used to practice the various embodiments disclosed herein. In some embodiments, such antibodies and antigen-binding fragments bind to an epitope within the prodomain, the epitope including one or more amino acid residues F147, Q149, L151, Y186, S168, K170, K205, and / or L207, numbered according to SEQ ID NO:52 (Dagbay et al. J Biol Chem. 2020 Apr 17;295(16):5404-5418).

[0139] In some embodiments, the anti-pro / latent myostatin antibodies or antigen-binding fragments thereof of the present disclosure and nucleic acid molecules of the present disclosure encoding such antibodies or antigen-binding fragments thereof comprise the CDR amino acid sequences shown in Tables 1G-1 to 1G-3.

[0140] [Table 7]

[0141] In Table 1, the single sequences of CDRH3 and CDRL3 reflect Kabat and IMGT.

[0142] [Table 8]

[0143] [Table 9]

[0144] [Table 10]

[0145] [Table 11]

[0146] [Table 12]

[0147] [Table 13]

[0148] [Table 14]

[0149] In some embodiments, an anti-pro / latent myostatin antibody or antigen binding portion thereof of the disclosure includes any antibody or antigen binding fragment thereof that includes CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or combinations thereof, as provided in any one of the antibodies shown in Tables 1G-1 through 1G-3. In some embodiments, an anti-pro / latent myostatin antibody or antigen binding portion thereof includes CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of any one of the antibodies shown in Tables 1G-1 through 1G-3. The disclosure also includes any nucleic acid sequence that encodes a molecule that includes CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3 as provided in any one of the antibodies shown in Tables 1G-1 through 1G-3. The antibody heavy and light chain CDR3 domains may play a particularly important role in the binding specificity / affinity of an antibody to an antigen. Accordingly, an anti-pro / latent myostatin antibody or antigen-binding portion thereof or nucleic acid molecule thereof of the present disclosure may comprise at least the heavy chain and / or light chain CDR3 of an antibody as shown in Tables 1G-1 to 1G-3.

[0150] An embodiment of the present disclosure relates to a monoclonal antibody or antigen-binding fragment that binds to pro / latent myostatin protein and comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3.

[0151] In some embodiments, CDRH1 comprises a sequence as set forth in any one of SEQ ID NOs: 1-3. In some embodiments, CDRH2 comprises a sequence as set forth in any one of SEQ ID NOs: 4-9. In some embodiments, CDRH3 comprises a sequence as set forth in any one of SEQ ID NOs: 10-11, 66, 71, 76, 81, 86, 91, 96, 101, 106, and 111. CDRL1 comprises a sequence as set forth in any one of SEQ ID NOs: 12-16, and 59. In some embodiments, CDRL2 comprises a sequence as set forth in any one of SEQ ID NOs: 60, 61, 131, and 64. In some embodiments, CDRL3 comprises a sequence as set forth in any one of SEQ ID NOs: 65, 23, 67, 72, 77, 82, 87, 92, 97, 102, 107, and 112.

[0152] In some embodiments (e.g., with respect to anti-pro / latent myostatin antibody Ab1 or its antigen-binding portion shown in Table 1G), CDRH1 comprises the sequence as set forth in SEQ ID NO: 1 or 2, CDRH2 comprises the sequence as set forth in SEQ ID NO: 4 or 5, CDRH3 comprises the sequence as set forth in SEQ ID NO: 10, CDRL1 comprises the sequence as set forth in SEQ ID NO: 12 or 13, CDRL2 comprises the sequence as set forth in SEQ ID NO: 60 or 61, and CDRL3 comprises the sequence as set forth in SEQ ID NO: 65, and the antibody or antigen-binding portion thereof binds to pro / latent myostatin.

[0153] In some embodiments (e.g., with respect to anti-pro / latent myostatin antibody Ab2 or its antigen binding portion shown in Table 1G), CDRH1 comprises the sequence as set forth in SEQ ID NO: 1 or 2, CDRH2 comprises the sequence as set forth in SEQ ID NO: 4 or 5, CDRH3 comprises the sequence as set forth in SEQ ID NO: 66, CDRL1 comprises the sequence as set forth in SEQ ID NO: 12 or 13, CDRL2 comprises the sequence as set forth in SEQ ID NO: 60 or 61, and CDRL3 comprises the sequence as set forth in SEQ ID NO: 67, and the antibody or antigen binding portion thereof binds to pro / latent myostatin.

[0154] In some embodiments (e.g., with respect to anti-pro / latent myostatin antibody Ab3 or its antigen binding portion shown in Table 1G), CDRH1 comprises the sequence as set forth in SEQ ID NO: 1 or 3, CDRH2 comprises the sequence as set forth in SEQ ID NO: 6 or 7, CDRH3 comprises the sequence as set forth in SEQ ID NO: 11, CDRL1 comprises the sequence as set forth in SEQ ID NO: 14 or 15, CDRL2 comprises the sequence as set forth in SEQ ID NO: 131 or 64, and CDRL3 comprises the sequence as set forth in SEQ ID NO: 23, and the antibody or antigen binding portion thereof binds to pro / latent myostatin.

[0155] In some embodiments (e.g., with respect to anti-pro / latent myostatin antibody Ab5 or its antigen binding portion shown in Table 1G), CDRH1 comprises the sequence as set forth in SEQ ID NO: 1 or 3, CDRH2 comprises the sequence as set forth in SEQ ID NO: 8 or 9, CDRH3 comprises the sequence as set forth in SEQ ID NO: 11, CDRL1 comprises the sequence as set forth in SEQ ID NO: 16 or 59, CDRL2 comprises the sequence as set forth in SEQ ID NO: 131 or 64, and CDRL3 comprises the sequence as set forth in SEQ ID NO: 23, and the antibody or antigen binding portion thereof binds to pro / latent myostatin.

[0156] In some examples, any of the anti-pro / latent myostatin antibodies or antigen-binding portions thereof of the present disclosure include any antibody or antigen-binding fragment having one or more CDR (e.g., CDRH or CDRL) sequences substantially similar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and / or CDRL3. For example, the antibody may comprise one or more CDR sequences as shown in Tables 1A to 1G-3 (SEQ ID NOs: 1 to 16, 18, 23, 59 to 61, 64 to 72, 76, 77, 81, 82, 86, 87, 91, 92, 96, 97, 101, 102, 106, 107, 111, 112, and 119 to 124), having up to 5, 4, 3, 2, or 1 amino acid residue mutations compared to the corresponding CDR region in any one of the SEQ ID NOs: 1 to 16, 18, 23, 59 to 61, 64 to 72, 76, 77, 81, 82, 86, 87, 91, 92, 96, 97, 101, 102, 106, 107, 111, 112, and 119 to 124) shown in Tables 1A to 1G-3.

[0157] In some embodiments, the anti-pro / latent myostatin antibodies or antigen-binding portions thereof described herein comprise three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:4 (HCDR2) and SEQ ID NO:10 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:12 (LCDR1), SEQ ID NO:60 (LCDR2) and SEQ ID NO:65 (LCDR3), as defined by the Kabat numbering system.

[0158] In some embodiments, the anti-pro / latent myostatin antibodies or antigen-binding portions thereof described herein comprise three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:2 (HCDR1), SEQ ID NO:5 (HCDR2) and SEQ ID NO:10 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:13 (LCDR1), SEQ ID NO:61 (LCDR2) and SEQ ID NO:65 (LCDR3), as defined by the IMGT numbering system.

[0159] In various embodiments, the anti-pro / latent myostatin antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:31.

[0160] In various embodiments, the anti-pro / latent myostatin antibody or antigen-binding portion thereof comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:50 and / or a light chain region comprising the amino acid sequence of SEQ ID NO:51.

[0161] In some embodiments, anti-pro / latent myostatin antibodies or antigen binding portions thereof of the present disclosure include any antibody that includes a heavy chain variable domain of any one of SEQ ID NOs: 24-29, 73, 78, 83, 88, 93, 98, 103, 108, 113, 125-128 or a light chain variable domain of any one of SEQ ID NOs: 30-35, 74, 79, 84, 89, 94, 99, 104, 109, 114, 129-130, 132, 133. In some embodiments, anti-pro / latent myostatin antibodies or antigen binding portions thereof of the present disclosure include any antibody that includes a heavy chain variable and light chain variable pair of SEQ ID NOs: 24 and 30; 25 and 31; 26 and 32; 27 and 33; 28 and 34; 29 and 35; 125 and 129; 126 and 132; 127 and 133; 128 and 130).

[0162] Aspects of the present disclosure provide anti-pro / latent myostatin antibodies or antigen-binding portions thereof having heavy chain variable and / or light chain variable amino acid sequences homologous to any of those described herein. In some embodiments, the anti-pro / latent myostatin antibodies or antigen-binding portions thereof comprise a heavy chain variable sequence or light chain variable sequence that is at least 75% (e.g., 80%, 85%, 90%, 95%, 98% or 99%) identical to any of the heavy chain variable sequences of SEQ ID NOs: 24-29, 73, 78, 83, 88, 93, 98, 103, 108, 113, 125-128 or any one of the light chain variable sequences of SEQ ID NOs: 30-35, 74, 79, 84, 89, 94, 99, 104, 109, 114, 129-130, 132, 133. In some embodiments, the homologous heavy and / or light chain variable amino acid sequences do not vary within any of the CDR sequences provided herein. For example, in some embodiments, a degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) may occur within the heavy and / or light chain variable sequences excluding any of the CDR sequences provided herein.

[0163] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0164] In some embodiments, conservative mutations may be introduced into CDR or framework sequences where the residues are not expected to be involved in interactions with pro / latent myostatin as determined based on the crystal structure. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants may be made according to methods for modifying polypeptide sequences known to those of skill in the art, such as those found in references that summarize such methods, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FMAusubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made within amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0165] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein contain mutations that confer desirable properties to the antibodies or antigen-binding fragments thereof. For example, to avoid potential complications due to Fab-arm exchange known to occur in natural IgG4 mAbs, the antibodies or antigen-binding fragments thereof provided herein may contain a stabilizing "Adair" mutation (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody," Mol Immunol 30, 105-108; 1993), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline resulting in an IgG1-like (CPPCP (SEQ ID NO: 58)) hinge sequence. Thus, any of the antibodies may contain a stabilizing "Adair" mutation or amino acid sequence (CPPCP (SEQ ID NO: 58)).

[0166] The anti-pro / latent myostatin antibodies or antigen-binding portions thereof of this disclosure may optionally include an antibody constant region or a portion thereof. For example, a VL domain may be attached at its C-terminus to a light chain constant domain like Cκ or Cλ. Similarly, a VH domain or a portion thereof may be attached to all or a portion 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, for example, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within the scope of this disclosure may include VH and VL domains or antigen-binding portions thereof combined with any suitable constant region.

[0167] In certain embodiments, the VH and / or VL domains can be reverted to their germline sequences, e.g., the FRs of these domains are mutated using conventional molecular biology techniques to correspond to those produced by germline cells. For example, the VH and / or VL domains can be reverted to the germline sequences of IgHV3-30 (SEQ ID NO: 36) and / or IgLV1-44 (SEQ ID NO: 37), respectively. It should be understood that any of the VH and / or VL domains can be reverted to any suitable germline sequence. In other embodiments, the FR sequences remain deviated from the consensus germline sequences.

[0168] IgHV3-30 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 36)

[0169] IgLV1-44 QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNG (SEQ ID NO: 37)

[0170] In some embodiments, the anti-pro / latent myostatin antibody or antigen-binding fragment may or may not include the framework regions of the antibodies set forth in SEQ ID NOs: 24-35. In some embodiments, the anti-pro-latent myostatin antibody is a murine antibody and includes murine framework region sequences.

[0171] In some embodiments, the anti-pro / latent myostatin antibody or antigen-binding fragment thereof has a relatively high affinity for pro / latent myostatin, e.g., 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 K less than M or lower DFor example, an anti-pro / latent myostatin antibody or antigen-binding fragment thereof can bind to pro / latent myostatin with an affinity of 5 pM to 500 nM, e.g., 50 pM to 100 nM, e.g., 500 pM to 50 nM. The invention also includes antibodies or antigen-binding fragments that compete with any of the antibodies described herein for binding to pro / latent myostatin and have an affinity of 50 nM or less (e.g., 20 nM or less, 10 nM or less, 500 pM or less, 50 pM or less, or 5 pM or less). The affinity and binding kinetics of anti-pro / latent myostatin antibodies can be tested using any suitable method, including, but not limited to, biosensor technology (e.g., OCTET or BIACORE). When binding profiles are measured by use of OCTET or BIACORE, the assay is typically performed according to the manufacturer's instructions unless otherwise specified.

[0172] In some embodiments, disclosed herein are antibodies or antigen-binding fragments thereof that specifically bind to pro / latent myostatin. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein bind to or near the toroid cleavage site or to or near the toroid docking site of pro / latent myostatin. In some embodiments, an antibody binds near the toroid cleavage site or binds near the toroid docking site if it binds within 15 amino acid residues or less from the toroid cleavage site or toroid docking site. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein bind within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues from the toroid cleavage site or toroid docking site. In some embodiments, the antibody binds to or near the toroid cleavage site of GDF8. For example, the antibody may bind to the amino acid sequence PKAPPLRELIDQYDVQRDDSSDGSLEDDDYHAT (SEQ ID NO: 62) as set forth in SEQ ID NO: 62. In other embodiments, any of the antibodies or antigen-binding fragments thereof provided herein bind to or near the proprotein convertase cleavage site or to or near the proprotein convertase docking site of pro / latent myostatin. In some embodiments, an antibody binds near the proprotein convertase cleavage site or to or near the proprotein convertase docking site if it binds within 15 amino acid residues or less from the proprotein convertase cleavage site or the proprotein convertase docking site. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein bind within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues from the proprotein convertase cleavage site or the proprotein convertase docking site. In some embodiments, an antibody binds to or near the proprotein convertase cleavage site of GDF8. For example, an antibody may bind to the amino acid sequence (GLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRC) as set forth in SEQ ID NO:63.

[0173] In some embodiments, the myostatin selective inhibitor of the present disclosure is an antibody or antigen-binding fragment thereof that optionally binds to an epitope comprising at least one amino acid residue of "KALDEN" (SEQ ID NO: 118) and / or "FVQILRLIKPMKDGTRYTGIRSLK" (SEQ ID NO: 57). In some embodiments, the antibody is apitegromab or a variant thereof, optionally, the variant is an Fc variant. In some embodiments, the antibody is not apitegromab or a variant thereof.

[0174] In some embodiments, the myostatin selective inhibitor of the present disclosure is an antibody or antigen-binding fragment thereof that optionally binds to an epitope that includes one or more amino acid residues of F147, Q149, L151, Y163, R167, S168, K170, K205, L207, E209 and N210 (based on the numbering of the human pro-GDF8 sequence as set forth in SEQ ID NO:52, which correspond to F170, Q172, L174, Y186, R190, S191, K193, K228, L230, E232 and N233, respectively, based on the numbering of Dagbay et al. J. Biol. Chem. (2020), 295(16):5404-5418). In preferred embodiments, such antibodies bind to an epitope comprising 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, or 3 or less of the amino acid residues set forth above. In some embodiments, the antibody is apitegromab or a variant thereof, optionally wherein the variant is an Fc variant. In some embodiments, the antibody is not apitegromab or a variant thereof.

[0175] In one example, the anti-pro / latent myostatin antibodies or antigen-binding fragments thereof described herein specifically bind to pro / latent myostatin relative to other forms of myostatin and / or other members of the TGFβ growth factor family, including, without limitation, AMH, ARTN, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDNF, INHA, INHBA, INHBB, INHBC, INHBE, LEFTY1, LEFTY2, NODAL, NRTN, PSPN, TGFβ1, TGFβ2, and TGFβ3 proteins. Such antibodies or antigen-binding fragments thereof may bind to pro / latent myostatin with much higher affinity (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1,000-fold higher) than other members of the TGFβ growth factor family. In some embodiments, such antibodies or antigen-binding fragments thereof may bind to pro / latent myostatin with at least 1000-fold higher affinity than other members of the TGFβ growth factor family. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein may bind to pro / latent myostatin with much higher affinity (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1,000-fold higher) than one or more forms of GDF11 or mature myostatin. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can bind to one or more forms of GDF11 (e.g., proGDF11, latent GDF11 or mature GDF11) or to pro / latent myostatin with at least 1,000-fold greater affinity compared to mature myostatin.Alternatively or in addition, the antibody or antigen-binding fragment thereof may exhibit much higher (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold higher) inhibitory activity against proteolytic cleavage (e.g., by proprotein convertases or toroidal proteases) of pro / latent myostatin compared to other members of the TGFβ family, such as pro / latent GDF11. In another embodiment, the antibody or antigen-binding fragment thereof disclosed herein does not bind to GDF11. This avoids potential toxicity issues associated with antibodies that cross-react with both myostatin and GDF11. One such example of potential toxicity relates to the loss of bone strength associated with GDF11 inhibition, as recently reported in Suh et al. Proceedings of the National Academy of Sciences Mar 2020, 117(9) 4910-4920, the contents of which are incorporated herein in their entirety.

[0176] In some embodiments, the antibody binds to the antigen but cannot effectively remove the antigen from plasma. Thus, in some embodiments, the concentration of the antigen in plasma can be increased by reducing the clearance of the antigen. However, in some embodiments, the antibody provided herein (e.g., sweeping antibody) has affinity for antigens that are sensitive to pH. Such pH-sensitive antibodies can bind to antigens in plasma with neutral pH and dissociate from antigens in acidic endosomes, thus reducing antibody-mediated antigen accumulation and / or promoting antigen clearance from plasma.

[0177] Aspects of the present disclosure relate to sweeping antibodies. As used herein, a "sweeping antibody" or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof that has both pH-sensitive antigen binding and at least a threshold level of binding to cell surface neonatal Fc receptor (FcRn) at neutral or physiological pH. In some embodiments, the sweeping antibody or antigen-binding portion thereof binds to neonatal Fc receptor FcRn at neutral pH. For example, the sweeping antibody may bind to FcRn at a pH ranging from 7.0 to 7.6. In some embodiments, the sweeping antibody or antigen-binding portion thereof binds to an antigen at an antigen-binding site and can bind to cellular FcRn via the Fc portion of the antibody. In some embodiments, the sweeping antibody or antigen-binding portion thereof can then be internalized and release the antigen in acidic endosomes, which can be degraded. In some embodiments, the sweeping antibody or antigen-binding portion thereof no longer binds to an antigen and can then be released by the cell (e.g., by exocytosis) back into the serum.

[0178] In some embodiments, the sweeping antibody or antigen-binding portion thereof has an increased half-life due to FcRn of the vascular endothelium (e.g., of a subject). In some embodiments, the vascular endothelial cells internalize the sweeping antibody or antigen-binding portion thereof, which in some embodiments binds to an antigen, such as myostatin (e.g., promyostatin, latent myostatin, or primed myostatin). In some embodiments, the sweeping antibody or antigen-binding portion thereof recirculates back into the bloodstream. In some embodiments, the sweeping antibody or antigen-binding portion thereof exhibits an increased half-life (e.g., in the serum of a subject) compared to its conventional counterpart. In some embodiments, the conventional counterpart of the sweeping antibody refers to the original antibody or antigen-binding portion thereof from which the sweeping antibody or antigen-binding portion thereof is derived (e.g., before the Fc portion of the conventional antibody was engineered to bind FcRn with higher affinity at pH 7). In some embodiments, the sweeping antibody or antigen-binding portion thereof exhibits a half-life in the serum of a subject that is at least 1%, 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 100%, 150%, 200% or 250% longer than its conventional counterpart.

[0179] 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 10 -3 M~10 -8 K in the M range D In some embodiments, the sweeping antibody binds to FcRn at a pH of 10 -3 M~10 -7 M, 10 -3 M~10 -6 M, 10 -3 M~10 -5 M, 10 -3 M~10 -4 M, 10 -4 M~10 -8 M, 10 -4 M~10 -7 M, 10 -4 M~10 -6 M, 10 -4 M~10 -5 M, 10-5 M~10 -8 M, 10 -5 M~10 -7 M, 10 -5 M~10 -6 M, 10 -6 M~10 -8 M, 10 -6 M~10 -7 M or 10 -7 M~10 -8 K in the M range D 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.

[0180] In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein are engineered to bind to FcRn with higher affinity. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein are engineered to bind to FcRn with higher affinity at pH 7.4. In some embodiments, increasing the affinity of an antibody or antigen-binding fragment thereof to FcRn extends its pharmacokinetic (PK) properties compared to its conventional counterpart. For example, in some embodiments, the sweeping antibody induces fewer adverse reactions because it is effective at lower doses. In some embodiments, the sweeping antibody or antigen-binding portion thereof is administered less frequently. In some embodiments, the transcytosis of the sweeping antibody or antigen-binding portion thereof to certain tissue types is increased. In some embodiments, the sweeping antibody or antigen-binding portion thereof enhances transplacental delivery efficiency. In some embodiments, the sweeping antibody or antigen-binding portion thereof is less expensive to produce.

[0181] In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein are engineered to bind to FcRn with lower affinity. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein are engineered to bind to FcRn with lower affinity at pH 7.4. In some embodiments, the reduced affinity of the sweeping antibody or antigen-binding portion thereof to FcRn shortens its pharmacokinetic (PK) profile compared to its conventional counterpart. For example, in some embodiments, the sweeping antibody or antigen-binding portion thereof is cleared more quickly for imaging and / or radioimmunotherapy. In some embodiments, the sweeping antibody or antigen-binding portion thereof promotes the clearance of endogenous pathogenic antibodies as a treatment for autoimmune diseases. In some embodiments, the sweeping antibody or antigen-binding portion thereof reduces the risk of adverse pregnancy outcomes that may be caused by transplacental transport of maternal-fetal specific antibodies.

[0182] In some embodiments, the sweeping antibody, or antigen-binding portion thereof, exhibits reduced affinity for the antigen at low pH compared to neutral or physiological pH (e.g., pH 7.4). In some embodiments, the sweeping antibody, or antigen-binding portion thereof, exhibits reduced affinity for the antigen at acidic pH (e.g., a pH in the range of 5.5 to 6.5) compared to physiological pH (e.g., pH 7.4).

[0183] It should be understood that any of the antibodies or antigen-binding fragments thereof provided herein may be engineered to dissociate from an antigen in response to a change in pH (e.g., a pH-sensitive antibody). In some embodiments, the sweeping antibodies or antigen-binding portions thereof provided herein are engineered to bind to an antigen in a pH-dependent manner. In some embodiments, the sweeping antibodies or antigen-binding portions thereof provided herein are engineered to bind to FcRn in a pH-dependent manner. In some embodiments, the sweeping antibodies or antigen-binding portions thereof provided herein are internalized by endocytosis. In some embodiments, the sweeping antibodies or antigen-binding portions thereof provided herein are internalized by FcRn binding. In some embodiments, the sweeping antibodies or antigen-binding portions thereof taken up by endocytosis release the antigen in an endosome. In some embodiments, the sweeping antibodies or antigen-binding portions thereof recycle back to the cell surface. In some embodiments, the sweeping antibodies remain bound to the cells. In some embodiments, the sweeping antibodies or antigen-binding portions thereof taken up by endocytosis recycle back to the plasma. It should be understood that any Fc portion of the antibody or antigen-binding fragment thereof provided herein can be engineered to have different FcRn binding activity. In some embodiments, the FcRn binding activity affects the clearance time of the antigen by the sweeping antibody. In some embodiments, the sweeping antibody can be a long-acting or fast-acting sweeping antibody.

[0184] In some embodiments, converting a conventional therapeutic antibody or antigen-binding portion thereof to a sweeping antibody or antigen-binding portion thereof reduces the effective dose. In some embodiments, converting a conventional therapeutic antibody or antigen-binding portion thereof to a sweeping antibody or antigen-binding portion thereof reduces the effective dose by at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%. In some embodiments, converting a conventional therapeutic antibody or antigen-binding portion thereof to a sweeping antibody or antigen-binding portion thereof reduces the effective dose by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 15-fold, 20-fold, 50-fold or 100-fold.

[0185] In some embodiments, the selection of the appropriate dose of the sweeping antibody or antigen-binding portion thereof for therapy may be performed empirically. In some embodiments, a high dose of the sweeping antibody or antigen-binding portion thereof may saturate FcRn, so that the antibody may stabilize the antigen in serum without being internalized. In some embodiments, a low dose of the sweeping antibody or antigen-binding portion thereof may not be therapeutically effective. In some embodiments, the sweeping antibody or antigen-binding portion thereof is administered once a day, once a week, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every eight weeks, once every ten weeks, once every twelve weeks, once every sixteen weeks, once every twenty weeks, or once every twenty-four weeks.

[0186] In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein may be modified or engineered to become a sweeping antibody. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein may be converted into a sweeping antibody using any suitable method. For example, suitable methods for making sweeping antibodies or antigen-binding portions thereof have been previously described in Igawa et al., (2013) "Engineered Monoclonal Antibody with Novel Antigen-Sweeping Activity In vivo," PLoS ONE 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 making sweeping antibodies or antigen-binding portions thereof as provided herein are not intended to be limiting. Thus, additional methods for making sweeping antibodies or antigen-binding portions thereof are within the scope of this disclosure.

[0187] Some aspects of the present disclosure include that any of the anti-pro / latent myostatin antibodies or antigen-binding fragments thereof provided herein can be used to detect apoptosis, such as AF (e.g., K D This is based on the recognition that the K (as expressed as K = K) of binding to pro / latent myostatin at relatively low pH (e.g., a pH range of 4.0 to 6.5) is sensitive to changes in pH. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have a K of binding to pro / latent myostatin at relatively high pH (e.g., a pH range of 7.0 to 7.4). D In some embodiments, the antibodies or antigen-binding fragments thereof provided herein exhibit an increase of 10 -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10-8 K for binding to pro / latent myostatin in the range of M D In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have a pH of 10 to 7.0. -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 K for binding to pro / latent myostatin in the range of M D In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have a K 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 higher at pH 4.0-6.5 compared to pH 7.0-7.4. D has.

[0188] In some embodiments, provided herein are pro / latent myostatin antibodies or antigen-binding fragments thereof that specifically bind to the same epitope as an antibody described in Table 2a, Table 11a, Table 11b or Table 13 of International Publication WO 2016 / 098357, which is based on International Patent Application No. PCT / JP2015 / 006323 published on June 23, 2016, and filed on December 18, 2015. In some embodiments, provided herein are pro / latent myostatin antibodies or antigen-binding fragments thereof that compete for binding with such antibodies.

[0189] In some embodiments, the pro / latent myostatin antibodies or antigen-binding fragments thereof provided herein do not specifically bind to the same epitope as an antibody described in Table 2a, Table 11a, Table 11b, or Table 13 of WO 2016 / 098357, which is based on International Patent Application No. PCT / JP2015 / 006323, published on June 23, 2016, and filed on December 18, 2015. In some embodiments, the pro / latent myostatin antibodies or antigen-binding fragments thereof provided herein do not compete or cross-compete for binding to the same epitope as an antibody described in Table 2a, Table 11a, Table 11b, or Table 13 of WO 2016 / 098357, which is based on International Patent Application No. PCT / JP2015 / 006323, published on June 23, 2016, and filed on December 18, 2015. In some embodiments, the pro / latent myostatin antibodies or antigen-binding fragments thereof provided herein do not specifically bind to the same epitope as an antibody comprising a VH and VL pair described in Table 2a, Table 11a, Table 11b or Table 13 of International Publication No. WO 2016 / 098357, which is based on the specification of International Patent Application No. PCT / JP2015 / 006323, published on June 23, 2016 and filed on December 18, 2015. In some embodiments, the pro / latent myostatin antibodies or antigen-binding fragments thereof provided herein do not compete or cross-compete for binding to the same epitope as an antibody comprising a VH and VL pair described in Table 2a, Table 11a, Table 11b or Table 13 of International Publication No. WO 2016 / 098357, which is based on the specification of International Patent Application No. PCT / JP2015 / 006323, published on June 23, 2016 and filed on December 18, 2015.

[0190] Antibodies and antigen-binding fragments that compete with anti-pro / latent myostatin antibodies or their antigen-binding fragments Aspects of the present disclosure relate to antibodies and antigen-binding fragments thereof that compete or cross-compete with any of the antibodies or antigen-binding fragments thereof provided herein. As used herein, the term "competes" with respect to an antibody means that a first antibody binds to an epitope of a protein (e.g., latent myostatin) in a manner sufficiently similar to the binding of a second antibody, such that the result of the binding of the first antibody to that epitope is detectably reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. Alternatively, the binding of the second antibody to that epitope may also be detectably reduced in the presence of the first antibody, but this need not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, if each antibody detectably inhibits the binding of the other antibody to its epitope or ligand, whether to the same extent, greater or less, the antibodies are said to "cross-compete" with each other for binding of their respective epitopes. Both competing and cross-competing antibodies are within the scope of the present disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), those skilled in the art will recognize that such competing and / or cross-competing antibodies can be encompassed and useful in the methods and / or compositions provided herein.

[0191] Aspects of the disclosure relate to antibodies or antigen-binding fragments thereof that compete or cross-compete with any of the antibodies or antigen-binding fragments thereof provided herein. In some embodiments, the antibodies or antigen-binding fragments thereof bind at or near the same epitope as any of the antibodies provided herein. In some embodiments, an antibody or antigen-binding fragment thereof binds near an epitope if it binds within 15 or fewer amino acid residues of the epitope. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein bind within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the epitope bound by any of the antibodies or antigen-binding fragments thereof provided herein. In preferred embodiments, such antibodies or antigen-binding fragments cross-compete with apitegromab for binding to human pro / latent myostatin. In some embodiments, such antibodies or antigen-binding fragments bind to an epitope within the prodomain of human myostatin, the epitope comprising one or more amino acid residues F147, Q149, L151, Y186, S168, K170, K205, and / or L207 (as numbered based on SEQ ID NO: 52 as disclosed herein). In some embodiments, such antibodies or antigen-binding fragments bind to an epitope within the prodomain of human myostatin, the epitope comprising one or more amino acid residues F147, Q149, L151, Y186, R167, S168, K170, K205, L207, E209, and / or N210, as numbered according to SEQ ID NO: 52. In some embodiments, such antibodies or antigen-binding fragments comprise an HCDR3 paratope that is a variant of SEQ ID NO: 10, the variant comprising up to two amino acid substitutions compared to SEQ ID NO: 10. In some embodiments, such antibodies or antigen-binding fragments comprise an HCDR3 sequence comprising a leucine at amino acid position 3 and a tryptophan at amino acid position 9 (as numbered based on SEQ ID NO:10).In some embodiments, such antibodies or antigen-binding fragments comprise an HCDR3 sequence comprising a leucine at amino acid position 3, a valine at amino acid position 4, a leucine at amino acid position 7, a tyrosine at amino acid position 8, and / or a tryptophan at amino acid position 9 (as numbered based on SEQ ID NO:10).

[0192] In another embodiment, the antibody or antigen-binding portion thereof has a binding affinity to any of the antigens provided herein (e.g., pro / latent myostatin), of at least 10 -8 The equilibrium dissociation constant K between the antibody and the protein is less than M D In other embodiments, the antibody or antigen-binding portion thereof competes or cross-competes at 10 -11 M~10 -8 K in the M range D compete or cross-compete with each other.

[0193] Aspects of the disclosure relate to antibodies or antigen-binding portions thereof that compete with any of the antibodies or antigen-binding fragments thereof provided herein for binding to pro / latent myostatin. In some embodiments, the antibodies or antigen-binding portions thereof bind to pro / latent myostatin at the same epitope as any of the antibodies or antigen-binding fragments thereof provided herein. For example, in some embodiments, any of the antibodies provided herein bind to or near the toroid cleavage site of pro / latent myostatin or bind to or near the toroid docking site. 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 bind to or near the proprotein convertase docking site. In another embodiment, the antibodies or antigen-binding portions thereof compete with any of the antibodies or antigen-binding fragments thereof for binding to pro / latent myostatin at the same epitope as any of the antibodies or antigen-binding fragments thereof provided herein. -6 The equilibrium dissociation constant, K, between an antibody or antigen-binding portion thereof and pro / latent myostatin is less than M D In other embodiments, an antibody or antigen-binding portion thereof that competes with any of the antibodies or antigen-binding portions thereof provided herein competes at 10-11 M~10 -8 K in the M range D It binds to pro / latent myostatin.

[0194] Any of the antibodies or antigen-binding fragments thereof provided herein may be characterized using any suitable method. For example, one method is to identify the epitope to which the antigen binds, or "epitope mapping". There are many suitable methods for mapping and characterizing the location of an epitope on a protein, including, for example, solving the crystal structure of an antibody-antigen complex as described in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999, competitive assays, gene fragment expression assays, and synthetic peptide-based assays. In an additional example, epitope mapping can be used to determine the sequence to which an antibody or antigen-binding fragment thereof binds. The epitope may be a linear epitope, i.e., contained in a stretch of amino acids, or may be a conformational epitope formed by three-dimensional interactions of amino acids that may not necessarily be contained in a stretch (primary structure linear sequence). Peptides of various lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used for binding assays with antibodies. In another example, the epitope to which an antibody or antigen-binding fragment thereof binds can be determined in a systematic selection by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody or antigen-binding fragment thereof. According to a gene fragment expression assay, an open reading frame encoding a target antigen is fragmented randomly or for a specific genetic configuration, and the reactivity of the antibody to be tested with the expressed fragments of the antigen is determined. Gene fragments can be generated, for example, by PCR, followed by in vitro transcription in the presence of radioactive amino acids and translation into protein. The binding of the antibody or antigen-binding fragment thereof to the radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis. Specific epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries).Alternatively, a defined library of overlapping peptide fragments can be tested for binding to a test antibody or antigen-binding fragment thereof in a simple binding assay. In an additional example, mutagenesis of the antigen-binding domain, domain swapping experiments and alanine scanning mutagenesis can be performed to identify sufficient and / or necessary residues required for epitope binding. For example, domain swapping experiments can be performed using mutants of the target antigen in which various fragments of the pro / latent myostatin polypeptide are replaced (swapped) with sequences from a closely related but antigenically distinct protein, such as another member of the TGFβ family (e.g., GDF11). By determining binding to mutant pro / latent myostatin by an antibody or antigen-binding portion thereof, the importance of binding of a particular antigen fragment to the antibody or antigen-binding portion thereof can be determined.

[0195] Alternatively, one can use other antibodies known to bind to the same antigen to perform competition assays to determine whether the antibody or antigen-binding portion thereof binds to the same epitope as the other antibody or antigen-binding portion thereof. Competition assays are well known to those of skill in the art.

[0196] By applying any suitable method, such as epitope mapping methods as described herein, it can be determined whether an anti-pro / latent myostatin antibody or antigen binding portion thereof binds to one or more of the specific residues / segments in pro / latent myostatin as described herein. Furthermore, the interaction of the antibody or antigen binding portion thereof with one or more of their defined residues in pro / latent myostatin can be determined by routine techniques. For example, a crystal structure can be determined and the distance between a residue in pro / latent myostatin and one or more residues in the antibody or antigen binding portion thereof can be determined accordingly. Based on such distance, it can be determined whether a certain specific residue in pro / latent myostatin interacts with one or more residues in the antibody or antigen binding portion thereof. Furthermore, by applying suitable methods such as competition assays and targeted mutagenesis assays, the preferential binding of a candidate anti-pro / latent myostatin antibody or antigen binding portion thereof to pro / latent myostatin compared to another target, such as mutant pro / latent myostatin, can be determined.

[0197] Preparation of anti-pro / latent myostatin antibodies or antigen-binding fragments thereof Numerous methods can be used to obtain the antibodies or antigen-binding fragments thereof of the present disclosure. For example, antibodies and antigen-binding fragments thereof can be produced using recombinant DNA methods. Monoclonal antibodies and antigen-binding fragments thereof can also be produced by hybridoma generation according to known methods (e.g., Kohler and Milstein (1975) Nature, 256:495-499). Hybridomas formed in this manner are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (e.g., OCTET or BIACORE) analysis, to identify one or more hybridomas that produce an antibody or antigen-binding fragment thereof that specifically binds to the specified antigen. Any form of the specified antigen can be used as an immunogen, such as recombinant antigens, naturally occurring forms, any variants or fragments thereof, as well as antigenic peptides thereof (e.g., any of the epitopes described herein as linear epitopes or within a framework as conformational epitopes). One exemplary method for producing antibodies and antigen-binding fragments thereof includes screening protein expression libraries, such as phage or ribosome display libraries, that express antibodies or fragments thereof (eg, scFv). Phage display is described, for example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222:581-597; WO 92 / 18619; WO 91 / 17271; WO 92 / 20791; WO 92 / 15679; WO 93 / 01288; WO 92 / 01047; WO 92 / 09690; and WO 90 / 02809.

[0198] In addition to the use of display libraries, a specific antigen (e.g., promyostatin) can be used to immunize a non-human animal, such as a rodent, such as a mouse, hamster, or rat. In one embodiment, the non-human animal is a mouse.

[0199] In another embodiment, monoclonal antibodies are obtained from non-human animals and then modified, for example, chimeric, using suitable recombinant DNA techniques.Various techniques for producing chimeric antibodies have been described.See, for example, Morrison et al., Proc.Natl.Acad.Sci.USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., EP 171496; EP 0173494, GB 2177096B.

[0200] For additional antibody production techniques, see Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988. This disclosure is not necessarily limited to any particular source, method of production or other particular characteristics of the antibodies.

[0201] Some aspects of the present disclosure relate to a host cell transformed with a polynucleotide or vector. The host cell may be a prokaryotic or eukaryotic cell. The polynucleotide or vector present in the host cell may be integrated into the genome of the host cell or it may be maintained extrachromosomally. The host cell may be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell. In some embodiments, the eukaryotic cell is, for example, of the genus Saccharomyces, particularly of the species S. cerevisiae. The term "prokaryotic" includes all bacteria that can be transformed or transfected with DNA or RNA molecules for the expression of an antibody or the corresponding immunoglobulin chain. Prokaryotic hosts may include gram-negative and gram-positive bacteria, such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. The term "eukaryotic" includes yeast, higher plants, insect and vertebrate cells, e.g., mammalian cells such as NSO and CHO cells. Depending on the host utilized in a recombinant production procedure, the antibody or immunoglobulin chain encoded by the polynucleotide may be glycosylated or non-glycosylated. The antibody or the corresponding immunoglobulin chain may also include an initial methionine amino acid residue.

[0202] In some embodiments, once the vector has been incorporated into a suitable host, the host may be maintained under conditions suitable for high level expression of the nucleotide sequence, followed by recovery and purification of the immunoglobulin light chain, heavy chain, light / heavy chain dimer or intact antibody, antigen-binding fragment or other immunoglobulin form as required; see Beychok, Cells of Immunoglobulin Synthesis, Academic Press, NY (1979). Thus, the polynucleotide or vector is then introduced into a cell that produces the antibody or antigen-binding fragment. Furthermore, the transgenic animal, preferably a mammal, comprising the host cell described above may also be used for large-scale production of antibodies or antibody fragments. Large-scale production typically refers to bioreactors (e.g., cell cultures) of 250 liters or more, such as 250L, 500L, 1000L, 1500L, 2000L, 3000L, 4000L, 5000L, 6000L or more.

[0203] Transformed host cells can be grown in fermenters and cultured using any suitable technique to achieve optimal cell growth. Once expressed, complete antibodies, their dimers, individual light and heavy chains, other immunoglobulin forms, or antigen-binding fragments can be purified according to standard means in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like; see Scopes, "Protein Purification", Springer Verlag, NY (1982). The antibodies or antigen-binding fragments can then be isolated from the growth medium, cell lysates, or cell membrane fractions. Isolation and purification of antibodies or antigen-binding fragments expressed, for example, in microorganisms, can be by any conventional means, including, for example, preparative chromatographic separations and immunological separations, including, for example, those involving the use of monoclonal or polyclonal antibodies directed against the constant regions of the antibodies.

[0204] Aspects of the present disclosure relate to hybridomas, which provide an indefinitely extended source of monoclonal antibodies. Instead of obtaining immunoglobulins directly from hybridoma cultures, immortalized hybridoma cells can be used as a source of rearranged heavy and light chain sites for subsequent expression and / or genetic engineering. Rearranged antibody genes can be reverse transcribed from appropriate mRNA to generate cDNA. In some embodiments, the heavy chain constant region can be replaced with that of a different isotype or removed entirely. Variable regions can be linked to encode a single chain Fv region. Multiple Fv regions can be linked to confer binding capabilities to two or more targets or chimeric heavy and light chain combinations can be utilized. Any suitable method can be used for cloning antibody variable regions and generating recombinant antibodies and antigen-binding portions thereof.

[0205] In some embodiments, suitable nucleic acids encoding the variable regions of the heavy and / or light chains are obtained and inserted into expression vectors that can be transfected into standard recombinant host cells. A variety of such host cells can be used. In some embodiments, mammalian host cells can be advantageous for efficient processing and production. Exemplary mammalian cell lines useful for this purpose include CHO, 293 or NSO cells. Production of the antibody or antigen-binding fragment can be carried out by culturing the modified recombinant host under culture conditions suitable for host cell growth and expression of the coding sequence. The antibody or antigen-binding fragment can be recovered by isolating it from the culture. The expression system can be designed to include a signal peptide so that the resulting antibody is secreted into the medium; however, intracellular production is also possible.

[0206] The present disclosure also includes polynucleotides encoding at least the variable regions of the immunoglobulin chains of the antibodies described herein. In some embodiments, the variable regions encoded by the polynucleotides are the VV of the variable regions of the antibodies produced by any one of the hybridomas described above. H and / or V L It comprises at least one complementarity determining region (CDR).

[0207] The polynucleotide encoding the antibody or antigen-binding fragment can be, for example, DNA, cDNA, RNA, or synthetically produced DNA or RNA, or a recombinantly produced chimeric nucleic acid molecule comprising any of these polynucleotides, alone or in combination. In some embodiments, the polynucleotide is part of a vector. Such vectors may further comprise genes, such as marker genes, that allow for the selection of the vector under suitable conditions in a suitable host cell.

[0208] In some embodiments, the polynucleotide is operably linked to an expression control sequence that allows expression in prokaryotic or eukaryotic cells. Expression of the polynucleotide includes transcription of the polynucleotide into a translatable mRNA. 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 facilitate initiation of transcription and optionally poly-A signals that facilitate termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional and translational enhancers and / or naturally associated or heterologous promoter regions. Suitable regulatory elements that allow expression in prokaryotic host cells include, for example, PL, Lac, Trp or Tac promoters in E. coli, and examples of regulatory elements that allow expression in eukaryotic host cells are AOX1 or GAL1 promoters in yeast or CMV-promoter, SV40-promoter, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or globin intron in mammalian and other animal cells.

[0209] In addition to elements involved in the initiation of transcription, such regulatory elements may also include transcription termination signals, such as an SV40-poly-A site or a tk-poly-A site downstream of the polynucleotide. Furthermore, depending on the expression system utilized, leader sequences capable of directing the polypeptide to an intracellular compartment or secreting it into the medium may be added to the coding sequence of the polynucleotide and have been previously described. The leader sequence is assembled at the appropriate time with translation, initiation and termination sequences and preferably a leader sequence capable of directing the secretion of the translated protein or a portion thereof, for example, into the extracellular medium. Optionally, a heterologous polynucleotide sequence may be used that encodes a fusion protein containing a C-terminal or N-terminal identification peptide that provides a desired characteristic, such as stabilization or simplified purification of the expressed recombinant product.

[0210] In some embodiments, the polynucleotides encoding at least the variable domains of the light and / or heavy chains may encode the variable domains of both or only one of the immunoglobulin chains. Similarly, the polynucleotides may be under the control of the same promoter and may be separately controlled for expression. Furthermore, some aspects relate to vectors, particularly plasmids, cosmids, viruses and bacteriophages, conventionally used in genetic engineering, including polynucleotides encoding the variable domains of the immunoglobulin chains of an antibody or antigen-binding fragment; optionally in combination with polynucleotides encoding the variable domains of other immunoglobulin chains of the antibody.

[0211] In some embodiments, the expression control sequences are provided as eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, although control sequences for prokaryotic hosts may also be used. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses may be used for delivery of polynucleotides or vectors to targeted cell populations (e.g., to engineer cells to express antibodies or antigen-binding fragments). A variety of suitable methods can be used to construct recombinant viral vectors. In some embodiments, polynucleotides and vectors can be reconstituted into liposomes for delivery to target cells. Vectors containing polynucleotides (e.g., sequences encoding heavy and / or light variable domains of immunoglobulin chains and expression control sequences) can be introduced into host cells by suitable methods that vary depending on the type of cellular host.

[0212] qualification The antibodies and antigen-binding fragments of the disclosure may be modified with a detectable label, including, but not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, non-radioactive paramagnetic metal ions, and affinity labels for detection and isolation of pro / latent myostatin. The detectable substance may be attached or conjugated directly to the polypeptide of the disclosure or indirectly through an intermediate moiety (e.g., a linker, etc.) using suitable techniques. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, or acetylcholinesterase; non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin, non-limiting examples of suitable fluorescent materials include biotin, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; non-limiting examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include radioactive metal ions, such as alpha, beta, ... Ar-emitters or ions such as iodine (131I, 125I, 123I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115mIn, 113mIn, 112In, 111In) and technetium (99Tc, 99mTc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (13 Other radioisotopes include 3Xe), fluorine (18F), 153Sm, Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 86R, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, and tin (113Sn, 117Sn).A detectable substance can be coupled or conjugated to an anti-pro / latent myostatin antibody or antigen-binding portion thereof of the present disclosure, either directly or indirectly through an intermediate (e.g., a linker, etc.) using suitable techniques. An anti-pro / latent myostatin antibody or antigen-binding portion thereof conjugated to a detectable substance can be used in diagnostic assays as described herein.

[0213] Biological Effects of Combining Myostatin Pathway Inhibitors with GLP-1 Pathway Activators Methods or treatments that include administering a myostatin pathway inhibitor (such as a myostatin antibody) in conjunction with a GLP-1 pathway activator are encompassed by the present disclosure and can be used as pharmaceuticals such that when each is administered to a subject in an effective amount, a beneficial effect (e.g., a therapeutic effect) occurs in the subject. Exemplary such beneficial biological effects are provided herein. A beneficial biological effect in a subject can be achieved by administration of a myostatin pathway inhibitor, such as an antibody or antigen-binding fragment thereof as described herein that specifically binds to pro / latent myostatin. In some embodiments, the myostatin pathway inhibitor and the GLP-1 pathway activator are administered in parallel, simultaneously, or sequentially. In some embodiments, the myostatin pathway inhibitor and the GLP-1 pathway activator are administered in an amount effective to cause two or more of the biological effects described below. In some embodiments, the myostatin pathway inhibitor and the GLP-1 pathway activator are administered in an amount effective to cause three or more of the biological effects described below. In some embodiments, the myostatin pathway inhibitor and GLP-1 pathway activator are administered in an amount effective to cause four or more of the biological effects described below. In some embodiments, the myostatin pathway inhibitor and GLP-1 pathway activator are administered in an amount effective to cause five or more of the biological effects described below. In some embodiments, the myostatin pathway inhibitor and GLP-1 pathway activator are administered in an amount effective to cause six or more of the biological effects described below. In some embodiments, the myostatin pathway inhibitor and GLP-1 pathway activator are administered in an amount effective to cause seven or more of the biological effects described below. In some embodiments, the myostatin pathway inhibitor and GLP-1 pathway activator are administered in an amount effective to cause eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen of the biological effects described below.

[0214] Effects on muscle tissue mass and / or function in human subjects In some embodiments, administration of a myostatin pathway inhibitor in conjunction with a GLP-1 pathway activator increases the amount and / or function of muscle tissue in a human subject. In some embodiments, the muscle tissue is selected from the group consisting of smooth muscle tissue, skeletal muscle tissue, and cardiac muscle tissue. Smooth muscle tissue is composed of long, tapered cells, is generally involuntary, and differs from striated muscle in that it has a much higher actin / myosin ratio, lacks prominent sarcomeres, and is capable of contracting to a much smaller percentage of its resting length. Smooth muscle cells are found especially in the walls of blood vessels, around the intestine, and in the uterus. Cardiac muscle tissue is striated, but involuntary tissue, and is responsible for the pumping activity of the vertebrate heart. Individual cardiac muscle cells are not fused together into a multinucleated structure as they are in striated muscle tissue. Skeletal muscle tissue is under voluntary control. The muscle fibers are syncytial, with myofibrils, tandemly arranged sarcomeres. There are two general types of skeletal muscle fibers based on their detailed myosin heavy chain (MHC) isoform expression: slow-twitch fibers (type I) and fast-twitch fibers (type II). Slow-twitch fibers are more suited to aerobic exercise and facilitate endurance feats such as long-distance running, while fast-twitch fibers fatigue quickly but are more suited to anaerobic exercise and are used for powerful explosive movements such as sprinting. The distinction between slow-twitch and fast-twitch fibers is based on histochemical staining for myosin adenosine triphosphatase (ATPase) and the type of myosin heavy chain. Slow-twitch fibers (type I fibers) are MHC isoform I, and the three fast-twitch isoforms (type II fibers) are MHC isoform IIa, MHC isoform IId, and MHC isoform IIb (S. Schiaffino, J. Muscle Res. Cell. Motil., 10 (1989), pp. 197-205).

[0215] In some embodiments, the mass and / or function of fast twitch muscle tissue is increased in a human subject. In other embodiments, the mass and / or function of slow twitch muscle tissue is increased in a human subject. Management of muscle mass is known to support weight loss, as observed, for example, with resistance training, in the context of treating obesity. Myostatin inhibition according to the present invention preferentially preserves fast twitch muscle fibers.

[0216] The biological effects of effective amounts of the pharmaceutical compositions and methods of treatment provided herein may be associated with a phenotypic change in muscle fiber type, a process called fiber type conversion. In some embodiments, fiber type conversion is initiated by events such as injury and starvation.

[0217] In one aspect, the disclosure provides a method of promoting fiber type conversion in a subject comprising administering to the subject a composition comprising a myostatin pathway inhibitor, e.g., an antibody or antigen binding fragment thereof that specifically binds to pro / latent myostatin and blocks release of mature myostatin, in an amount effective to promote fiber type conversion, thereby promoting fiber type conversion in the subject.

[0218] In another aspect, the disclosure provides a method of preferentially increasing type II or fast twitch fibers over type I or slow twitch fibers in a subject, the method comprising administering to the subject a myostatin pathway inhibitor, e.g., a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin and blocks release of mature myostatin, in an amount effective for fiber type conversion to preferentially increase type II or fast twitch fibers over type I or slow twitch fibers, thereby preferentially increasing type II or fast twitch fibers over type I or slow twitch fibers in the subject.

[0219] In some embodiments, administration of an effective amount of each of the myostatin pathway inhibitor and GLP-1 pathway activator as described herein to a subject can result in an increase in muscle mass and / or muscle function. Preferably, such an increase in muscle mass is clinically meaningful to benefit or otherwise improve the subject's health status. For example, a clinically meaningful change in muscle mass can improve a patient's exercise capacity, self-care, metabolism, etc. In some embodiments, the increase in muscle mass is an increase in one or more lean muscles. In some embodiments, such an increase in muscle mass is a systemic effect such that the entire body or substantially the entire body of muscle shows a measurable effect. In some embodiments, lean muscle is a muscle that is densely packed with contractile tissue and has a low fat and connective tissue content. In other embodiments, the effect is localized to certain groups / types of muscle. In some embodiments, muscle tissue mass, e.g., lean muscle tissue mass, 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 mass, e.g., lean muscle tissue mass, is increased by at least 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 may be estimated or measured by any suitable known method, including measurement of cross-sectional area by MRI (e.g., forearm cross-section), circumference, diaphragm width (e.g., by ultrasound), qNMR, DXA, etc.

[0220] In some embodiments, administering an effective amount of a combination of a myostatin pathway inhibitor and a GLP-1 pathway activator to a subject can result in increased muscle function. Muscle function can be measured by a variety of measures, including, but not limited to, force generation, grip strength (e.g., maximal grip strength), endurance, muscle oxidative capacity, dynamic grip endurance, and the like. In some embodiments, serum creatinine level is used as a validated biomarker that is indicative of muscle mass, although it has limited sensitivity.

[0221] In some embodiments, muscle tissue function is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, muscle tissue function is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%. In some embodiments, increased muscle function includes, for example, an improvement in a rating 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.

[0222] In some embodiments, the use of myostatin pathway inhibitors alone or in combination with GLP-1 pathway activators in the methods of the invention may increase muscle tissue mass and / or function in subjects suffering from pathology resulting from, for example, spinal cord injury. In some embodiments, the subject is in the acute phase of spinal cord injury immediately following injury, where diagnosis between complete and incomplete injury is generally difficult. In other embodiments, the subject is in the subacute phase of spinal cord injury, where there is a distinctive difference between complete and incomplete spinal cord injury, and where recovery through ongoing rehabilitation is possible. In yet another embodiment, the subject is in the chronic phase of spinal cord injury. The chronic phase of SCI occurs approximately 4-6 months after the date of injury, when the patient shows a substantial decline in the rate of recovery, or when rehabilitation efforts reach a plateau despite ongoing standard treatment efforts.

[0223] In some embodiments, the amount and / or function of muscle tissue underlying a lesion is increased in a subject suffering from a lesion, e.g., spinal cord injury. In other embodiments, the amount and / or function of muscle tissue overlying a lesion is increased in a subject suffering from a lesion, e.g., spinal cord injury. In some embodiments, the muscle is selected from the group consisting of soleus, gastrocnemius, biceps, and triceps. In some embodiments, the amount of muscle tissue is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, the amount of muscle tissue is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%. In some embodiments, the function of muscle tissue is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, muscle tissue function is increased by at least 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%.

[0224] In some embodiments, administration of a myostatin pathway inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, alone or in combination with a GLP-1 pathway activator, increases locomotor function in a human subject, e.g., a subject suffering from a pathology. In some embodiments, the locomotor function of the human subject is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, the locomotor function of the human subject is increased by at least 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%.

[0225] In some embodiments, administration of a myostatin pathway inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds pro / latent myostatin, alone or in combination with a GLP-1 pathway activator, increases motor coordination and balance in a human subject, e.g., a subject suffering from a pathology. In some embodiments, motor coordination and balance in a human subject is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, the motor coordination and balance of the human subject is increased by at least 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%.

[0226] In another embodiment, administration of a myostatin pathway inhibitor alone or in combination with a GLP-1 pathway activator increases muscle strength in a human subject, such as a subject suffering from a pathology. In some embodiments, the muscle strength of a human subject is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the muscle strength of a human subject is increased by at least 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%.

[0227] In some embodiments, administration of a myostatin pathway inhibitor alone or in combination with a GLP-1 pathway activator can result in clinically meaningful changes in muscle function corresponding to increased functionality in the patient. In some embodiments, increased functionality can include improvements in the patient's exercise capacity, self-care, metabolism, and the like. In some embodiments, administration of an effective amount of a myostatin pathway inhibitor, such as an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, promotes or accelerates recovery from conditions, such as injury, surgery, and other medical procedures. Suitable such conditions can involve conditions associated with nerve injury (whether resulting from injury or surgery or other clinical procedures).

[0228] For example, suitable subjects include generally healthy individuals, such as those who: i) have sustained an acute injury involving nerve damage that affects muscle function; ii) are scheduled to undergo a surgical procedure (therapeutic or corrective) that may cause unintended nerve damage (e.g., motor neuron injury); iii) have undergone a surgical procedure that has caused unintended muscle dysfunction; iv) are undergoing a treatment that involves immobilization of a particular muscle or muscle group (e.g., a cast, etc.); and v) are on a ventilator (e.g., as a result of an acute injury). Administration of a myostatin pathway inhibitor as described herein may accelerate the recovery of such patients. In some embodiments, such administration may be prophylactic. For example, an antibody may be administered to prevent muscle dysfunction before or immediately after undergoing a surgical procedure that may cause nerve damage and associated muscle dysfunction. Prevention includes reducing or alleviating the severity of such dysfunction. In these embodiments, administration may be localized to the affected area, e.g., at or near the site of injury, surgery, etc.

[0229] Effects on muscle protein catabolism and / or muscle amino acid release in human subjects In some embodiments, administration of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor), alone or in combination with a GLP-1 pathway activator, reduces muscle protein catabolism and / or muscle amino acid release in a human subject. In some embodiments, muscle protein catabolism and / or muscle amino acid release is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, muscle protein catabolism and / or muscle amino acid release is reduced by at least 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%.

[0230] Efficacy in preventing muscle loss or atrophy in human subjects In some embodiments, administration of an effective amount of a combination of myostatin pathway inhibitors (e.g., myostatin inhibitors, e.g., myostatin selective inhibitors), alone or in combination with a GLP-1 pathway activator, prevents muscle loss or muscle atrophy in a human subject at risk of developing muscle loss and / or muscle atrophy. In some embodiments, muscle loss or muscle 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 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, treatment results in, for example, loss of body fat mass, while muscle mass is preserved (i.e., no gain or loss of muscle mass). In some embodiments, subjects receiving combination therapy may exhibit an increase in muscle mass. In some embodiments, treatment with combination therapy maintains an increase in muscle mass compared to muscle mass prior to treatment.

[0231] In some embodiments, suitable subjects are those who have not yet developed atrophy but are considered to be at risk of developing atrophy. In some embodiments, the subject is in a weight loss program, such as a program that includes a diet and / or exercise regimen. In some embodiments, the subject is in a weight loss program that includes a diet regimen of at least 20%, such as at least 30%, 35%, 40%, 45% or 50% calorie reduction. In some embodiments, the subject has type 2 diabetes. In some embodiments, the subject has cardiovascular disease. In some embodiments, the subject has a disease or condition associated with a neurological defect that impairs motor neuron function. In some embodiments, such a condition is caused by muscular dystrophy or muscle atrophy. In some embodiments, the neurological defect is caused by nerve damage. In some embodiments, the nerve damage involves partial denervation of motor neurons that causes partial functional impairment of affected muscles. In some embodiments, such a condition is caused by SCI. In some embodiments, the subject with SCI is in the acute or subacute phase of SCI (e.g., has not yet reached the chronic phase).

[0232] In some embodiments, when a composition comprising an effective amount of a myostatin signaling inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) is administered to a patient population at risk for developing muscle atrophy associated with partial denervation of motor neurons, the composition i) prevents the onset or worsening of muscle atrophy in a statistically significant proportion of the patient population; or ii) reduces the severity of muscle atrophy in a statistically significant proportion of the patient population. In some embodiments, the myostatin signaling inhibitor is administered in conjunction with a GLP-1 pathway activator as described herein.

[0233] Prevention of muscle loss or atrophy by use of a myostatin signaling inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor), alone or in combination with a GLP-1 pathway activator as described herein, can be readily monitored or determined by any suitable method for assessing muscle mass or motor function associated with the affected muscles.

[0234] In some embodiments, administration of an effective amount of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) also prevents or alleviates neuropathy, such as diabetic neuropathy or early onset axonal polyneuropathy in affected limbs.

[0235] Effects on intramuscular fat infiltration in human subjects In some embodiments, administration of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor), alone or in combination with a GLP-1 pathway activator, reduces intramuscular fat infiltration in a human subject. In some embodiments, intramuscular fat infiltration is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, intramuscular fat infiltration is reduced by at least 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%.

[0236] Effects on body adipose tissue levels in human subjects In some embodiments, administration of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) alone or in combination with a GLP-1 pathway activator affects body adipose tissue levels in human subjects. As used herein, the term "body adipose tissue" refers to fat, including connective tissue that stores fat. Body adipose tissue originates from preadipocytes. Its main role is to store energy in the form of lipids, but it also provides cushioning and protection for the body. The two types of body adipose tissue are white adipose tissue (WAT), which stores energy, and brown adipose tissue (BAT), which generates body heat.

[0237] Brown adipose tissue (BAT) is known to function in dissipating chemical energy in response to cold or excessive feeding, and also has the ability to regulate energy balance. Activation of brown adipose tissue has been shown to improve glucose homeostasis and insulin sensitivity in humans, suggesting that anyone with impaired insulin function may benefit from BAT activation (Stanford et al., J Clin Invest. 2013, 123(1):215-223).

[0238] Beige adipose tissue is generated as a result of the browning of WAT, also known as beigification. This occurs when adipocytes within WAT depots develop characteristics of BAT. Beige adipocytes take on a multilocular appearance (containing several lipid droplets) and increase expression of uncoupling protein 1 (UCP1). In doing so, these normal energy-storing white adipocytes become energy-releasing adipocytes (Harms et al. Nature Medicine. 2013,19(10):1252-63).

[0239] Visceral or abdominal fat (also known as organ fat or intraperitoneal fat) is located inside the abdominal cavity and packed between the organs (stomach, liver, intestines, kidneys, etc.). Visceral fat is different from subcutaneous fat, which is located under the skin, and intramuscular fat, which is scattered among skeletal muscles. Fat in the lower body, such as in the thighs and buttocks, is subcutaneous and is a non-uniformly spaced tissue, while fat in the abdomen is mostly visceral and is semi-liquid. Excessive amounts of visceral fat are known as central obesity, "central adiposity" or "abdominal fat," with excessive abdominal protrusion, and new developments such as the Body Volume Index (BVI) are specifically designed to measure abdominal volume and abdominal fat. Excess visceral fat has also been linked to type 2 diabetes, insulin resistance, inflammatory diseases, and other obesity-related disorders (Mokdad et al., JAMA: The Journal of the American Medical Association. 2001, 289(1):76-9).

[0240] Body adipose tissue mass can be determined by any method known to those of skill in the art. For example, body adipose tissue can be measured by qNMR, dual energy X-ray absorptiometry (DXA), and other methods known in the art.

[0241] In some embodiments, administration of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) increases brown adipose tissue levels and / or beige adipose tissue levels in a human subject. In some embodiments, administration of a myostatin pathway inhibitor, e.g., an anti-pro / latent myostatin antibody or antigen-binding portion thereof, decreases white adipose tissue and visceral adipose tissue levels in a human subject. In some embodiments, administering includes administering a GLP-1 pathway activator in combination with the myostatin inhibitor.

[0242] In some embodiments, brown or beige adipose tissue levels are increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, brown or beige adipose tissue levels are increased by at least 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%.

[0243] In some embodiments, white or visceral adipose tissue levels are reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, white or visceral adipose tissue levels are reduced by at least 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%.

[0244] In some embodiments, myostatin inhibition has different effects during weight loss compared to weight maintenance. In some embodiments, inhibiting the myostatin pathway promotes greater loss of body fat mass during weight maintenance compared to loss of body fat mass during active weight loss. In some embodiments, inhibiting the myostatin pathway promotes greater loss of body fat mass under moderate calorie restriction (e.g., calorie restriction of 30% or less, e.g., 20% calorie restriction) compared to extreme calorie restriction (e.g., calorie restriction of 30% or more).

[0245] In some embodiments, the rate of fat gain slows or stops compared to the rate before treatment, hi some embodiments, the treatment maintains a reduced rate of fat gain compared to a faster rate of fat gain before treatment.

[0246] Effect on fat-to-muscle tissue ratio in human subjects In some embodiments, administration of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor), alone or in combination with a GLP-1 pathway activator, reduces fat to muscle tissue ratio in a human subject. In some embodiments, the fat to muscle tissue 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 tissue ratio is reduced by at least 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, administration of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor), alone or in combination with a GLP-1 pathway activator, maintains a reduction in the ratio of body adipose tissue to muscle tissue in a human subject who has previously achieved a reduction in fat-to-muscle tissue ratio.

[0247] In some embodiments, administration of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor), alone or in combination with a GLP-1 pathway activator, increases muscle tissue to fat ratio in a human subject. In some embodiments, muscle tissue to fat ratio 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 to fat ratio is increased by at least 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, administration of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor), alone or in combination with a GLP-1 pathway activator, maintains an increase in muscle tissue to fat ratio in a human subject who has previously achieved an increase in such ratio.

[0248] Hormonal control Leptin is a hormone produced and secreted by body adipose tissue and plays a role in regulating food intake and stimulating energy expenditure. Defects in leptin production have been reported to cause severe genetic obesity in rodents and humans. In addition to its effects on body weight, leptin also has a variety of other functions, including regulating hematopoiesis, angiogenesis, wound healing, and immune and inflammatory responses. Leptin acts through the leptin receptor, a single transmembrane domain receptor of the cytokine receptor family, which is found in several alternatively spliced ​​forms in many tissues. The LEP gene is the human homolog of the gene (ob) mutant in the mouse "obese" phenotype. Leptin levels in a subject refer to the amount of circulating leptin in the body of a subject, e.g., a mammalian subject, and a reduction in leptin levels refers to a reduction in the amount of circulating leptin, e.g., as a result of treatment, compared to a baseline measurement.

[0249] In some embodiments, the therapeutic efficacy of treating a subject (e.g., a mammalian subject) with a myostatin pathway inhibitor (e.g., alone or in combination with a GLP-1 pathway activator) may be determined by measuring a reduction in leptin levels in the subject's blood after administration of the myostatin inhibitor compared to before administration. In some embodiments, therapeutic efficacy may be determined by (i) determining the subject's blood leptin levels before administration of the myostatin inhibitor; (ii) administering the myostatin inhibitor; and (iii) determining the subject's blood leptin levels after administration of the myostatin inhibitor; a reduction in blood leptin levels is indicative of therapeutic efficacy.

[0250] Adiponectin is an adipokine involved in the control of fat metabolism and insulin sensitivity with direct antidiabetic, antiatherogenic and anti-inflammatory activities. Adiponectin stimulates AMPK phosphorylation and activation in liver and skeletal muscle to enhance glucose utilization and fatty acid burning, and inhibits endothelial NFκ-B signaling through cAMP-dependent pathways. Adiponectin may be involved in brown adipocyte differentiation. As used herein, the term "adiponectin level" refers to the amount of circulating adiponectin, e.g., total adiponectin or glycosylated adiponectin. The plasma adiponectin level of a subject, e.g., a mammalian subject, may be measured, for example, by a total adiponectin assay (ELISA kit EZHADP-61K, Millipore, St. Charles, Missouri, USA), which captures all forms of circulating adiponectin with a sensitivity of 0.78ng / mL and intra- and inter-batch coefficients of variation of 1.8% and 6.2%, respectively. An increase or decrease in adiponectin levels refers to a greater or lesser amount of circulating adiponectin detected in a subject (eg, after treatment of a metabolic disease or disorder) compared to a baseline measurement.

[0251] Ghrelin is an appetite-regulating hormone that has appetite-stimulating effects, induces fat accumulation, and stimulates gastric secretion. Plasma ghrelin concentrations increase in fasting states and decrease after habitual feeding. Normal ghrelin concentrations in plasma samples in humans are 10-20 fmol / ml for n-octanoyl ghrelin and 100-150 fmol / ml for total ghrelin, including both acyl-modified and desacyl ghrelin. Ghrelin levels can be measured, for example, using Ghrelin Human ELISA kid, catalog number BMS2192 (Invitrogen).

[0252] Effects on metabolic rate in human subjects In some embodiments, administration of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor), alone or in combination with a GLP-1 pathway activator, increases the metabolic rate of a human subject. In some embodiments, administration may increase the basal metabolic rate of the subject. Metabolic rate can be calculated by any method known in the art, such as by examining oxygen input and carbon dioxide output or by indirect calorimetry. In some embodiments, metabolic rate is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, the metabolic rate is increased by at least 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] Effects on glucose uptake in human subjects In some embodiments, administration of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor), alone or in combination with a GLP-1 pathway activator, affects glucose uptake by tissues in a human subject. In some embodiments, glucose uptake by muscle tissue is increased. In some embodiments, glucose uptake by muscle tissue is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In some embodiments, glucose uptake by muscle tissue is increased by at least 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%.

[0254] In other embodiments, glucose uptake by white adipose tissue, liver tissue and / or vascular tissue is decreased. In some embodiments, glucose uptake by white adipose tissue, liver tissue and / or vascular tissue is decreased 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 / or vascular tissue is decreased by at least 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%.

[0255] In some embodiments, treating a subject with a myostatin pathway inhibitor, alone or in combination with a GLP-1 pathway activator, reduces non-fasting glucose levels in the subject's serum compared to a pre-treatment baseline.

[0256] The term "non-fasting glucose level" refers to the glucose content in a subject's blood measured without the need to fast or abstain from eating and drinking for an extended period of time, e.g., 8 hours. Blood glucose can be measured using a hemoglobin A1C test, also known as A1C, HbA1C, glycated hemoglobin or glycosylated hemoglobin test. Traditional homoglucose monitoring can also be used to measure blood glucose. The term "postprandial insulin level" refers to the insulin concentration in a subject's blood immediately after eating (e.g., 2 hours after eating). The term "postprandial glucose level" refers to the glucose concentration in a subject's blood immediately after eating (e.g., 2 hours after eating). Postprandial insulin and glucose levels can be influenced by carbohydrate absorption, insulin and glucagon secretion and their coordinated effects on glucose metabolism in hepatic and peripheral tissues. The magnitude and time of peak plasma insulin or glucose concentration depends on a variety of factors, including the timing, portion size and composition of the meal. In nondiabetic human subjects, plasma glucose concentrations peak approximately 60 minutes after the start of the meal and return to preprandial levels within 2-3 hours. Postprandial insulin levels may be measured using a postprandial insulin test, such as a 2 hour postprandial insulin test.Postprandial glucose levels may be measured using a postprandial blood glucose test, such as a 2 hour postprandial blood glucose test.

[0257] Effect on insulin sensitivity in human subjects In some embodiments, administration of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor), alone or in combination with a GLP-1 pathway activator, increases insulin sensitivity in a human subject. Methods for measuring insulin sensitivity are known in the art, such as glucose tolerance tests and fasting insulin or glucose tests. In a glucose tolerance test, a fasting patient takes a 75 gram oral dose of glucose, and then blood glucose levels are measured over the next two hours. A blood glucose level below 7.8 mmol / L (140 mg / dl) is considered normal, a blood glucose level between 7.8 and 11.0 mmol / L (140 and 197 mg / dl) is considered impaired glucose tolerance (IGT), and a blood glucose level above 11.1 mmol / L (200 mg / dl) is considered diabetes mellitus. For fasting insulin testing, fasting serum insulin levels above 25mIU / L or 174pmol / L are considered insulin resistant. To measure postprandial insulin levels, subjects may follow a diet containing at least 150g of carbohydrates per day for two weeks. After an overnight fast of more than 10 hours, patients undergo an oral glucose tolerance test, with samples taken at baseline and again at 30, 60, 120, and 180 minutes. If the sum of the insulin at the second and third hours is less than 60μU / mL, it is considered within the normal range. If the sum of the insulin at the second and third hours is greater than or equal to 60 but less than 100, borderline hyperinsulinemia or borderline insulin resistance is indicated.Hyperinsulinemia may be indicated when the sum of 2nd and 3rd hour insulin is greater than 100, when there is a delayed insulin peak in the 2nd or 3rd hour, or when fasting insulin is greater than 50 (see, e.g., DiNicolantonio, JJ, et. al. Postprandial insulin assay as the earliest biomarker for diagnosing pre-diabetes, type 2 diabetes and increased cardiovascular risk. Published online November 27. Available at: http: / / openheart.bmj.com / content / openhrt / 4 / 2 / e000656.full.pdf Accessed November 28,2017.).

[0258] Effects on insulin-dependent glycemic control in human subjects In some embodiments, administration of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor), alone or in combination with a GLP-1 pathway activator, increases insulin-dependent glycemic control in a human subject. In some embodiments, insulin-dependent glycemic control is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, insulin-dependent glycemic control is increased by at least 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%.

[0259] Effect on preventing the onset of metabolic disorders in subjects In some embodiments, administration of an effective amount of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor), alone or in combination with a GLP-1 pathway activator as described herein, may prevent the development of a metabolic disorder in a subject, e.g., a human subject. In some embodiments, the development of a metabolic disorder 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 incidence of metabolic disorders is reduced by at least 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%.

[0260] In some embodiments, administration of an effective amount of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) reduces the risk of cardiovascular disease, for example, by reducing cholesterol levels and / or LDL / HDL ratios. Cholesterol includes both low-density lipoprotein (LDL) cholesterol and high-density lipoprotein (HDL) cholesterol. Total cholesterol level refers to a measure of the total amount of cholesterol (e.g., LDL and HDL) in a blood sample taken from a subject. LDL cholesterol contains low-density lipoproteins, which make up most of the cholesterol found in the body. High LDL levels can cause lipid deposits (e.g., plaque) in arteries, which may be associated with or linked to increased risk of cardiovascular conditions such as heart disease and stroke. In contrast, HDL cholesterol contains high-density lipoproteins, which absorb cholesterol and transport it back to the liver. In general, high HDL cholesterol levels are associated with lower heart disease and stroke risks. Cholesterol levels can be measured using blood tests, including blood tests commonly known as a complete cholesterol test, a lipid panel or a lipid profile.

[0261] In some embodiments, suitable subjects are those who have not fully developed metabolic disease, but are considered to be at risk of developing such a condition. In some embodiments, the subject has a disease or condition related to muscle dysfunction. In some embodiments, the muscle dysfunction is related to partial denervation of motor neurons, which causes partial dysfunction in affected muscles. In some embodiments, such a condition is caused by muscular dystrophy or muscle atrophy. In some embodiments, such a condition is caused by SCI. In some embodiments, the subject with SCI is in the acute or subacute phase of SCI (e.g., has not yet reached the chronic phase).

[0262] In some embodiments, when a composition comprising an effective amount of a myostatin signaling inhibitor described herein (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) is administered to a patient population at risk of developing a metabolic disorder associated with muscle dysfunction, the composition i) prevents the onset or exacerbation of the metabolic disorder in a statistically significant proportion of the patient population; or ii) reduces the severity of the metabolic disorder in a statistically significant proportion of the patient population. In some embodiments, the inhibitor of myostatin signaling is administered in conjunction with a GLP-1 pathway activator.

[0263] In some embodiments, metabolic effects may be monitored or measured by insulin resistance, lipid panels / markers (e.g., leptin, adiponectin, ghrelin), inflammatory markers and oxidative stress markers, including but not limited to, IL-6, TNF, CRP, plasma total antioxidant status, lipid oxidation and erythrocyte glutathione peroxidase activity.

[0264] Pharmaceutical Compositions The myostatin pathway inhibitor and GLP-1 pathway activator described herein may be formulated into one or more pharmaceutical compositions suitable for administration in a human or non-human subject. In some embodiments, the myostatin pathway inhibitor and the GLP-1 pathway activator are co-formulated into one composition. For example, the myostatin pathway inhibitor and the GLP-1 pathway activator may be co-formulated for intravenous (iv) or subcutaneous (sc) administration. In other embodiments, the myostatin pathway inhibitor and the GLP-1 pathway activator are formulated for separate administration to a human or non-human subject (e.g., a mammal). For example, the myostatin pathway inhibitor may be formulated for iv administration and the GLP-1 pathway activator may be formulated for sc or oral administration. In some embodiments, the myostatin pathway inhibitor is formulated for sc administration. In some embodiments, the dosing schedule of the myostatin pathway inhibitor and the GLP-1 pathway activator may require administration on different days or at different times. In some embodiments, the myostatin pathway inhibitor and the GLP-1 pathway activator may be administered in parallel, separately or simultaneously.

[0265] Such pharmaceutical compositions may be intended for therapeutic or prophylactic use. One or more of the myostatin pathway inhibitors and / or GLP-1 pathway activators may be mixed with pharma- ceutically acceptable carriers (excipients), including buffers, to form a pharmaceutical composition for administration to a patient who may benefit from reduced myostatin signaling and GLP-1 pathway activation in vivo. By "pharma-ceutically acceptable" it is meant that the carrier must be compatible with the active ingredients of the composition (and preferably have the ability to stabilize the active ingredients) and not be harmful to the subject to be treated. Examples of pharma-ceutically acceptable excipients (carriers), including buffers, will be apparent to those skilled in the art and have been previously described. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); polypeptides of low molecular weight (less than about 10 residues); serum albumin; the hydrophilic polymer such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Pharmaceutically acceptable excipients are further described herein.

[0266] In some cases, the pharmaceutical compositions described herein include emulsion-based or lipid-based formulations, such as liposomes containing myostatin inhibitors and / or GLP-1 pathway activators, which can be prepared by any suitable method, such as those described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be prepared by reverse phase evaporation with lipid compositions including phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Extrusion of liposomes through filters of defined pore size yields liposomes with the desired diameter.

[0267] The myostatin inhibitor and / or GLP-1 pathway activator may also be entrapped in microcapsules, such as hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules, respectively, prepared, for example, by coacervation techniques or interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Exemplary techniques have been previously described, see, for example, Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0268] In other examples, the pharmaceutical compositions described herein can be formulated in sustained release format.Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing myostatin inhibitors and / or GLP-1 pathway activators, and such matrices are in the form of shaped articles, such as films or microcapsules.Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0269] Pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0270] The pharmaceutical compositions described herein comprising a myostatin inhibitor and a GLP-1 pathway activator may be in unit dosage form (either separately or together) such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

[0271] To prepare solid compositions such as tablets, the active ingredient can be mixed with a pharmaceutical carrier, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gums, and other pharmaceutical diluents, such as water, to form a solid preformulated composition containing a homogeneous mixture of the compound of the present disclosure or its pharma- ceutically acceptable non-toxic salts. When these preformulated compositions are referred to as homogeneous, it means that the active ingredient is uniformly dispersed throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid preformulated composition is then subdivided into unit dosage forms of the type described above containing from 0.1 mg to about 500 mg of the active ingredient of the present disclosure. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage that provides the advantage of sustained action. For example, the tablet or pill can include an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be released after a delay. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0272] Suitable surfactants include, in particular, non-ionic agents such as polyoxyethylene sorbitans (e.g., Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Compositions containing a surfactant may conveniently contain 0.05-5% surfactant, and may be 0.1-2.5%. It will be appreciated that other ingredients, such as mannitol or other pharma- ceutically acceptable vehicles, may be added as required.

[0273] Suitable emulsions may be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient may either be dissolved in a premixed emulsion composition, or alternatively, it may be dissolved in an emulsion formed upon mixing with oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and phospholipids (e.g., egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be appreciated that other ingredients, such as glycerol or glucose, may be added to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, e.g., 5-20%.

[0274] The emulsion composition can be prepared by mixing anti-promyostatin antibody with Intralipid™ or its components (soybean oil, egg phospholipids, glycerol and water).

[0275] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable, aqueous or organic solvents, or mixtures thereof, as well as powders. The liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as set out above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0276] Compositions, preferably in sterile pharma- ceutically acceptable solvents, may be nebulized by the use of a gas. Nebulized solutions may be breathed directly from the nebulizer device or the nebulizer device may be attached to a face mask, tent or intermediate positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices that deliver the formulation in an appropriate manner.

[0277] kit The present disclosure also provides kits (including disposable and / or self-administered kits) for use in alleviating diseases / disorders associated with metabolic disorders. Such kits may include one or more containers containing a myostatin pathway inhibitor (e.g., a myostatin inhibitor, such as a myostatin selective inhibitor) and a GLP-1 pathway activator, such as any of those described herein. In some embodiments, the myostatin pathway inhibitor and the GLP-1 pathway activator are provided in the same container. In some embodiments, the container is a syringe. In some embodiments, the myostatin pathway inhibitor and the GLP-1 pathway activator are provided in separate containers.

[0278] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include instructions for administration of a myostatin pathway inhibitor and a GLP-1 pathway activator to treat or delay the onset or alleviate a target disease, such as those described herein. The kit may further include instructions for selecting an individual suitable for treatment based on identifying whether the individual has a target disease. In yet other embodiments, the instructions include instructions for administration of an antibody and a GLP-1 pathway activator (e.g., a peptide hormone mimetic) to an individual at risk for a target disease.

[0279] Instructions for use of myostatin pathway inhibitors (e.g., myostatin inhibitors, e.g., myostatin selective inhibitors) and GLP-1 pathway activators generally include information regarding dosage, dosing schedules, and routes of administration for the intended treatment. Containers may be unit dose, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions provided in kits of the present disclosure are typically written instructions on a label or insert (e.g., paper included with the kit), although machine-readable forms of instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0280] The label or package insert indicates that the composition is used for treating, delaying the onset of, and / or ameliorating a disease or disorder associated with metabolic syndrome, diabetes, or obesity. Instructions can be provided for practicing any of the methods described herein.

[0281] The kits of the present disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar® or plastic bags), and the like. Packaging for use in combination with special devices, such as inhalers, nasal administration devices (e.g., atomizers), or infusion devices, such as mini-pumps, are also contemplated. The kits may have a sterile access port (e.g., the container may be an intravenous fluid bag or vial with a stopper pierceable by a hypodermic needle). The containers may also have a sterile access port (e.g., the container may be an intravenous fluid bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an anti-pro / latent myostatin antibody or antigen-binding fragment thereof, such as those described herein.

[0282] The kit may optionally provide additional components, such as buffers and interpretive information. Typically, the kit includes a container and one or more labels or package inserts on or associated with the container. In some embodiments, the disclosure provides an article of manufacture that includes the contents of the kit described above.

[0283] subject The pharmaceutical compositions and treatment regimens described herein are suitable for administration in human or non-human subjects, e.g., mammalian subjects. Accordingly, treatment regimens including myostatin inhibitors in conjunction with GLP-1 pathway activators (e.g., GLP-1 analogs such as liraglutide) are useful for administration to subjects who are expected to benefit from reduced myostatin signaling and increased insulin production. In some embodiments, suitable subjects include healthy individuals who may nevertheless benefit from increased muscle mass / muscle function and improved metabolism. In some embodiments, suitable subjects have an existing muscle pathology and / or associated metabolic dysfunction. In some embodiments, suitable subjects are at risk of developing one or more such pathologies. In some embodiments, suitable subjects are those undergoing treatment with a therapy that includes another therapeutic agent for treating a muscle / metabolic pathology, but that is associated with adverse effects or toxicity. In some embodiments, the subject is a human patient from birth to less than 18 years of age. In some embodiments, the subject is a human subject (child or adolescent) aged 2 to 19 years of age. In some embodiments, the subject is a human subject aged 12 years or older (eg, 12 to 17 years old).

[0284] In some embodiments, preferred subjects include obese subjects (e.g., mammalian subjects) or subjects who are overweight, e.g., subjects with a body mass index (BMI) of 27 kg / m 2 The obese subject includes subjects with at least one weight-related condition (such as diabetes mellitus type 2, hypertension, cardiovascular disease, or high cholesterol), including overweight subjects. In some embodiments, obese subjects have a BMI between 28 and 40, inclusive. In some embodiments, obese subjects have a BMI between 30 kg / m 2 In some embodiments, the European Medicines Agency Guideline on Clinical Evaluation of Medicinal Products Used in Weight Management provides a weight management target of 27 kg / m 2A subject with a BMI of 30 kg / m2 or more is considered overweight, and a subject with a BMI of 30 kg / m2 or more is considered obese. In some embodiments, the subject is on a low-calorie diet and / or exercise regimen. In some embodiments, the subject has a metabolic disorder. In some embodiments, the subject has excess abdominal fat.

[0285] Tables 4 and 5 below show BMI ranges that may be used for overweight and obesity. Online tools for measuring BMI in adults can be found at https: / / www.cdc.gov.

[0286] [Table 15]

[0287] Younger individuals (e.g., children and adolescents) grow at different rates at different times, so it is not always easy to tell if a child is overweight. The CDC BMI growth curves can be used to compare a child's or adolescent's BMI to other children or adolescents of the same sex and age. Online tools for measuring BMI in children and teenagers can be found at https: / / www.cdc.gov / healthyweight / bmi / calculator.html.

[0288] [Table 16]

[0289] In some embodiments, a subject with obesity is obese and healthy, the subject is overweight but does not have any comorbidities or risk factors for comorbid conditions and does not have any impairment in their everyday feeling or function. In other embodiments, a subject with obesity is obese with risk factors, the subject is overweight and still does not have any comorbidities, but the subject has measurable risk factors for comorbid conditions and / or impairment in their everyday feeling or function. For example, the subject may be at risk for insulin resistance, glucose intolerance, hypertension, cardiovascular disease, dyslipidemia, hyperuricemia, type 2 diabetes, stroke, fatty liver disease, kidney disease, and other health problems. In other embodiments, a subject with obesity is obese and sick, the subject is overweight and has one or more comorbidities and impairment in their everyday feeling or function that can be attributed to obesity.

[0290] In some embodiments, subjects with obesity are assessed using the Edmonton Obesity Staging System (EOSS), a 5-point ranking classification system that takes into account comorbidities and functional status, as shown in Table 6.

[0291] [Table 17]

[0292] In some embodiments, the pharmaceutical agents disclosed herein are suitable for administration in pediatric, adult and / or geriatric populations.

[0293] In some embodiments, such medicaments are suitable for administration in subjects between the ages of 2 and 19 years.

[0294] In some embodiments, such medicaments are suitable for administration in subjects 12 years of age or older (eg, 12 to 17 years of age).

[0295] A population in need of a myostatin inhibitor in combination with a GLP-1 pathway agonist as described herein may range from 0-6 months, 0-12 months, 0-18 months, 0-24 months, 0-36 months, 0-72 months, 6-36 months, 6-36 months, 6-72 months, 12-36 months, 12-72 months. In some embodiments, a pediatric population suitable for administration of a myostatin inhibitor, such as an antibody or antigen-binding fragment, as described herein and expected to benefit from such treatment may range from 0-6 years, 0-12 years, 3-12 years, 0-17 years. In some embodiments, the population is at least 5 years of age, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 years of age. In some embodiments, the pediatric population may be less than 18 years of age. In some embodiments, the pediatric population may be (a) at least 5 years of age, and (b) under 18 years of age.

[0296] An adult population in need of a therapy comprising a myostatin inhibitor and a GLP-1 pathway activator as described herein can be at least 20 years of age, such as at least 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65 years of age. In some embodiments, the adult population can be under 65 years of age. In some embodiments, the adult population can be under 75 years of age.

[0297] Elderly populations in need of therapy comprising a myostatin inhibitor and a GLP-1 pathway activator as described herein may be aged 65 years or older (i.e., ≧65 years), for example, at least 70, 75 or 80 years of age.

[0298] In some embodiments, a human subject who is expected to benefit from a treatment comprising a myostatin inhibitor and a GLP-1 pathway activator may be a human patient who has, is at risk of developing, or is suspected of having a metabolic disease / disorder. A subject who has a metabolic disease or disorder (e.g., obesity, type 2 diabetes mellitus (T2DM), etc.) may be identified by routine examination, such as laboratory tests, organ function tests, CT scans, or ultrasound. A subject who is suspected of having any of such diseases / disorders may exhibit one or more symptoms of a metabolic disease / disorder. A subject who is at risk of a metabolic disease / disorder may be a subject who has one or more risk factors for the metabolic disease / disorder.

[0299] In some embodiments, a human subject who is expected to benefit from a treatment comprising a myostatin inhibitor and a GLP-1 pathway activator may be a human subject with T2DM with a hemoglobin A1c (HbA1c) of 5% to 15% (e.g., greater than or equal to 6.5% and less than or equal to 10%).

[0300] In some embodiments, a human subject who is expected to benefit from a treatment comprising a myostatin inhibitor and a GLP-1 pathway activator may be a human subject with T2DM who has been undergoing anti-diabetic treatment for at least 3 months. In some embodiments, the subject has been undergoing anti-diabetic treatment for about 3 months. In some embodiments, the subject has been undergoing anti-diabetic treatment for at least 6 months.

[0301] In some embodiments, a human subject who is expected to benefit from a treatment comprising a myostatin inhibitor and a GLP-1 pathway activator may be a human subject having a body weight of at least 70 kg. In some embodiments, the subject has a body weight of at least 80 kg. In some embodiments, the subject has a body weight of at least 80 kg and no more than 140 kg. In some embodiments, the subject has a body weight of more than 140 kg.

[0302] A control subject, as described herein, is a subject that provides a suitable reference for evaluating the effectiveness of a particular treatment or intervention in a test subject or subject. A control subject may be a subject of similar age, race, sex, weight, height and / or other characteristics, or any combination thereof, as the test subject.

[0303] In some embodiments, a myostatin assay (e.g., myostatin ELISA) is used to identify subjects in need of myostatin inhibitor and GLP-1 pathway activator treatment. For myostatin assay methods, see Lakshman et al. Molecular and Cell Endocrinology (2009) 302:26-32 (myostatin ELISA) and Bergen et al. Skeletal Muscle (2015) 5:21 (liquid chromatography and tandem mass spectrometry, both of which are incorporated herein by reference).

[0304] In some embodiments, a GLP-1 assay (e.g., measuring circulating GLP-1) is used to identify subjects in need of treatment with myostatin inhibitors and GLP-1 pathway activators. Circulating levels of GLP-1 and other incretin hormones can be determined by enzyme-linked immunosorbent assay (ELISA) and / or other methods known in the art. Circulating levels of GLP-1 can be determined, for example, by using commercially available quantitative methods.

[0305] Total ELISA kit (EMD Millipore, Billerica, MA, USA), microplate absorbance reader (Bio-Rad) set at 450 nm. In some embodiments, a method is provided for improving muscle performance in a subject. The subject may or may not have or be at risk of having a pathology 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 exert a force (e.g., on an external object). In some embodiments, muscle performance may relate to the ability of a muscle to consume energy. For example, in some embodiments, muscle performance may relate to the ability of a muscle to produce and / or consume adenosine triphosphate (ATP) molecules to facilitate muscle contraction. In some embodiments, muscle performance refers to the ability of a muscle to repeatedly contract for a particular duration. In some embodiments, muscle performance refers to the ability of a muscle to exert a force on an object, for example to move the object over a measurable distance. In some embodiments, muscle performance refers to the ability of a muscle to exert a force on an object for a particular duration (e.g., to move that object a measurable distance in a particular duration).

[0306] In some embodiments, a combination of a myostatin inhibitor and a GLP-1 pathway activator described herein is administered to a subject in need of treatment in an amount sufficient to inhibit proteolytic activity of pro / latent myostatin to active myostatin in vivo by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more). In other embodiments, a myostatin inhibitor, such as an antibody or antigen-binding portion thereof, is administered in an amount effective to reduce pro / latent myostatin or latent myostatin levels by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more).

[0307] In some embodiments, a combination of a myostatin inhibitor and a GLP-1 pathway activator described herein is administered to a subject in need of treatment in an amount sufficient to inhibit proteolytic activity of pro / latent myostatin to active myostatin in vivo by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) and / or in an amount sufficient to enhance insulin secretion or inhibit glucagon secretion by at least 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10.0 times.

[0308] In some embodiments, a myostatin inhibitor described herein, such as an anti-pro / latent myostatin antibody or antigen binding portion thereof, is administered to a subject who would benefit from increased muscle mass. In some embodiments, a combination of a myostatin inhibitor and a GLP-1 pathway activator is administered to a subject who would benefit from increased muscle to fat ratio. In some embodiments, a combination described herein is administered to a subject who would benefit from increased muscle function. In some embodiments, the subject may or may not have or be at risk of having a condition associated with reduced muscle mass and / or reduced muscle function. In some embodiments, the subject has or is at risk of having a condition associated with reduced muscle mass and / or reduced muscle function.

[0309] The methods of the present invention further include selecting a patient (subject) or patient population likely to benefit from the combination or adjunctive (add-on) therapy described herein.

[0310] In some embodiments, the subject suffers from or is at risk of developing a muscle condition or disorder. In some embodiments, the subject suffers from or is at risk of developing a metabolic disorder. In some embodiments, the subject suffers from or is at risk of developing a disease or disorder associated with impaired neurological signaling.

[0311] Selection of a patient may be based on the patient's responsiveness to another therapy aimed at treating a metabolic disease such as obesity or type 2 diabetes. Patients who respond poorly to such therapy may show enhanced response to the therapy when used in conjunction with, supplemented with, adjunctive to, or add-on to a myostatin pathway inhibitor. Thus, in some embodiments, a myostatin pathway inhibitor is used in treating a metabolic disease in a patient, the treatment comprising administration of a myostatin pathway inhibitor to a patient who has received another therapy aimed at treating a metabolic disease but has failed to achieve the intended therapeutic goal or expected therapeutic outcome, optionally, the myostatin pathway inhibitor is a myostatin selective inhibitor, and further, optionally, the metabolic disease is obesity / overweight and / or type 2 diabetes. In some embodiments, the "another therapy aimed at treating a metabolic disease" is or comprises a GLP-1 receptor agonist. Examples of other therapies, including those that contain GLP-1 receptor agonists, include, without limitation, albiglutide, taspoglutide, semaglutide, exenatide, BPI-3016, GW002, glutazumab, exendin-4, exenatide, GLP-1(7-36)NH2, everestumab, liraglutide, lixisenatide, tirzepatide, dulaglutide, danugliplon (Pfizer).In some embodiments, other therapies comprising a GLP-1 receptor agonist include, but are not limited to, a GLP-1 receptor agonist / GIP receptor antagonist combination, such as AMG 133 (Amgen); a GLP-1 / GIP dual agonist, such as tirzepatide (LY3298176; Eli Lilly) and CT-388; an amylin / GLP-1 combination, such as a caglilintide / semaglutide combination (Novo Nordisk); a GLP-1 / glucagon combination, such as DD01 (Neuraly); a GLP-1 / glucagon combination, such as ALT-801 (Altimmune), a GLP-1 / GIP, such as CT-388 (Carmot); an IBI362 (LY-330567 (Innovent / Eli and GLP-1 / GIP / Glucagon triple receptor agonists such as LY3437943 (Eli Lilly). In some embodiments, the intended therapeutic goal or expected therapeutic outcome is at least 5% or at least 10% weight loss (percent change from baseline), reduction in BMI, reduction in waist circumference, reduction in fat mass, increase in lean to fat mass ratio and / or improved insulin sensitivity.

[0312] Selection of a patient may be based on the patient's ability to implement or comply with a therapeutic regimen of weight management treatment. In some embodiments, a myostatin pathway inhibitor is used to treat a metabolic disorder in a patient, the treatment including administration of a myostatin pathway inhibitor to a patient who is on a weight management regimen, such as a GLP-1 pathway activator, and who is unable or fails to implement or adhere to a reduced calorie regimen and / or exercise regimen intended as part of the GLP-1 pathway activator therapy.

[0313] The selection of the patient may be based on one or more background therapies to which the patient is receiving. The background therapy may be weight management and / or standard care medication for diabetes. In some embodiments, a myostatin selective inhibitor is used to treat the patient's obesity / overweight, and the treatment includes administering a myostatin selective inhibitor to a patient on a background therapy containing a biguanide, such as metformin or a derivative thereof. Non-limiting examples of metformin include Fortamet, Glucophage, Glucophage XR, Glumetza, Riomet, Obimet, Gluformin, Dianben, Diabex, Diaformin, Metsol, Siofor, Metforgamma, and Glifor, as well as metformin-containing medications including additional active ingredients. Examples include, but are not limited to, thiazolidinediones (glitazones) and rosiglitazone.

[0314] In some embodiments, a myostatin selective inhibitor is used to treat an overweight or obese patient, and the treatment comprises administering the myostatin selective inhibitor to a patient on medication for type 2 diabetes. The antidiabetic medication can be an alpha-glucosidase inhibitor; a dipeptidyl peptidase-4 (DPP-4) inhibitor, such as alogliptin, linagliptin, saxagliptin, or sitagliptin; a sodium-glucose cotransporter-2 (SGLT-2) inhibitor, such as dapagliflozin, canagliflozin, empagliflozin, or ertugliflozin; a sulfonylurea; or a thiazolidinedione.

[0315] Route of administration To practice the methods disclosed herein, an effective amount of the pharmaceutical composition described above can be administered to a subject (e.g., a mammalian subject) in need of treatment by a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation, or topical routes. In some embodiments, administration is subcutaneous. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, may also be useful for administration. Liquid formulations can be directly nebulized, and lyophilized powders can be nebulized after reconstitution. Alternatively, anti-pro / latent myostatin antibodies can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized milled powder.

[0316] Depending on the type of disease or site of disease to be treated, the pharmaceutical composition can be administered to the subject using conventional methods known to those skilled in the art of medicine. The composition can also be administered by other conventional routes, such as orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or from an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject from an injectable depot administration route, such as using a 1-month, 3-month, or 6-month depot injectable or biodegradable material and method.

[0317] In a preferred embodiment, the composition comprising a myostatin pathway inhibitor (such as a myostatin inhibitor) is formulated for subcutaneous administration. In some embodiments, the composition contains both a myostatin pathway inhibitor and a GLP-1 pathway activator in the same formulation. In some embodiments, separate formulations may be used, for example, for adjunctive therapy.

[0318] Injectable compositions may contain a variety of carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injections, water-soluble antibodies can be administered by drip infusion, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients can include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. For intramuscular preparations, for example, a sterile formulation of the antibody in a suitable soluble salt form can be administered dissolved in a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% glucose solution.

[0319] In one embodiment, the myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody or antigen-binding portion thereof, is administered by a site-specific or targeted local delivery technique. Examples of site-specific or targeted local delivery techniques include various implantable depot sources or local delivery catheters, such as infusion catheters, indwelling catheters or needle catheters, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site-specific carriers, direct injection or direct application of the myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody or antigen-binding portion thereof. See, e.g., WO 00 / 53211 and U.S. Pat. No. 5,981,568.

[0320] The specific dosing regimens used in the methods described herein, e.g., dose, timing and repetition, will depend on the particular subject and the subject's medical history as well as the formulation and pharmacokinetic properties of each drug administered, e.g., myostatin inhibitors and GLP-1 pathway activators.

[0321] The efficacy of a treatment for a disease / disorder associated with metabolic syndrome can be determined using any suitable method.

[0322] In some embodiments, with respect to an increase in promyostatin levels in a target muscle, the increase is at least 1 fold, 1.2 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold or more (or any range bracketed by any of these values) compared to a control promyostatin level. In one embodiment, the increase in promyostatin levels in a target muscle is in the range of 1-3 fold, 1.2-10 fold, 2-9 fold, 3-8 fold, 4-7 fold, 2-7 fold, etc. increase compared to a control promyostatin level.

[0323] The term "control level" refers to a generally accepted or predetermined level of a biological marker, e.g., a marker level obtained prior to treatment or disease onset or prior to administration of a drug, e.g., an antibody or antigen-binding portion thereof, that is found in a subject or population of subjects exhibiting one or more particular characteristics, e.g., the presence or absence of a particular disease or condition.

[0324] In some embodiments, with respect to the increase in latent myostatin in the target muscle following 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 (or within any time range bracketed by any of the recited durations) following the administration step. In one embodiment, the increase in latent myostatin in the target muscle following the administration step is detectable for at least 5 days, 7 days, 14 days, 21 days, 28 days, or 30 days (or within any time range bracketed by any of the recited durations) following the administration step. In one embodiment, the increase in latent myostatin levels in the target muscle following the administration step is at least 1-fold, 1.2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more (or any range bracketed by any of these values) compared to the latent myostatin levels in the target muscle prior to the administration step. In one embodiment, the increase in latent myostatin levels in the target muscle after the administration process is in the range of 1-fold to 3-fold, 1.2-fold to 10-fold, 2-fold to 9-fold, 3-fold to 8-fold, 4-fold to 7-fold, 2-fold to 7-fold, etc. compared to the latent myostatin levels in the target muscle before the administration process.

[0325] In some embodiments, with respect to an 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 (or within any time range bracketed by any of the recited durations) after the administration step. In one embodiment, the increase in circulating latent myostatin after the administration step is detectable for at least 5 days, 7 days, 14 days, 21 days, 28 days, or 30 days (or within any time range bracketed by any of the recited durations) after the administration step. In one embodiment, the increase in circulating latent myostatin levels after the administration step is at least 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold or more (or any range bracketed by any of these values) compared to circulating latent myostatin levels before the administration step. In one embodiment, the increase in latent myostatin levels in the target muscle after the administration process is in the range of 1-fold to 3-fold, 1.2-fold to 10-fold, 2-fold to 9-fold, 3-fold to 8-fold, 4-fold to 7-fold, 2-fold to 7-fold, etc. compared to the latent myostatin levels in the target muscle before the administration process.

[0326] In some embodiments, in relation to the reduction of circulating latent myostatin levels, the reduction is at least 1-fold, 1.2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more (or any range bracketed by any of these values) compared to control levels of latent myostatin. In one embodiment, the reduction in circulating latent myostatin levels is in the range of 1-fold to 3-fold, 1.2-fold to 10-fold, 2-fold to 9-fold, 3-fold to 8-fold, 4-fold to 7-fold, 2-fold to 7-fold, etc., reduction compared to control latent myostatin levels. In relation to administration of a myostatin inhibitor and a GLP-1 pathway activator, the myostatin inhibitor can be an adjunct therapy, an add-on therapy, that is used in combination with or supplements the GLP-1 pathway activator.

[0327] In some embodiments, in the context of administering a combination therapy comprising a myostatin inhibitor and a GLP-1 pathway activator to a subject (e.g., a mammalian subject), an effective amount is an amount effective to increase the mass of a target muscle in a subject compared to a control muscle mass. In some embodiments, the mass of a muscle treated with an effective amount of the combination therapy is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, etc., compared to a control muscle not treated with an effective amount of the combination. In some embodiments, such an increase in muscle mass is achieved in a selected muscle group or muscle type in the subject. In some embodiments, an effective amount is an amount effective to maintain muscle mass in a subject (e.g., a mammalian subject) throughout treatment with a myostatin inhibitor and a GLP-1 pathway activator. For example, a subject receiving the combination therapy may lose body fat mass while retaining muscle mass, hi some embodiments, an effective amount is an amount effective to reduce loss of target muscle mass in a subject compared to a control muscle mass during and / or after treatment with the combination therapy.

[0328] The term "control" with respect to a control sample refers 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 may cause or predispose the subject to a certain disease or condition, a subject having a disease or condition of interest, a sample from a subject treated with a pharmaceutical carrier, a sample from a subject prior to treatment, a sham or buffer treated subject or sample, an untreated subject or sample, and the like.

[0329] In some embodiments, in the context of administering a myostatin inhibitor and a GLP-1 pathway activator to a subject, an effective amount is an amount effective to reduce overall body weight. In some embodiments, the body weight of a subject treated with an effective amount of the combination therapy is reduced by 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 a subject receiving a GLP-1 pathway activator alone. In some embodiments, the reduction in body weight of a subject treated with an effective amount of the combination therapy is primarily due to a reduction in body fat mass as determined by qNMR.

[0330] In some embodiments, in the context of administering a combination of a myostatin inhibitor with a GLP-1 pathway activator to a subject, an effective amount is an amount effective to convert the subject's fiber type. In some embodiments, an effective amount of the combination therapy can promote fiber type conversion from type I to type II. In some embodiments, an effective amount of the combination therapy can promote fiber type conversion from type I to type IIB. In some embodiments, an effective amount of the combination therapy can promote type II fibers compared to other fiber types. In some embodiments, an effective amount of the combination therapy can promote type IIB fibers compared to other fiber types. In some embodiments, such fiber phenotype conversion can occur without a significant change in overall muscle mass. In other embodiments, such fiber phenotype conversion can occur concomitantly with an increase in overall muscle mass.

[0331] In some embodiments, in the context of administering a combination of a myostatin inhibitor and a GLP-1 pathway activator to a subject, an effective amount is an amount effective to increase the diameter of a muscle fiber in the subject as compared to a control muscle fiber. In some embodiments, the increase in muscle fiber diameter is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 4-fold, at least 5-fold or more as compared to a control muscle fiber. In some embodiments, the increase in muscle fiber diameter is in the range of 1-fold to 5-fold, 2-fold to 10-fold, 1-fold to 1.5-fold, 1-fold to 2-fold, etc. as compared to a control muscle fiber.

[0332] In some embodiments, in the context of administering a combination of a myostatin inhibitor and a GLP-1 pathway activator (e.g., a GLP-1R agonist) to a subject, an effective amount is an amount effective to increase the muscle-to-fat ratio of the subject as compared to a control muscle mass. In some embodiments, the increase in muscle-to-fat ratio is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 4-fold, at least 5-fold or more as compared to a control subject. In some embodiments, the increase in muscle-to-fat ratio is in the range of 1-fold to 5-fold, 2-fold to 10-fold, 1-fold to 1.5-fold, 1-fold to 2-fold, etc. as compared to a control subject.

[0333] In some embodiments, in the context of administering a myostatin inhibitor and a GLP-1 pathway activator to a subject, an effective amount is an amount effective to reduce intramuscular fat infiltration in the subject as compared to a control muscle mass. In some embodiments, the reduction in intramuscular fat infiltration is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 4-fold, at least 5-fold or more as compared to a control subject. In some embodiments, the reduction in intramuscular fat infiltration is in the range of 1-5-fold, 2-10-fold, 1-1.5-fold, 1-2-fold, etc. as compared to a control subject.

[0334] In some embodiments, a method of preventing muscle mass loss and / or increasing muscle mass in a human subject comprises administering to the subject a myostatin inhibitor in combination with a GLP-1 pathway activator, where the combination therapy inhibits the formation of mature myostatin following proteolysis by toroid proteases. In one embodiment, inhibition of cleavage of promyostatin or latent myostatin following proteolysis by toroid proteases results in a progressive or sustained increase in muscle mass. In one embodiment, the subject exhibits a progressive increase in muscle mass over at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, or 20 weeks (or any range bracketed by any of these values). In some embodiments, a method of preventing muscle mass loss and / or increasing muscle mass in a human subject comprises administering to the subject more than two doses of a myostatin inhibitor, such as a pro / latent myostatin antibody or antigen-binding fragment thereof. In one embodiment, administering the combination therapy includes at least a first dose and a second dose, wherein the first dose and the second dose are administered to the subject at least about 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, or 12 weeks apart.

[0335] As used herein, the term "control muscle mass" refers to a reference standard useful for assessing the effect of a disease state (e.g., treatment with a myostatin inhibitor and a GLP-1 pathway activator) on the mass of a target muscle in a subject. In some embodiments, the target muscle is the gastrocnemius muscle. In some embodiments, the control muscle mass is a predetermined value. In some embodiments, the control muscle mass is experimentally determined. In some embodiments, the control muscle mass is the mass of a target muscle in a subject who has not been administered a myostatin inhibitor, e.g., a pro / latent myostatin antibody or antigen-binding fragment thereof. In some embodiments, the control muscle mass is the mass (e.g., the average mass) of a target muscle in a population of subjects who have not been administered the combination therapy. In some embodiments, the control muscle mass is the amount of target muscle in a subject prior to (e.g., immediately prior to) administration of the combination therapy. In some embodiments, the control muscle mass is the amount of target muscle in a subject administered a conventional antibody (e.g., of the same isotype as the anti-myostatin antibody) obtained from an animal that has not been exposed to the antigen against which the anti-myostatin antibody or antigen-binding fragment thereof is directed, instead of a myostatin inhibitor (e.g., an anti-myostatin antibody). In some embodiments, the control muscle mass is the amount of target muscle in a subject administered a vehicle, e.g., saline, instead of a myostatin inhibitor, e.g., an anti-myostatin antibody or antigen-binding fragment thereof.

[0336] Dosage Empirical considerations, such as half-life, will generally contribute to the determination of dosage. For example, antibodies and antigen-binding portions thereof that are compatible with the human immune system, such as humanized or fully human antibodies, may be used to extend the half-life of the antibody and prevent the antibody from being attacked by the host's immune system. The pharmacokinetic properties of small peptides, such as peptide hormones, such as incretins, may be improved by tethering the peptide to a bulkier molecule (such as BSA) or by modifying the recognition site of a peptidase, such as DPP-IV, for example, by acylation or amino acid substitution. The frequency of administration may be determined and adjusted over the course of therapy and is generally, but not necessarily, based on the treatment and / or suppression and / or improvement and / or delay of a disease / disorder associated with metabolic disorders, such as obesity, diabetes, or metabolic syndrome. Alternatively, a sustained continuous release formulation of the myostatin inhibitor and / or GLP-1 pathway activator may be appropriate. A variety of formulations and devices for achieving sustained release will be apparent to one skilled in the art and are within the scope of this disclosure.

[0337] Dosage regimens for GLP-1 pathway activators, such as GLP-1 mimetics, may depend in part on the mode of administration, as well as on the pharmacokinetics of the particular drug substance. For example, exenatide (based on exendin-4) may be administered subcutaneously to mammals (e.g., humans) at an initial dose of 5 μg twice daily, with maintenance doses of 5-10 μg twice daily, within 1 hour before breakfast and dinner, approximately 6 hours or more apart. Extended release exenatide (exenatide ER) may be administered subcutaneously to mammals (e.g., humans) at a dose of 2 mg once daily, with or without food. Lixisenatide (based on exendin-4) may be administered subcutaneously to mammals (e.g., humans) at an initial dose of 10 μg once daily, with maintenance doses of 20 μg once daily, within 1 hour before the first meal of the day. Liraglutide (modified human GLP-1) may be administered subcutaneously to a mammal (e.g., human) at a starting dose of 0.6 mg and a maintenance dose of 1.2 or 1.8 mg once daily, with or without food. Dulaglutide (modified human GLP-1) may be administered subcutaneously at a starting dose of 0.75 mg and a maintenance dose of 0.75 or 1.5 mg once daily (with or without food). Semaglutide (modified human GLP-1) may be administered subcutaneously at a starting dose of 0.25 mg and a maintenance dose of 0.5 or 1.0 mg once daily (with or without food). Several clinically approved GLP-1R agonists are reviewed in Cornell, J Clin Pharm Ther. 2020;45(Suppl 1):17-27.

[0338] In one embodiment, as part of a treatment regimen that includes a myostatin inhibitor and a GLP-1 pathway activator, an obese or overweight subject is given a GLP-1 mimetic by subcutaneous injection once a day. In another example, the GLP-1 pathway activator (e.g., a GLP-1 mimetic) is given once a week. In some examples, the starting dose of the GLP-1 mimetic is lower than the maintenance dose of the GLP-1 mimetic. In some examples, the dose (either the starting dose or the maintenance dose or both) of the GLP-1 mimetic (or other GLP-1 pathway activator) administered in the combination therapy is lower than the FDA approved dose.

[0339] In one embodiment, the dosage of a myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody or antigen-binding fragment thereof, as described herein, can be empirically determined in an individual given one or more administrations of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof. The individual is given increasing dosages of an antagonist. Indicators of disease / disorder can be followed to determine the effectiveness of the antagonist.

[0340] In general, an initial dosage candidate for administration of any of the antibodies or antigen-binding fragments thereof described herein may be about 2 mg / kg. For purposes of this disclosure, a typical daily dosage may range from about 0.1 μg / kg to 3 μg / kg to 30 μg / kg to 300 μg / kg, to 3 mg / kg to 30 mg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the condition, treatment is continued until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate the disease or disorder associated with pro / latent myostatin or its symptoms. An exemplary titration regimen includes administering an initial dose of about 2 mg / kg of the antibody or antigen-binding fragment thereof, followed by a weekly maintenance dose of about 1 mg / kg, or followed by a biweekly maintenance dose of about 1 mg / kg. However, other dosage regimens may be useful depending on the pharmacokinetic decay pattern the practitioner wishes to achieve. For example, administration 1 to 4 times per week is contemplated. In some embodiments, administrations ranging from about 3 μg / kg to about 2 mg / kg (such as about 3 μg / kg, about 10 μg / kg, about 30 μg / kg, about 100 μg / kg, about 300 μg / kg, about 1 mg / kg, and about 2 mg / kg) may be used. In some embodiments, the frequency of administration is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 or 3 months, every 4 months, every 5 months, every 6 months, every 8 months, every 10 months, every year, or longer. The progress of this therapy is easily monitored by conventional techniques and assays. The titration regimen (including the antibody used) may vary over time.

[0341] In some embodiments, administration of any of the myostatin inhibitors, such as the antibodies or antigen-binding fragments thereof described herein, comprises a single dose. In some embodiments, administration of any of the myostatin inhibitors, such as the antibodies or antigen-binding fragments thereof described herein, comprises multiple doses (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses). Administering can include more than two doses. In some embodiments, administration comprises at least a first dose and a second dose of a therapeutically effective amount of a myostatin inhibitor, such as an antibody or antigen-binding portion thereof. In one embodiment, the first and second doses are administered to the subject at least about 4 weeks, 6 weeks, 8 weeks, or 12 weeks apart.

[0342] For purposes of this disclosure, the appropriate dosage of the myostatin inhibitor and GLP-1 pathway activator will depend on the specific myostatin inhibitor (or composition thereof) and GLP-1 pathway activator used, the type and severity of the disease / disorder, whether the administration of the combination therapy is for prophylactic or therapeutic purposes, previous therapy, the patient's current pharmacotherapy profile, the patient's medical history and response to the combination therapy, the possibility of synergistic effects of the drug combination, and the discretion of the attending physician. In some embodiments, the clinician will administer the combination therapy (either together or each drug individually) until a dosage is reached that achieves the desired result for the combination or each drug individually. The administration of the combination therapy may be continuous or intermittent depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art. The administration of the myostatin inhibitor and GLP-1 pathway activator may be essentially continuous over a preselected period of time, or may be in a series of spaced doses, for example, before, during, or after the onset of a metabolic disease or disorder.

[0343] As used herein, the term "treating" refers to the application or administration of a composition comprising one or more active agents to a subject having a myopathy-related disease / disorder, a symptom of the disease / disorder, or a predisposition to the disease / disorder, with the intent to eradicate, cure, alleviate, palliate, alter, repair, enhance, ameliorate or affect the disorder, symptom of the disease, or predisposition to the disease / disorder.

[0344] Alleviating a metabolic disease / disorder includes delaying the onset or progression of the disease or reducing disease severity. Alleviating a disease does not necessarily require a curative outcome. As used herein, "delaying" the onset of a metabolic disease / disorder means delaying, preventing, delaying, retarding, stabilizing, and / or postponing the progression of the disease. This delay can be for a variety of lengths of time, depending on the history of the disease and / or the individual being treated. A method of "delaying" or alleviating the progression of a disease or delaying the onset of a disease is a method that reduces the probability of one or more symptoms of the disease progressing within a given time frame and / or reduces the severity of the symptoms within a given time frame, compared to not using the method. Such comparisons are usually based on clinical trials with a sufficient number of subjects to obtain statistically significant results. Measurement of improvement in pathology associated with a metabolic disease or disorder may be quantified, for example, by qNMR assessment of circulating metabolic biomarkers (examples of such biomarkers are discussed, for example, in Robberecht et al., Metab Syndr Relat Disord. 2016 Mar;14(2):47-93 and Belhayara et al., Nutrients. 2020 Mar;12(3):727, each of which is incorporated herein by reference in its entirety). "Onset" or "progression" of a disease refers to the initial onset of symptoms and / or subsequent progression of a disease. Onset of a disease can be detected and determined using standard clinical techniques. However, onset also refers to progression that may be undetectable. For purposes of this disclosure, onset or progression refers to the biological course of a symptom. "Onset" includes onset, recurrence, and onset. As used herein, "onset" or "onset" of a disease / disorder associated with metabolic disturbance includes initial onset and / or recurrence.

[0345] Combination Therapy The present invention encompasses pharmaceutical compositions and related methods used as combination therapy to treat subjects who may benefit from myostatin inhibition and GLP-1 pathway activation in vivo. In any of these embodiments, such subjects may receive combination therapy that includes a first composition that includes at least one myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) in combination with a second composition that includes at least one additional therapeutic agent intended to treat the same or overlapping disease or clinical condition. The first and second compositions may both act on the same cellular target or may act on separate cellular targets. In some embodiments, the first and second compositions may treat or alleviate the same or overlapping set of symptoms or aspects of a disease or clinical condition. In some embodiments, the first and second compositions may treat or alleviate a separate set of symptoms or aspects of a disease or clinical condition. Such combination therapy may be administered in conjunction with each other. The phrase "in conjunction with" in reference to combination therapy means that the therapeutic effect of the first therapy overlaps in time and / or space with the therapeutic effect of the second therapy in a subject receiving the combination therapy. Thus, the combination therapy can be formulated as a single formulation for parallel administration, or can be formulated as separate formulations for simultaneous or sequential administration of the therapies. The term "parallel administration" as used herein can include co-formulated therapeutics being administered together, or separately formulated therapeutics being administered at the same time, e.g., simultaneously or in some embodiments, on the same day. The term "sequential administration" as used herein includes a treatment regimen in which the therapeutics are administered at separate times. In some embodiments, a subject receives parallel administration of two or more therapies when the subject is under therapy with one or more first therapeutics at the start of the treatment regimen, and one or more second therapeutics are added to the regimen at a particular time during the treatment.

[0346] In some embodiments, the combination therapy produces a synergistic effect in the treatment of a disease. The term "synergy" refers to an effect (e.g., greater efficacy) that is greater than the additive effect of the sum of each monotherapy. In some embodiments, the combination therapy produces an additive effect in the treatment of a disease.

[0347] In some embodiments, a combination therapy including the methods described herein (e.g., administration of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) and a GLP-1 pathway activator) provides overall efficacy equivalent to that provided by another therapy (e.g., monotherapy of an additional agent), but is associated with fewer undesirable adverse effects or less severe toxicity associated with the second agent compared to monotherapy of the second agent. In some embodiments, such combination therapy allows for reduced dosage of the second agent while maintaining overall efficacy. Such combination therapy may be particularly suitable for patient populations requiring long-term treatment and / or involving pediatric patients, patients aged 2-19 years, or patients aged 12 years or older (e.g., 12 to 17 years).

[0348] Thus, the present invention provides pharmaceutical compositions and methods for use in combination therapy for increasing lean mass to fat mass ratio and treating or preventing diseases associated with impaired neurological signaling, including metabolic disease or metabolic syndrome, diabetes (e.g., T2DM), obesity, and spinal cord injury. In some embodiments, the method or pharmaceutical composition for use comprises a first therapy. In some embodiments, the method or pharmaceutical composition for use further comprises a second therapy. In some embodiments, the first therapy may be useful for increasing lean mass to fat mass ratio. In some embodiments, the second therapy may be useful for treating or preventing metabolic disease or diseases associated with impaired neurological signaling. In some embodiments, the second therapy may reduce or treat at least one symptom associated with the targeted disease. The first and second therapies may exert their biological effects through similar or unrelated mechanisms of action. In some embodiments, either or both of the first and second therapies may exert their biological effects through multiple mechanisms of action.

[0349] In some embodiments, the first therapy is a GLP-1 pathway activator and the second therapy is a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor). In some embodiments, the GLP-1 pathway activator is administered in conjunction with a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor). In some embodiments, the myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) is administered to a subject receiving GLP-1 pathway activator therapy. In some embodiments, the myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) is administered to a subject who has not previously received GLP-1 pathway activator therapy. In some embodiments, the GLP-1 pathway activator and the myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) are administered concurrently. In some embodiments, the GLP-1 pathway activator and the myostatin pathway inhibitor (e.g., a myostatin inhibitor, such as a myostatin selective inhibitor) are administered sequentially. In some embodiments, the GLP-1 pathway activator and the administered myostatin pathway inhibitor (e.g., a myostatin inhibitor, such as a myostatin selective inhibitor) are administered separately.

[0350] It should be understood that the pharmaceutical compositions described herein, for each embodiment described, may have the first and second therapies in the same pharma- ceutically acceptable carrier or in different pharma- ceutically acceptable carriers. It should further be understood that within the embodiments described, the first and second therapies may be administered simultaneously or sequentially.

[0351] The combination of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) and a GLP-1 pathway activator according to the methods disclosed herein may be used in combination with one or more additional therapeutic agents. In some embodiments, the myostatin pathway inhibitor is a myostatin selective inhibitor. In some embodiments, the myostatin pathway inhibitor inhibits pro / latent myostatin. In some embodiments, the myostatin pathway inhibitor does not inhibit GDF11. In some embodiments, the myostatin pathway inhibitor inhibits myostatin and does not inhibit any other members of the TGFβ superfamily. Examples of additional therapeutic agents that may be used with the combination therapy include, but are not limited to, additional diabetes therapeutic agents, diabetic complication therapeutic agents, cardiovascular disease therapeutic agents, antihyperlipidemic agents, blood pressure lowering or antihypertensive agents, antiobesity agents, chemotherapeutic agents, immunotherapeutic agents, immunosuppressants, and the like. Such combination therapy may advantageously utilize lower dosages of administered therapeutic agents, thus avoiding potential toxicities or complications associated with various monotherapies. In some embodiments, the additional therapeutic agent is administered in the same treatment regimen as the combination therapy including a myostatin pathway inhibitor and a GLP-1 pathway activator. In other embodiments, a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) and a GLP-1 pathway activator are administered to a subject already receiving one or more therapeutic agents, such as those described above. In some embodiments, treatment with a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) in conjunction with a GLP-1 pathway activator reduces symptoms of type 2 diabetes. In some embodiments, treatment with a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) in conjunction with a GLP-1 pathway activator induces remission of T2DM. In some embodiments, remission of T2DM refers to a return of blood glucose levels to normal or healthy levels, eg, the blood glucose levels of a non-diabetic individual, eg, an A1C level of less than 6.5%.

[0352] In some embodiments, the combination of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) and a GLP-1 pathway activator according to the methods disclosed herein may be used in combination with one or more diet and / or exercise treatment regimens. In some embodiments, a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) and a GLP-1 pathway activator are administered to a subject on a diet and / or exercise regimen for weight loss or improvement or reduction of symptoms of one or more metabolic diseases or disorders associated with impaired neurological signaling, including metabolic syndrome, diabetes (e.g., T2DM), obesity, and spinal cord injury. In some embodiments, diet regimens include, but are not limited to, dieting through calorie restriction (e.g., reduced caloric intake or reduced absorption of calories), altered nutritional content (e.g., high protein, low fat, low carb, keto, paleo, etc.), or altered food timing (e.g., intermittent fasting, increased frequency of eating, etc.) or a combination of altered timing, portion size, and nutritional content (e.g., eating smaller, more frequent meals that include higher protein, lower fat, and / or other nutritional content restrictions).

[0353] Examples of drugs for treating diabetes mellitus (e.g., T2DM) 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), drugs that enhance insulin sensitivity, pharma- ceutical acceptable salts, hydrates, or solvates thereof (e.g., pioglitazone, troglitazone, rosiglitazone, netoglitazone, balaglitazone, rivoglitazone, tesaglitazar, farglitazar, CLX-0921, R-483, NIP-221, NIP-223, DRF-2189, GW-7282, TAK-559, T-131, RG-12525, LY-510929, LY-519818, BMS-298585, DRF-2725, GW-1536, GI -262570, KRP-297, TZD18 (Merck), DRF-2655, etc.), α-glycosidase inhibitors (e.g., voglibose, acarbose, miglitol, emiglitate, etc.), biguanides (e.g., phenformin, metformin, buformin, etc.) or sulfonylureas (e.g., tolbutamide, glibenclamide, gliclazide, chlorpropamide, tolazamide, acetohexamide, glyclopyramide, glimepiride, etc.), as well as other insulin secretagogues (e.g., repaglinide, senaglinide, nateglinide, mitiglinide, GLP-1 pathway activators, etc.), amylin agonists (e.g., pramlintide, etc.), phosphotyrosine phosphatase inhibitors (e.g., vanadate, etc.), etc.

[0354] Examples of GLP1-R pathway activators include, but are not limited to, incretin mimetics such as albiglutide, taspoglutide, semaglutide, exenatide, BPI-3016, GW002 (a GLP1 mimetic with an albumin moiety that extends half-life; for a discussion of extending the half-life of GLP-1, see, e.g., Eur J Pharmacol. 2021 Jan 5;890:173650), glutazumab, exendin-4, exenatide, GLP-1(7-36)NH2, everestumab, liraglutide, lixisenatide, tirzepatide, XW003, neuiglutide, MEDI0382 or dulaglutide.

[0355] Further examples of GLP1R pathway activators include DPPIV inhibitors, non-limiting examples of which are sitagliptin, vildagliptin, saxagliptin, linagliptin, gemigliptin, anagliptin, teneligliptin, alogliptin, trelagliptin, omarigliptin, evogliptin, gosogliptin, dutogliptin and berberine. Examples of drugs for treating diabetic complications include, but are not limited to, aldose reductase inhibitors (e.g., tolrestat, epalrestat, zenarestat, zopolrestat, minalrestat, fidarestat, SK-860, CT-112, etc.), neurotrophic factors (e.g., NGF, NT-3, BDNF, etc.), PKC inhibitors (e.g., LY-333531, etc.), advanced glycation end products (AGE) inhibitors (e.g., ALT946, pimagedine, pyridoxamine, phenacylthiazolium bromide (ALT766), etc.), active oxygen quenchers (e.g., thioctic acid or a derivative thereof, bioflavonoids including the following, flavones, isoflavones, flavonones, procyanidins, anthocyanidins, pycnogenol, lutein, lycopene, vitamin E, coenzyme Q, etc.), cerebral vasodilators (e.g., tiapride, mexiletine, etc.).

[0356] In one embodiment, remission of diabetes may be induced by administering a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) in combination with a GLP-1 pathway activator, optionally in combination with a calorie restricted diet or other diet. In some embodiments, an exercise routine is included depending on the physical capabilities of the subject. In some embodiments, subjects with T2DM in remission may be able to reduce or eliminate other diabetes medications.

[0357] In some embodiments, a combination of a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) and a GLP-1 pathway activator may be used in combination with one or more hyperlipidemic agents, immunotherapeutic agents, anti-obesity agents, chemotherapeutic agents, immunotherapeutic agents, immunosuppressants, or agents that improve cachexia, in accordance with the methods disclosed herein.

[0358] Examples of antihyperlipidemic agents include statin compounds which are cholesterol synthesis inhibitors (e.g., pravastatin, simvastatin, lovastatin, atorvastatin, fluvastatin, rosuvastatin, etc.), squalene synthetase inhibitors or fibrate compounds having triglyceride lowering effects (e.g., fenofibrate, gemfibrozil, bezafibrate, clofibrate, simfibrate, clinofibrate, etc.), niacin, PCSK9 inhibitors, triglyceride lowering agents, and cholesterol sequestering agents.

[0359] Examples of antihypertensive agents include angiotensin-converting enzyme inhibitors (e.g., captopril, enalapril, delapril, benazepril, cilazapril, enalapril, enalaprilat, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, trandolapril, etc.), angiotensin II antagonists (e.g., losartan, candesartan cilexetil, olmesartan medoxomil, eprosartan, valsartan, telmisartan, irbesartan, tasosartan, pomisartan, lipisartan, forasartan, etc.), calcium channel blockers (e.g., amlodipine), and aspirin.

[0360] Anti-obesity agents include, for example, agents that increase energy expenditure and / or fat metabolism and weight loss through neural and chemical regulation. Anti-obesity agents include biopharmaceutical agents, such as antibodies, fusion proteins, nanobodies, etc., and small molecule agents. Drugs include those that target GLP-1 and its receptor (GLP-1R) including GLP-1R agonists or other GLP-1 pathway activators, biologics such as glutazumab, everestumab, exenatide, liraglutide, lixisenatide, tirzepatide, albiglutide, XW003, neuiglutide, MEDI0382 and dulaglutide, and small molecules such as topiramate, dexfenfluramine, fenfluramine, phentermine, phenmetrazine, sibutramine, amfepramone, dexamphetamine, mazindol, diethylpropion, orlistat, cathine, oleoylestrone, perflubron, benfluorex, setomelanotide, aclimostat, cetilistat, amlintide, rimonabant, phenylpropanolamine, clobenzorex. Medications also include insulin and insulin secretagogues, gastrointestinal lipase inhibitors (e.g., orlistat, etc.), sodium glucose cotransporter 2 (SGLT-2) inhibitors, β3-adrenergic receptor agonists (e.g., CL-316243, SR-58611-A, UL-TG-307, SB-226552, AJ-9677, BMS-196085, etc.), peptide-based appetite suppressants (e.g., leptin, CNTF, etc.), cholecystokinin agonists (e.g., lynchtript, FPL-15849, etc.), oxytocin and oxytocin analogs (e.g., carbetocin, etc.).

[0361] Examples of chemotherapeutic agents include alkylating agents (e.g., cyclophosphamide, ifosfamide, etc.), metabolic antagonists (e.g., methotrexate, 5-fluorouracil, etc.), anticancer antibiotics (e.g., mitomycin, adriamycin, etc.), plant-derived anticancer agents (e.g., vincristine, vindesine, taxol, etc.), cisplatin, carboplatin, etoposide, etc. Among these substances, 5-fluorouracil derivatives such as furtulon and neofurtulon are particularly preferred.

[0362] Examples of immunotherapeutic agents include microbial or bacterial components (e.g., muramyl dipeptide derivatives, picibanil, etc.), polysaccharides with immune enhancing activity (e.g., lentinan, schizofiran, krestin, etc.), cytokines obtained by genetic engineering techniques (e.g., interferon, interleukin (IL), etc.), and colony-stimulating factors (e.g., granulocyte-colony stimulating factor, erythropoietin, etc.). Among these substances, particularly preferred ones are IL-1, IL-2, IL-12, etc.

[0363] Examples of immunosuppressants include cyclosporine (Sandimmune, Gengraf, Neoral), tacrolimus (Prograf, FK506), ASM 981, sirolimus (RAPA, rapamycin, Rapamune) or its derivative SDZ-RAD, glucocorticoids (prednisone, prednisolone, methylprednisolone, dexamethasone, etc.), purine synthesis inhibitors (mycophenolate mofetil, MMF, CellCept (registered trademark), azathioprine, cyclophosphamide), interleukin antagonists (basiliximab, daclizumab, deoxyspergualin), lymphocyte depletion agents such as antithymocyte globulin (thymoglobulin, lymphoglobuline), and anti-CD3 antibodies (OKT3).

[0364] In addition, agents with established cachexia-ameliorating effects in animal models or at clinical stages may also be used in combination with the anti-myostatin antibodies of the present invention, such as cyclooxygenase inhibitors (e.g., indomethacin, etc.), progesterone derivatives (e.g., megestrol acetate, etc.), glucosteroids (e.g., dexamethasone, etc.), metoclopramide-based agents, tetrahydrocannabinol-based agents, lipid metabolism-ameliorating agents (e.g., eicosapentaenoic acid, etc.), growth hormone, IGF-1, antibodies against TNF-α, LIF, IL-6, and oncostatin M. Additional therapeutic agents for use in treating diseases or conditions related to metabolic disorders and / or neurological signaling disorders will be apparent to those of skill in the art and are within the scope of the present disclosure.

[0365] In some embodiments, additional agents suitable for administration as a combination therapy with a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) / GLP-1 pathway activator combination described herein include anti-fibrotic agents, such as TGFβ1 inhibitors. In some embodiments, a second agent suitable for administration as a combination therapy with an antibody described herein is a modulator (e.g., agonists and antagonists) of certain members of the TGFβ growth factor superfamily, such as BMP6, BMP7, GDF11, TGFβ2, TGFβ3, etc. In some embodiments, the additional therapy includes an inhibitor of RGMc, such as an anti-RGMc antibody.

[0366] Any of the above-mentioned agents can be administered in combination with a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) / GLP-1 pathway activator to treat a metabolic disease or disorder, such as metabolic syndrome, obesity, T2DM, or T2DM associated with obesity. In some embodiments, any of the above-mentioned agents can be administered in combination with a myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin selective inhibitor) and / or a GLP-1 pathway activator to treat a metabolic disease or disorder, such as metabolic syndrome, obesity, T2DM, or T2DM associated with obesity, where these agents are administered in conjunction with a diet and / or exercise regimen. Examples of dietary regimens include, but are not limited to, dieting through calorie restriction (e.g., reduced caloric intake or reduced absorption of calories), altered nutritional content (e.g., high protein, low fat, low carbohydrate, keto, paleo, etc.) or altered food timing (e.g., intermittent fasting, increased frequency of eating, etc.) or a combination of altered timing, portion size and nutritional content (e.g., eating smaller, more frequent meals that include higher protein, lower fat and / or other nutritional content restrictions).

[0367] Use of a combination of myostatin pathway inhibitors and GLP-1 pathway activators for the treatment of metabolic diseases / disorders The pharmaceutical compositions described herein are suitable for administration to human patients for the treatment or prevention of diseases and conditions in which it is desirable to r...

Claims

1. A pharmaceutical composition comprising a myostatin selective inhibitor for use in the treatment of a metabolic disorder in a subject, wherein the treatment comprises administering the myostatin selective inhibitor to the subject in combination with a GLP-1 analog, and the myostatin selective inhibitor and the GLP-1 analog are administered in an amount sufficient to treat the metabolic disorder; optionally, the metabolic disorder is type 2 diabetes mellitus (T2D), overweight, obesity, obesity associated with T2D or metabolic syndrome, the myostatin selective inhibitor is a myostatin selective antibody or an antigen-binding fragment thereof that binds to myostatin and exhibits no detectable binding or potency against GDF11 or activin A, the GLP-1 analog binds to and can activate the GLP-1 receptor and comprises the amino acid sequence EGTFTSD (SEQ ID NO: 116) and / or the amino acid sequence HXXGXFTXD (SEQ ID NO: 117; wherein X is any amino acid residue) pharmaceutical composition.

2. A pharmaceutical composition comprising a GLP-1 analog for use in the treatment of a metabolic disorder in a subject, wherein the treatment comprises administering the GLP-1 analog to the subject in combination with a myostatin selective inhibitor, and the myostatin selective inhibitor and the GLP-1 analog are administered in an amount sufficient to treat the metabolic disorder; optionally, the metabolic disorder is type 2 diabetes mellitus (T2D), overweight, obesity, obesity associated with T2D or metabolic syndrome, the myostatin selective inhibitor is a myostatin selective antibody or an antigen-binding fragment thereof that binds to myostatin and exhibits no detectable binding or potency against GDF11 or activin A, the GLP-1 analog binds to and can activate the GLP-1 receptor and comprises the amino acid sequence EGTFTSD (SEQ ID NO: 116) and / or the amino acid sequence HXXGXFTXD (SEQ ID NO: 117; wherein X is any amino acid residue) pharmaceutical composition. A pharmaceutical composition comprising a myostatin selective inhibitor and a GLP-1 analog for use in the treatment of a metabolic disorder of a subject, wherein the treatment comprises administering the myostatin selective inhibitor to the subject in combination with the GLP-1 analog, and the myostatin selective inhibitor and the GLP-1 analog are administered in an amount sufficient to treat the metabolic disorder; optionally, the metabolic disorder is type 2 true diabetes (T2D), overweight, obesity, obesity associated with T2D or metabolic syndrome, the myostatin selective inhibitor is a myostatin selective antibody or an antigen-binding fragment thereof that binds to myostatin and exhibits no detectable binding or potency against GDF11 or activin A, the GLP-1 analog binds to and can activate the GLP-1 receptor and comprises the amino acid sequence EGTFTSD (SEQ ID NO: 116) and / or the amino acid sequence HXXGXFTXD (SEQ ID NO: 117; wherein X is any amino acid residue) pharmaceutical composition. A pharmaceutical composition for use according to any one of claims 1 to 3, a. the GLP-1 analog is an Fc conjugate or multifunctional molecule comprising the GLP-1 analog; and / or b. the GLP-1 analog is semaglutide, exenatide ER, liraglutide, lixisenatide, tirzepatide, XW003, niruglutide, MEDI0382, dulaglutide, albiglutide, AMG 133, CT-388, a combination of cagrilintide / semaglutide, DD01, ALT-801, or IBI362, pharmaceutical composition. A pharmaceutical composition for use according to any one of claims 1 to 3, a. the myostatin selective inhibitor is trevoglutumab, GYM329, MST1032, or an antibody or antigen-binding fragment that competes or cross-competes with trevoglutumab, GYM329, MST1032 or apiteglumab; and / or b. The myostatin selective inhibitor comprises at least one amino acid mutation that increases the binding affinity for FcRn. Pharmaceutical composition.

6. A pharmaceutical composition for use according to any one of claims 1 to 3, wherein the myostatin selective inhibitor is an antibody or an antigen-binding fragment thereof that binds to an epitope comprising one or more amino acid residues of KALDEN (SEQ ID NO: 118) and / or FVQILRLIKPMKDGTRYTGIRSLK (SEQ ID NO: 57); the antibody is not aptigromab, Pharmaceutical composition.

7. A pharmaceutical composition for use according to any one of claims 1 to 3, wherein the myostatin selective inhibitor is an antibody or an antigen-binding fragment thereof that selectively binds to pro- and / or latent myostatin; optionally, the antibody or an antigen-binding fragment thereof does not bind to mature myostatin, Pharmaceutical composition.

8. A pharmaceutical composition for use according to any one of claims 1 to 3, a. The subject is an adult subject having a BMI of 25 or more or a pediatric or adolescent aged 2 to 19 years (e.g., 12 years or older, e.g., 12 to 17 years) having a BMI at or above the 85th percentile value on the CDC growth curve; b. The subject has received at least one therapy for obesity, overweight, or weight-related morbidities but has not been able to achieve the intended clinical outcome, which is a weight loss of at least 5% or 10% compared to the baseline weight before the start of the at least one therapy, and optionally, the subject has been unable to implement or continue a diet regimen and / or an exercise regimen as part of weight management; c. The subject has one or more of central adiposity, cardiovascular disease, kidney disease, fatty liver disease, sleep apnea, hypertension, high blood triglyceride levels, high blood cholesterol levels, low high-density lipoprotein (HDL) levels, and a hemoglobin A1c (HbA1c) level of 6% or more (e.g., 6.5% - 10%), and optionally, the subject has a body mass index (BMI) greater than 25, and further optionally, the subject has a BMI of 28 - 40 including the endpoints; and / or d. The subject is on a calorie-restricted diet and / or an exercise regimen. Pharmaceutical composition. **Claim 9** A pharmaceutical composition for use according to any one of claims 1 - 3, a. The myostatin selective inhibitor and the GLP-1 analog are administered in parallel, simultaneously, or sequentially; optionally, the myostatin selective inhibitor and the GLP-1 analog are formulated as a single formulation, as part of a single molecular construct, or as separate formulations, and / or b. The myostatin selective inhibitor is administered subcutaneously or intravenously. Pharmaceutical composition. **Claim 10** A pharmaceutical composition for use according to any one of claims 1 - 3, a. The treatment reduces triglyceride, total cholesterol, LDL cholesterol, and / or non-fasting glucose levels compared to baseline levels measured before the start of the treatment, and optionally, the treatment reduces triglyceride, total cholesterol, LDL cholesterol, and / or non-fasting glucose levels compared to treatment with the GLP-1 analog alone, and / or b. The treatment reduces body fat mass, maintains fat-free mass, or improves insulin sensitivity and / or insulin secretion compared to baseline levels measured before the start of the treatment, and optionally, the treatment improves insulin sensitivity and / or insulin secretion compared to treatment with the GLP-1 analog alone. Pharmaceutical composition. **Claim 11** A composition comprising a myostatin selective inhibitor and a GLP-1 analog, wherein the myostatin selective inhibitor is a myostatin selective antibody or an antigen-binding fragment thereof that binds to myostatin and exhibits no detectable binding or potency against GDF11 or activin A, wherein the GLP-1 analog binds to and can activate the GLP-1 receptor and comprises the amino acid sequence EGTFTSD (SEQ ID NO: 116) and / or the amino acid sequence HXXGXFTXD (SEQ ID NO: 117; wherein X is any amino acid residue), optionally, the GLP-1 analog is semaglutide, exenatide ER, liraglutide, lixisenatide, tildatide, XW003, neiraglutide, MEDI0382, dulaglutide or albiglutide, Composition.

12. The composition according to claim 11, a. the myostatin selective inhibitor is an antibody or an antigen-binding fragment thereof that binds to an epitope comprising one or more amino acid residues of KALDEN (SEQ ID NO: 118) and / or FVQILRLIKPMKDGTRYTGIRSLK (SEQ ID NO: 57); the antibody is not apiteglumab, or b. the myostatin selective inhibitor is an antibody or an antigen-binding fragment thereof that competes or cross-competes with apiteglumab for antigen binding, the antibody is not apiteglumab; optionally, the antibody or antigen-binding fragment comprises an HCDR3 paratope comprising up to two amino acid substitutions compared to SEQ ID NO: 10; further optionally, the antibody or antigen-binding fragment is numbered according to SEQ ID NO: 52 and binds to an epitope comprising one or more of the amino acid residues F147, Q149, L151, Y186, S168, K170, K205 and / or L207, Composition.

13. A pharmaceutical composition comprising a myostatin selective inhibitor for use as an adjuvant therapy for weight management in the treatment of obesity or overweight in a subject, The therapy includes administration of a myostatin selective inhibitor in an amount effective to treat obesity or overweight, and the subject is treated with a GLP-1 analog; The myostatin selective inhibitor is a myostatin selective antibody or an antigen-binding fragment thereof that binds to myostatin and exhibits no detectable binding or potency against GDF11 or activin A, The GLP-1 analog can bind to and activate the GLP-1 receptor and includes the amino acid sequence EGTFTSD (SEQ ID NO: 116) and / or the amino acid sequence HXXGXFTXD (SEQ ID NO: 117; wherein X is any amino acid residue), Optionally, the subject has at least one weight-related condition; further optionally, the subject has been treated with the GLP-1 analog for at least 3 months, Pharmaceutical composition.

14. A pharmaceutical composition comprising a GLP-1 analog for use as an adjuvant therapy for weight management in the treatment of obesity or overweight in a subject, The treatment includes administration of an effective amount of the GLP-1 analog to treat obesity or overweight, and the subject is treated with a myostatin selective inhibitor; The myostatin selective inhibitor is a myostatin selective antibody or an antigen-binding fragment thereof that binds to myostatin and exhibits no detectable binding or potency against GDF11 or activin A, The GLP-1 analog can bind to and activate the GLP-1 receptor and includes the amino acid sequence EGTFTSD (SEQ ID NO: 116) and / or the amino acid sequence HXXGXFTXD (SEQ ID NO: 117; wherein X is any amino acid residue) Optionally, the subject has at least one weight-related condition; further optionally, the subject has been treated with the myostatin selective inhibitor for at least 3 months, Pharmaceutical composition.

15. The composition according to any one of claims 1 to 3, 11, 13 and 14, The myostatin selective inhibitor is not apiteglomarub. Composition.

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