Therapies for muscular dystrophies

Abstract

Disclosed herein are improved methods for treating muscular dystrophy, such as DMD, BMD, or FSHD. Various combination therapies and monotherapies are provided.

Description

[0001] Attorney Docket No. 15094.0067-00304

[0002] THERAPIES FOR MUSCULAR DYSTROPHIES

[0003] RELATED APPLICATIONS

[0004] [1] This Application claims the benefit of and priority to U.S. Provisional Applications 63 / 740,155 filed December 30, 2024, and entitled “COMBINATION THERAPIES FOR TREATING MUSCULAR DYSTROPHIES”; 63 / 750,476 filed January 28, 2025, and entitled “THERAPIES FOR MUSCULAR DYSTROPHIES”; 63 / 769,674 filed March 10, 2025, and entitled “THERAPIES FOR MUSCULAR DYSTROPHIES”; 63 / 784,918 filed April 7, 2025, and entitled “THERAPIES FOR MUSCULAR DYSTROPHIES”; 63 / 796,297 filed April 28, 2025, and entitled “THERAPIES FOR MUSCULAR DYSTROPHIES”; 63 / 822,073 filed June 11 , 2025, and entitled “THERAPIES FOR MUSCULAR DYSTROPHIES”; and 63 / 938,129 filed December 10, 2025, and entitled “THERAPIES FOR MUSCULAR DYSTROPHIES,” the contents of which are expressly incorporated herein by reference in their entirety.

[0005] SEQUENCE LISTING

[0006] [2] The instant application contains a Sequence Listing which has been submitted electronically in ST.26 XML format and is hereby incorporated by reference in its entirety. Said ST.26 XML copy, created on December 11 , 2025, is named 15094.0067-00304.xml and is 351 ,574 bytes in size.

[0007] FIELD OF THE INVENTION

[0008] [3] This invention relates generally to improved methods for treating muscular dystrophies.

[0009] BACKGROUND

[0010] [4] Muscular dystrophies are a class of muscle disorders in which mutations in the dystrophin gene lead to reduced levels of functional dystrophin, resulting in progressive muscle degeneration and weakness. Among muscular dystrophies, Duchenne muscular dystrophy (DMD) is a common and severe form of muscular dystrophy that predominantly affects boys.

[0011] [5] DMD is a devastating disease affecting approximately 1 in every 3,500 male births worldwide. Multiple mutations in the dystrophin gene have been implicated as underlying causes of DMD. However, there remains no cure for patients with DMD to date. The natural history of DMD causes progressive loss of muscle strength, loss of ambulation skills (mean age of 10.0 years), and associated impaired cardiac (reduced left ventricular ejection fraction) and respiratory / lung function (decreased forced vital capacity). Life expectancy in this population was reported in early adulthood (premature death in their twenties usually due to cardiorespiratory failure).

[0012] [6] The affected muscle tissue in DMD is characterized by muscular atrophy, fibrosis, and fat accumulation. Loss of dystrophin leads to weakened, damaged muscle that is replaced by non-contractile fibrotic tissue. The disease phenotype in DMD is severe, whereas BMD is associated with mild to moderate phenotype with significant variability.Attorney Docket No. 15094.0067-00304

[0013] [7] Currently available treatment for DMD include dystrophin restorers (also referred to as dystrophin upregulators or correctors), including 4 exon skipping ASOs (Exondys 51® [eteplirsen, 2016], Vyondys 53® [golodirsen, 2019], Viltepso® [viltolarsen, 2020], Amondys® 45 [casimersen, 2021]) and gene therapy (ELEVIDYS®, delandistrogene moxeparvovec (2023)). Exon skippers and gene therapy have shown only modest increases in dystrophin; as such, some high responders may have significant functional benefit. Generally, however, the degree of increase in Dystrophin levels shown by these therapies is limited.

[0014] [8] Myostatin has been established as a negative regulator of muscle mass, inhibition of which has been shown to cause muscle enhancement in multiple species including human. However, previous myostatin pathway inhibitors, including domagrozumab and Taldefgrobep alfa (T-alfa), failed to show functional benefit in placebo-controlled phase II trials in DMD boys.

[0015] [9] Previous anti-myostatin trials failed due to safety concerns or lack of efficacy. Insights from these trials suggest that these studies were not conducted in combination with dystrophin corrector therapies or newer anti-inflammatory or muscle reparative agents. Furthermore, these studies enrolled older pediatric patients in declining trajectory without dystrophin-targeted therapies, as outlined below.

[0016]

[0010] Ramatercept (ACE-031 ; Acceleron) is a ligand trap, ACVR2B-Fc for myostatin, GDF11 , activins and BMPs. Ramatercept was tested in a phase 2 clinical trial in DMD. Twenty-four subjects with DMD aged 4+ years (mean 9.8 years ± 3.2 years) and able to ambulate 10 meters in <12 seconds, and on background glucocorticoid therapy for at least 12 months, were treated with ramatercept or placebo via subcutaneous administration every 2 weeks or every 4 weeks for 24 weeks. This trial was discontinued for safety reasons (terminated due to epistaxis, telangiectasias). However, ramatercept showed trends for pharmacodynamic effects on lean mass, fat mass, bone mineral density and 6MWT. In this study, the subjects’ older age with advanced disease progression may account for lack of efficacy. Data were suggestive of positive effect, but limited sample size and AEs led to discontinuation.

[0017]

[0011] Domagrozumab (PF-06252616; Pfizer) is a non-selective monoclonal antibody for myostatin and GDF11. Domagrozumab was tested in a phase 2 clinical trial in 120 ambulatory DMD subjects aged 6-15 years old (8.7 years mean ± 2.0 years) on background glucocorticoid therapy for at least 6 months. Subjects were treated with domagrozumab or placebo via intravenous administration every 4 weeks for two 49-week periods. This study was also discontinued for efficacy reasons (primary endpoint of 4-stair climb showed no improvement over placebo). Secondary endpoints showed positive trends in thigh volume and North Star Ambulatory Assessment (NSAA), but no change in 10-meter walk / run test and 6-minute walk test distance. In this study, the subjects’ older age with advanced disease progression and no dystrophin corrector may account for lack of efficacy.

[0018]

[0012] Taldefgrobep alfa (RG6206; Roche) is a non-selective adnectin for MSTN and GDF11. Taldefgrobep alfa was tested in a phase 2 / 3 trial (SPITFIRE) in 166 ambulatory DMD subjects aged 6-11 (mean 8.4 ± 1 .7 years) on background glucocorticoid therapy. Subjects were treated with taldefgrobep alfa or placebo once a week for 48 weeks. While subjects did show increases in lean body mass and contractile volume, thisAttorney Docket No. 15094.0067-00304

[0019] trial was terminated at interim futility analysis. In this study, the subjects’ older age with advanced disease progression and no dystrophin corrector may account for lack of efficacy.

[0020]

[0013] Dystrophin correcting therapies and muscle reparative therapy (givinostat) have modest to moderate functional benefit showing mitigation of functional decline, but not stabilization of motor function.

[0021]

[0014] Additional pipeline therapeutics show greater promise but remain to be clinically proven. There remains a significant unmet need for improved treatment for dystrophic patients.

[0022]

[0015] FSHD is one of the most common forms of inherited muscle diseases in adults, with an estimated mean prevalence of 5 (range: 0.79 to 12) affected per 100000 individuals (Deenen 2025 Prevalence and incidence rates of 17 neuromuscular disorders: An updated review of the literature. J Neuromuscul Dis.

[0023] 2025;12(6):713-722.). A genetic disorder caused by the abnormal expression of the DUX4 transcription factor gene in muscle tissue, FSHD is characterized by progressive facial muscle weakness that spreads to the shoulders and lower limbs. While the disorder typically manifests in adolescence or early adulthood as a skeletal muscle disease, severe and rapidly progressing early-onset forms may affect infants and young children. FSHD progresses slowly in most patients, with fluctuating periods of high disease activity and stabilization. Impaired mobility, difficulty using hands and arms, fatigue, and pain may significantly reduce physical functioning, quality of life, and the ability to live independently.

[0024]

[0016] About 95% of patients with FSHD have Type 1 disease (FSHD1), which is usually inherited in an autosomal dominant manner (about 10% to 30% of individuals with FSHD 1 have de novo mutations). About 5% of patients with FSHD have Type 2 disease (FSHD2), which is inherited in a digenic manner (Preston 1999 [Updated 2025 Jul 10], In: Adam MP, Feldman J, Mirzaa GM, et al., editors. GeneReviews® [Internet], Seattle (WA): University of Washington, Seattle; 1993-2025). Although the genetics and molecular mechanisms underlying FSHD1 and FSHD2 are different, both result in the abnormal expression of the DUX4 gene, which leads to muscle cell death and atrophy (Wagner 2019 Facioscapulohumeral Muscular Dystrophies. Continuum (Minneap Minn). 2019;25(6):1662-1681).

[0025]

[0017] Despite recent progress in developing treatments that inhibit DUX4 expression using small molecules or by targeting DUX4 mRNA, there is no Food and Drug Administration-approved pharmacological treatment to prevent or stop the muscle wasting and weakness that characterize FSHD. Available therapies include pharmacological interventions that are used to treat pain, depression, and other symptoms of FSHD. Management typically also involves supportive care including physical therapy to maintain muscle function and quality of life. However, in severe cases, assistive devices (e.g., ankle-foot orthoses, wheelchairs), surgical interventions (e.g., scapular fixation), or non-invasive ventilation for respiratory complications may be necessary to address functional limitations (Preston 1999 [Updated 2025 Jul 10], In: Adam MP, Feldman J, Mirzaa GM, et al., editors. GeneReviews® [Internet], Seattle (WA): University of Washington, Seattle; 1993-2025; Tawil 2015 Guideline Development, Dissemination, and Implementation Subcommittee of the American Academy of Neurology; Practice Issues Review Panel of the American Association of Neuromuscular & Electrodiagnostic Medicine. Evidence-based guideline summary: Evaluation, diagnosis, and management of facioscapulohumeral muscular dystrophy: Report of theAttorney Docket No. 15094.0067-00304

[0026] Guideline Development, Dissemination, and Implementation Subcommittee of the American Academy of Neurology and the Practice Issues Review Panel of the American Association of Neuromuscular & Electrodiagnostic Medicine. Neurology. 2015;85(4):357-64.).

[0027]

[0018] More recently, myostatin is being evaluated as a therapeutic target for treating FSHD patients with moderate to severe disease phenotypes. For example, MANOEUVRE (NCT05548556) is a Phase 2 study of the myostatin inhibitor RO7204239 (GYM329), and according to the sponsor, its purpose is to test whether RO7204239 can “help muscles grow”.

[0028]

[0019] A substantial unmet medical need for FSHD patients remains to stop muscle atrophy and improve muscle function. A muscle-targeted therapy is therefore needed to address pathological changes that underlie FSHD. An effective muscle-targeted therapy that can improve muscle strength and motor function can provide additional functional gains and reduce disease burden for these patients.

[0029] SUMMARY

[0030]

[0020] Each motor unit is comprised of a motor neuron and the skeletal muscle fibers innervated by the neuron's axon terminals. In certain neuromuscular disorders, such as SMA, the underlining defect is in the motor neuron, where mutations in the motor neuron protein SMN cause denervation of the target muscle. However, the muscle itself is inherently relatively normal. It is in this context that Applicant previously demonstrated that a myostatin-selective inhibitor used in conjunction with an upregulator of SMN protein expression, can enhance skeletal muscle and its function. This has been more recently validated in a clinical setting (SAPPHIRE, NCT05156320).

[0031]

[0021] Thus, the combination approach was aimed at targeting both components of the motor unit, namely, the motor neuron and the target muscle, by upregulating SMN expression in the motor neuron to address the genetic defect, while concurrently enhancing muscle by inhibiting myostatin to address atrophy in the target muscle.

[0032]

[0022] Here, we instead sought, inter alia, to interrogate whether a selective inhibitor of myostatin could enhance muscle function when used in conjunction with another muscle-targeting agent intended to increase Dystrophin expression in a muscular dystrophy model (of DMD) and whether a selective inhibitor of myostatin could improve muscle function a muscular dystrophy model (of FSHD) as a monotherapy.

[0033]

[0023] For the use of a selective-myostatin inhibitor and a Dystrophin upregulator in conjunction for treating a muscular dystrophy, the rationale for this approach is two-fold: First, dystrophic muscle that is stabilized with a dystrophin corrector may be amenable to treatment with a myostatin-selective inhibitor such as apitegromab as a means of increasing muscle strength; and secondly, myostatin-selective inhibitor treatment of muscle with partial dystrophin expression may further increase dystrophin levels (Myostatin is a negative regulator of protein synthesis). These insights led to the therapeutic hypothesis that Apitegromab treatment in DMD patients with meaningful dystrophin correction, or in a narrowly defined BMD population, may have meaningful positive effects on muscle mass and function.

[0034]

[0024] Data disclosed herein show that inhibition of the myostatin signaling pathway (e.g., myostatin-selective inhibitors) can lead to synergistic improvement in muscle function in DMD mice treated with aAttorney Docket No. 15094.0067-00304

[0035] dystrophin upregulator. Surprisingly, the enhancement of muscle function was found to be mediated at least in part by an increase in specific force. To the best knowledge of Applicant, this is the first such demonstration, in which myostatin inhibition is tied directly to increased specific force, ratherthan enhanced muscle strength merely due to larger muscle. In addition to the observed effects on enhanced muscle function, myostatin inhibition was surprisingly shown to augment dystrophin protein expression in DMD mice treated with dystrophin upregulator, providing added advantage of the combination approach. This is particularly noteworthy because the existing dystrophin upregulators have been shown to produce only limited dystrophin expression that is modest at best. Thus, the data presented herein suggest that myostatin inhibitors (myostatin-selective inhibitors in particular) can exert dual actions of both improving muscle quality and increasing dystrophin expression, resulting in enhanced motor function.

[0036]

[0025] Inhibiting myostatin is hypothesized to enhance muscle function in DMD in the presence of a dystrophin restorer or muscle repair agent by driving growth of restored, functional muscle. Apitegromab has shown improved motor function in SMA; it is therefore hypothesized to enhance function of retained muscle with restored dystrophin in DMD (effect could be limited by continued muscle deterioration due to suboptimal correction) and to drive muscle growth. In addition, apitegromab is hypothesized to increase dystrophin expression.

[0037]

[0026] The present disclosure also includes selection and identification of patients who are likely to benefit from myostatin inhibitor therapy. In this regard, the inventors of the present application recognized that selecting for higher functioning patients on dystrophin corrector (or upregulator) therapies may therefore have a greater potential for effectively treating DMD. Previous myostatin inhibitors failed in earlier trials in DMD where the subjects tended to be older and further along in their disease progression. In the context of the present invention, using a selective myostatin inhibitor to treat DMD, age and disease stage (and / or levels of dystrophin expression) can be helpful patient selection criteria for more successful treatment of DMD. When considering age of subjects in DMD, natural history shows functional declines by ~7-8 years old. Previous anti-myostatin DMD studies enrolled patients with rapidly declining motor function. Therefore, a 4-7 year old DMD population would be likely to have least minimal functional decline, and selection of DMD subjects aged 4+ years old with enrichment for stabilized patients with sufficient dystrophin correction may be a viable approach. In addition, selection of DMD patients for treatment by assessing baseline muscle function can also improve potential for success in treatment. North Star Ambulatory Assessment (NSAA) and other prognostic factors are more predictive of motor function trajectory across mutational phenotypes. Therefore, baseline NSAA (e.g. > 17) may be used to select a more homogeneous population across larger age range with high motor function and similar disease trajectory. Baseline time to rise (TTR) of 4 - 8 seconds may also be used to select such a population for treatment.

[0038]

[0027] Furthermore, the standard of care (SOC) for DMD often includes corticosteroids, which have been suggested to cause interference with the myostatin pathway. It is contemplated herein that a myostatin inhibitor used for carrying out the combination therapy disclosed herein should be selected at least on the basis of the ability to prevent glucocorticoid-induced muscle atrophy.

[0039]

[0028] The invention further includes the use of an anti-fibrotic agent, preferably a TGFpl inhibitor, in addition to or in lieu of corticosteroids to address inflammation and fibrosis of affected muscle. In preferredAttorney Docket No. 15094.0067-00304

[0040] embodiments, the TGFpl inhibitor is a TGFpl -selective inhibitor, and more preferably a select inhibitor of LTBP-associated TGFpl .

[0041]

[0029] Accordingly, the present disclosure provides therapeutic use of myostatin inhibitors forthe treatment of DMD. In one aspect, myostatin inhibitors may be used in the treatment of DMD in a subject who is diagnosed with DMD and has retained motor function as measured by NSAA of 20 or higher (e.g., 20, 21 , 22, 23, 24 or 25). In some embodiments, the subject has received a background therapy comprising a dystrophin upregulator therapy. Additionally or alternatively, the subject is 8 years of age or younger.

[0042]

[0030] The inventors of the present disclosure recognized that the timing of myostatin inhibitor-based intervention may be a key factor in optimizing the effect of myostatin inhibition in DMD patients. More specifically, the natural history of the disease progression in DMD patients shows that younger patients continue to develop muscle until it reaches its plateau at the age of about 6-7, as determined by NSAA. Thereafter, the motor function rapidly declines. By age 10, on average, patients have an NSAA score of below 20 (e.g., 17-19). Because myostatin is a negative regulator of muscle growth, the inventors reasoned that inhibition of this pathway may be most effective during the growing phase leading to plateau, and the following phase prior to the onset of rapid decline. Based on these insights, in some embodiments according to the present disclosure, patients under the age of 8-9 (e.g., 7 and under) are selected for myostatin inhibitor therapy. In preferred embodiments, the patient is between the age of 4-7. In some embodiments, the patient is on a dystrophin upregulator therapy. Preferably, the myostatin inhibitor is a myostatin-selective inhibitor, most preferably apitegromab.

[0043]

[0031] In preferred embodiments, the myostatin inhibitor is a myostatin-selective inhibitor (such as apitegromab, GYM329, or trevogrumab). In some embodiments, the dystrophin upregulator therapy is a splice modifier or an exon-skipping agent. In some embodiments, the dystrophin upregulator therapy is a gene therapy. In some embodiments, the dystrophin upregulator therapy is a protein therapy comprising a truncated form of dystrophin.

[0044]

[0032] The present disclosure includes combination therapy for use in the treatment of a muscular dystrophy, such as DMD in a subject. According to the invention, the combination therapy comprises at least two of the following pharmacological agents: i) a myostatin inhibitor, preferably a myostatin-selective inhibitor; ii) a Dystrophin upregulator; and, iii) an anti-fibrotic agent. Patients who receive the combination therapy may be further treated with background therapy (e.g., SOC), such as anti-inflammatory (e.g., corticosteroids). Combination therapies according to the present disclosure are not limited to particular modalities of therapeutic agents.

[0045]

[0033] In some embodiments, the combination therapy for treating a muscular dystrophy such as DMD is comprised of a myostatin inhibitor and a Dystrophin upregulator. Optionally, the combination therapy further comprises an anti-inflammatory, wherein further optionally the anti-inflammatory is a corticosteroid.

[0046]

[0034] In some embodiments, the combination therapy for treating a muscular dystrophy such as DMD is comprised of a myostatin inhibitor and an anti-fibrotic agent. Optionally, the combination therapy further comprises an anti-inflammatory, wherein further optionally the anti-inflammatory is a corticosteroid.Attorney Docket No. 15094.0067-00304

[0047]

[0035] In some embodiments, the combination therapy for treating a muscular dystrophy such as DMD is comprised of a Dystrophin upregulatorand an anti-fibrotic agent. Optionally, the combination therapy further comprises an anti-inflammatory, wherein further optionally the anti-inflammatory is a corticosteroid.

[0048]

[0036] In some embodiments, the combination therapy for treating a muscular dystrophy such as DMD is comprised of a myostatin inhibitor, a Dystrophin upregulator, and an anti-fibrotic agent. Optionally, the combination therapy further comprises an anti-inflammatory, wherein further optionally the antiinflammatory is a corticosteroid.

[0049]

[0037] In a further aspect, the present disclosure provides myostatin inhibitor, either as monotherapy or combination therapy, for use in treating neuromuscular disorders in which the affected muscle tissue comprises a mosaic or mixed pattern of genetic abnormality. The muscle tissue may benefit from the ability of a myostatin inhibitor to enhance muscle function in the unaffected muscle fibers to compensate for the muscle atrophy of the nearby affected fibers. In some embodiments, such neuromuscular disorders include FSHD. FSHD myocytes comprise sporadic DUX4 expression (e.g., mixed pattern of DUX4-expressing and DUX4-non-expressing groups). FSHD is therefore the result of a toxic gain-of-function from de-repression of the DUX4 gene, a gene not normally expressed in skeletal muscle. Based on this recognition, in some embodiments, the invention includes the use of a myostatin inhibitor (preferably myostatin-selective inhibitor) to treat FHSD as monotherapy. As demonstrated herein, this approach achieved surprising effects on improving the quality of muscle, as evidenced by enhanced specific force, despite not directly addressing the underlying genetic defects in the FSHD model. These findings also suggest that assessments of changes in muscle or motor function may be preferred measures of efficacy, as opposed to muscle growth / size.

[0050]

[0038] The stochastic expression of DUX4 in skeletal muscle affected by FSHD leads to islands of necrotic fibers surrounded by an otherwise healthy population of muscle fibers. These fibers are able to respond to treatment with a myostatin inhibitor monotherapy (e.g., apitegromab) even in the absence of corrector therapy, as would normal wild-type muscle, and potentially compensate for the reduced contractile capacity of DUX4-positive fibers, as shown herein.

[0051]

[0039] In a nonclinical FLExD murine model of FSHD herein, muSRK-015, the murine analog of apitegromab, demonstrated a significant increase in skeletal muscle mass and concomitant increases in muscle force and endurance, demonstrating the potential for a myostatin inhibitor as monotherapy in FSHD.

[0052]

[0040] In a clinical setting, it is contemplated herein that FSHD patients with less severe disease presentation (e.g., early in disease progression and / or milder disease symptoms) may particularly benefit from myostatin inhibitor monotherapy. On the other hand, combination therapy comprising a myostatin inhibitor and a gene corrector (such as agents aimed to suppress DUX4 expression) may be considered for more severe disease presentation (e.g., patients manifesting moderate to severe FSHD disease phenotypes).

[0053]

[0041] Accordingly, in some embodiments, the present disclosure provides a myostatin inhibitor for use in the treatment of facioscapulohumeral muscular dystrophy (FSHD) in a patient in need thereof, wherein the treatment comprises administration of a myostatin inhibitor to the patient in an amount effective to treat FSHD, and wherein the patient has a 10-meter walk / run test (10MWRT) time of less than or equal to 5Attorney Docket No. 15094.0067-00304

[0054] seconds and / or a Ricci Clinical Severity Scale score of 0.5 to 4 (e.g., 1.5 to 3.0, 1.5 to 2.5) at baseline. In some aspects, the present disclosure provides a myostatin inhibitor for use in the treatment of FSHD in a patient in need thereof, wherein the treatment comprises administration of a myostatin inhibitor to the patient in an amount effective to treat FSHD, and wherein the patient has at least two of the following at baseline: a) a Ricci Clinical Severity Scale score of 0.5 to 4 (e.g., 1.5 to 3.0; 1.5 to 2.5); b) a 10-meter walk / run test (10MWRT) time of less than or equal to 5 seconds; and c) a timed-up-and-go (TUG) time of 20 seconds or less (e.g., 16 seconds or less, 14 seconds or less, 12 seconds or less).

[0055]

[0042] In some embodiments, the patient is 16-65 years of age (e.g., 16-60 years of age or 18-60 years of age) at screening or at initiation of the administration of the myostatin inhibitor. In some embodiments, the patient has a baseline Ricci Clinical Severity Scale score of less than 2.5, e.g., about 1.5 to 2.4, and / or has about 4 to about 10 D4Z4 repeat units in at least one allele of the DUX4 gene. In some embodiments, the patient has mild-to-moderate or later-onset FSHD. In some embodiments, the patient is diagnosed with, exhibits, or progressed into a moderate to severe phenotype of FSHD, and / or the patient has early-onset (childhood-onset or infantile) FSHD. In some embodiments, the patient has been genetically diagnosed with FSHD1 or FSHD2.

[0056]

[0043] In some embodiments, the myostatin inhibitor is a myostatin-selective inhibitor, wherein optionally the myostatin-selective inhibitor is selected from: i) an antibody that selectively binds to myostatin but does not bind to Activin A or GDF11 ; ii) an antibody that selectively binds to mature myostatin; iii) an antibody that selectively binds to pro / latent myostatin, thereby inhibiting myostatin activation; or iv) a nucleic acidbased agent that blocks expression of endogenous myostatin. In some embodiments, the myostatin-selective inhibitor is selected from apitegromab, GYM329, trevogrumab, SRK-439, or a variant thereof. In some embodiments, the myostatin-selective inhibitor is apitegromab. In some embodiments, the apitegromab is administered to the patient at a dose of 2-20 mg / kg (e.g., 10 mg / kg). In some embodiments, the apitegromab is administered to the patient once every four weeks or once a month. In some embodiments, the myostatin inhibitor is administered to the patient intravenously.

[0057]

[0044] In some embodiments, the myostatin inhibitor is administered to the patient as a monotherapy. In some embodiments, the patient is further administered a DUX4 inhibitor or wherein the patient is on a DUX4 inhibitor therapy, optionally wherein the DUX4 inhibitor is an siRNA that blocks DUX4 expression (e.g., delpacibart braxlosiran or a TfR1-binding Fab conjugated to an siRNA against DUX4 (e.g., DYNE-302)).

[0058]

[0045] In some embodiments, the treatment increases total lean muscle volume (LMV) in the patient as compared to baseline, optionally wherein the total LMV is measured by a whole-body Magnetic Resonance Imaging (MRI) scan. In some embodiments, the treatment improves motor function in the patient as compared to baseline, optionally wherein motor function is measured by a quantitative myometry test, a 10-meter walk / run test (10WMRT), a Timed Up and Go test, a 5x sit-to-stand test (5x STS), and / or an exercise questionnaire. In some embodiments, the treatment increases exercise capacity and / or endurance in the patient as compared to baseline, optionally wherein the increase in exercise capacity and / or endurance is measured by a graded treadmill test, e.g. as measured by time and / or distance to exhaustion. In some embodiments, the treatment increases muscle function in the patient as compared to baseline, wherein optionally the increase in muscle function comprises an increase in a max force level of a muscleAttorney Docket No. 15094.0067-00304

[0059] (e.g., tibialis anterior (TA) muscle) in the patient. In some embodiments, the treatment increases muscle mass in the patient as compared to baseline, optionally wherein the treatment increases the muscle mass of a gastrocnemius muscle in the patient. In some embodiments, the amount effective to treat FSHD is an amount sufficient to achieve any one or more of the following: i) increase muscle mass in the patient as compared to baseline, ii) increase muscle function in the patient as compared to baseline, iii) increase exercise capacity and / or endurance in the patient as compared to baseline, iv) increase total lean muscle volume (LMV) in the patient as compared to baseline, and v) delay disease progression.

[0060]

[0046] In some embodiments, the patient has mild-to-moderate FSHD. In some embodiments, the patient has later-onset FSHD.

[0061]

[0047] In some aspects, the present disclosure provides a myostatin inhibitor for use in the treatment of Becker muscular dystrophy (BMD) in a patient in need thereof, wherein the treatment comprises administration of a myostatin-selective inhibitor in an amount effective to treat BMD, wherein the myostatin-selective inhibitor is administered to the patient as a monotherapy. In some aspects, the present disclosure provides a myostatin inhibitor for use in the treatment of Duchenne muscular dystrophy (DMD) in a patient in need thereof, wherein the treatment comprises administration of a myostatin-selective inhibitor in an amount effective to treat DMD, wherein the patient is treated with a dystrophin upregulator, and wherein the patient has a dystrophin expression level at screening or initiation of administration of the myostatin-selective inhibitor sufficient to retain motor function, optionally wherein the retention of motor function is as measured by a North Star Ambulatory Assessment (NSAA) score of 17 or greater prior to the administration of the myostatin-selective inhibitor and / or a TTR (“time to rise”) of 4-8 seconds prior to the administration of the myostatin-selective inhibitor, and wherein optionally the patient is 7 years of age or younger (e.g., between 4-7 years old) at the time of screening for or initiation of a myostatin inhibitor therapy. In some embodiments, the dystrophin upregulator comprises a pharmaceutical agent aimed to increase dystrophin expression, wherein optionally the dystrophin upregulator comprises an exon-skipping agent or a gene therapy.

[0062]

[0048] In some aspects, the present disclosure provides a myostatin inhibitor for increasing exercise capacity and / or endurance in a subject suffering from a muscular dystrophy, comprising administration of a myostatin-selective inhibitor to the subject in an amount effective to increase the exercise capacity and / or endurance of the subject.

[0063]

[0049] In some embodiments, the myostatin inhibitor is a myostatin-selective inhibitor, wherein further optionally the myostatin-selective inhibitor is selected from apitegromab, GYM329, trevogrumab, SRK-439, or a variant thereof.

[0064]

[0050] In some embodiments, the treatment increases muscle mass in the patient as compared to baseline, optionally wherein the treatment increases the muscle mass of a gastrocnemius or tibialis anterior (TA) muscle in the patient. In some embodiments, the treatment increases muscle function in the patient as compared to baseline, optionally wherein the increase in muscle function comprises an increase in a max force level of a muscle (e.g., tibialis anterior (TA) muscle) in the patient. In some embodiments, the amount effective to treat DMD is an amount sufficient to delay disease progression and / or an amount effective to enhance dystrophin expression as compared to dystrophin upregulator as monotherapy. In someAttorney Docket No. 15094.0067-00304

[0065] embodiments, the patient undergoes moderate strength training and / or aerobic exercise as part of the treatment regimen.

[0066] BRIEF DESCRIPTION OF THE FIGURES

[0067]

[0051] The patent or application file contains at least one drawing executed in color. Copies of this patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0068]

[0052] FIG.1 provides a set of eight immunofluorescent images collected from tissue sections of D2.mdx and wild type control mice, either with or without dystrophin upregulator (PMO) treatment. The left six images are of gastroc, and the right two are of TA. Increased Dystrophin immunoreactivity confirms that M23D Vivo Morpholino (PMO) partially restores Dystrophin expression in D2.mdx model.

[0069]

[0053] FIGs.2A-2C provide data showing effects of myostatin-selective inhibitor in D2.mdx mice, either alone or in combination with Dystrophin upregulator. FIG.2A provides a graph showing some improvement in muscle quality with PMO treatment in gastroc. FIG.2B and FIG.2C provide graphs showing that combination of myostatin-selective inhibitor and PMO results in synergistic or additive increase in the size of gastroc and tibialis anterior (TA), respectively. ****P<0.0001 ; *P<0.05. Statistical analysis was done using one-way ANOVA.

[0070]

[0054] FIGs.3A-3F provide data showing effects of myostatin-selective inhibitor in D2.mdx mice, either alone or in combination with Dystrophin upregulator, on muscle strength. Force generation in tibialis anterior was enhanced in mice treated with combination therapy (FIG.3A; ****P<0.0001 based on two-way ANOVA analysis), but the same degree of force generation enhancement was not seen in gastroc (FIG.3B). FIG.

[0071] 3C provides data showing effects of myostatin-selective inhibitor and PMO treatments on muscle force normalized to muscle weight (“specific force” in mN / g) for tibialis anterior (TA) compared to wild-type and (vehicle) control. ****P<0.0001 based on two-way ANOVA analysis. FIG. 3D provides data showing effects of myostatin-selective inhibitor and PMO treatments on mean circulating total latent myostatin concentrations in serum of D2.mdx mice treated with muSRK-015P with and without PMO. Data are shown as mean (± SD) ng / mL. SD, standard deviation. FIG. 3E provides data on effects of myostatin-selective inhibitor and PMO treatments on cumulative frequency of fiber sizes (myofiber cross-sectional area sizes) of gastroc muscle from D2.mdx mice treated with muSRK-015P or PMO alone and in combination, compared to control treatment. FIG. 3F provides data showing effects of myostatin-selective inhibitor and PMO treatments on relative (percent of wild-type levels) dystrophin protein expression determined by western blot of samples from D2.mdx mice treated with muSRK-015P or PMO alone and in combination, compared to control treatment. Data shown are means (± SD) relative to wild-type dystrophin levels. SD, standard deviation. Dotted line at 100 corresponds to mean wild-type level. **P< 0.01 based on one-way ANOVA analysis

[0072]

[0055] FIGs. 4A-4B provide data showing effects of myostatin-selective inhibitor treatment on body weight in a murine model of FSHD. FIG. 4A shows changes in whole body weight over the course of the study duration (28 days). FIG. 4B shows terminal body weight in three study groups.Attorney Docket No. 15094.0067-00304

[0073]

[0056] FIGs. 5A-5B provide data showing effects of myostatin-selective inhibitor treatment on muscle weight (TA and Gastroc) in a murine model of FSHD. FIG. 5A shows Gastroc muscle weight at study end in three study groups. **P<0.01 based on t-test. FIG. 5B shows TA muscle weight at study end in three study groups.

[0074]

[0057] FIGs. 6A-6D provide data showing effects of myostatin-selective inhibitor treatment on TA force generation in a murine model of FSHD. Animals treated with muSRK-015P significantly increased TA force as determined by: absolute / measured max force (FIG. 6A; **P<0.01 based on two-way ANOVA analysis); max force normalized to body weight (“Max / BW,” FIG. 6B; *P<0.05 based on two-way ANOVA analysis); max force normalized to muscle weight, known as “specific force” (“Max / MW,” FIG. 6C); and max force normalized to muscle length (“Max / Length,” FIG. 6D; *P<0.05 based on two-way ANOVA analysis).

[0075]

[0058] FIGs.7A-7D provide data showing effects of myostatin-selective inhibitor treatment on Gastroc force generation in a murine model of FSHD. Animals treated with muSRK-015P slightly increased Gastroc force as determined by: absolute / measured max force (FIG. 7A); max force normalized to body weight (“Max / BW,” FIG. 7B); max force normalized to muscle weight (“Max / MW,” FIG. 7C); and max force normalized to muscle length (“Max / Length,” FIG. 7D).

[0076]

[0059] FIG.8 provides data from FLExDUX4.CRE mice treated with muSRK-015P. Serum exposures of the antibody are consistent on Days 7 and 28 (FIG. 8, Left panel). Circulating total latent myostatin levels are increased in muSRK-015P-treated mice, indicating target engagement (FIG. 8, Right panel). Data are shown as mean (± SD) ng / mL. SD, standard deviation.

[0077]

[0060] FIG. 9 provides data from a second study in 8-month old female mice in a FLExDUX4.CRE murine model of FSHD, showing effects of myostatin-selective inhibitor treatment on muscle weight (Gastroc and TA). FIG.9, Left panel shows Gastroc muscle weight at study end in three study groups. FIG. 9, Right panel shows TA muscle weight at study end in three study groups.

[0078]

[0061] FIG. 10 provides data from a second study in a murine model of FSHD showing effects of myostatin-selective inhibitor treatment on Gastroc force generation. Gastroc force was similar in mSRK-015P- and control-treated mice as determined by: measured max force (FIG. 10, Left panel); and specific force (max force normalized to muscle weight, “Max / muscle weight,” FIG. 10, Right panel).

[0079]

[0062] FIG. 11 provides data from a second study in a murine model of FSHD showing effects of myostatin-selective inhibitor treatment on TA force generation. Animals treated with muSRK-015P slightly increased TA force as determined by: measured max force (FIG.11, Left panel; **P<0.001 based on two-way ANOVA analysis); and max force normalized to muscle weight, known as “specific force” (“Max / MW,” FIG.11 , Right panel).

[0080]

[0063] FIG. 12 provides data from a second study in an aged murine model of FSHD, showing effects of myostatin-selective inhibitor treatment on fatigue and exercise performance as measured by treadmill distance (FIG. 12, Left panel) and treadmill time to exhaustion (FIG. 12, Right panel). *P<0.05 based on t-test.

[0081]

[0064] FIG. 13 provides data from a second study (8-month-old FLExDUX4.CRE and wild-type CRE mice) in a murine model of FSHD showing serum exposures of the antibody muSRK-015P. Serum exposures ofAttorney Docket No. 15094.0067-00304

[0082] the antibody are consistent on Days 7 and at end of study (“Term”) (FIG. 13, Left panel). Circulating total latent myostatin levels are increased in muSRK-015P-treated mice, indicating target engagement (FIG. 13, Right panel). Data are shown as means (± SD): ug / mL for muSRK-015P levels; ng / mL for total latent myostatin; SD, standard deviation. These results are consistent with those from younger mice in the previous study.

[0083]

[0065] FIGs. 14A-14D provide data from two studies in 8-10-week-old and 8-month old FLExDUX4.CRE mice (“FLExD”), showing increases in both percentage of myocytes with central nuclei (FIG. 14A) and percentage of total area staining with Sirius red for fibrosis (FIG. 14B) relative to wild-type mice (WT). FIG.

[0084] 14C and FIG. 14D provide data on myofiber cross-sectional area (CSA) of muscles from studies in FLExDUX4.CRE mice aged 8 to 10 weeks, or 8 months old, treated with muSRK-015P or isotype control antibody at 20 mg / kg, dosed weekly for 4 weeks. WT animals with CRE driver (“CRE WT”) were also analyzed as a control group. FIG. 14C shows myofiber CSA in the gastrocnemius at the end of the study in 8 to 10 week old FLExDUX4.CRE mice, n = 10 / group. FIG. 14D shows myofiber CSA in the TA at the end of the study, n = 7 to 8 / group.

[0085]

[0066] FIGs. 15A-15B provide data from studies in 8-10-week-old (FIG. 15A) and 8-month-old (FIG. 15B) FLExDUX4.CRE FSHD model mice, showing levels of macrophages (F4 / 80 positive cells) and satellite cells (Pax7 positive cells) in FLExDUX4.CRE FSHD treated with muSRK-015P or isotype control compared to wild-type (WT). Treatment with muSRK-015P resulted in reduced macrophages (FIG. 15A, *P<0.05 based on unpaired t-test) and increased satellite cell number (FIG. 15A, *P<0.05 based on unpaired t-test) in the 8-10-week-old mice. Levels in 8-month-old mice (FIG. 15B) did not have a statistically significant difference between muSRK-015P and isotype control treated mice.

[0086]

[0067] FIG. 16 provides a study schema for clinical study SRK-015-009, which includes a Screening Period (~4 weeks) and a Treatment Period (~52 weeks). Participants are randomized 1 :1 (stratified by sex) in a double-blinded manner to receive study drug (apitegromab monotherapy or placebo) via IV infusion at Visit 1 (Day 1) and approximately every 4 weeks thereafter. The last dose is administered at Visit 13 (Day 337). Upon completion of the Treatment Period, participants are eligible to enroll in an open-label study (OLE) study or remain in the current study for the Safety Follow-Up Period. Abbreviations: IV, intravenous; Q4W, once every 4 weeks; R, randomization

[0087]

[0068] FIG. 17 provides data from a study in 8-10-week old male and female mice in a FLExDUX4.CRE murine model of FSHD, showing effects of myostatin-selective inhibitor treatment on muscle weight (Gastroc and TA). FIG. 17, Left panel shows Gastroc and TA muscle weights for female mice at study end in three study groups. FIG. 17, Right panel shows Gastroc and TA muscle weights for male mice at study end in three study groups. **** P value < 0.0001 , ** P value = 0.0095, and * P value = 0.0168 based on Welch’s T-test.

[0088]

[0069] FIG. 18 provides data from a study in 8-10-week old male and female mice in a FLExDUX4.CRE murine model of FSHD showing effects of myostatin-selective inhibitor treatment on Gastroc force generation. FIG. 18, Left panel shows Gastroc max force and specific force in female mice, while FIG. 18, Right panel shows Gastroc max force and specific force in male mice. Gastroc force was similar in muSRK-Attorney Docket No. 15094.0067-00304

[0089] 015P- and control-treated mice as determined by: measured max force (FIG. 18, Top plots); and specific force (max force normalized to muscle weight, “Max / muscle weight,” FIG. 18, Bottom plots).

[0090]

[0070] FIG. 19 provides data from a study in male and female mice in a FLExDUX4.CRE murine model of FSHD showing effects of myostatin-selective inhibitor treatment on TA force generation. FIG. 19, Left panel shows TA max force and specific force in female mice, while FIG. 19, Right panel shows TA max force and specific force in male mice. Animals treated with muSRK-015P slightly increased TA force as determined by: measured max force (FIG. 19, Top plots); and max force normalized to muscle weight, known as “specific force” (FIG. 19, Bottom plots). P values based on 2-way ANOVA.

[0091]

[0071] FIG. 20 provides data from a study in male and female mice in a FLExDUX4.CRE murine model of FSHD, showing effects of myostatin-selective inhibitor treatment on max force (force at 150 Hz stimulation) for TA and Gastroc muscles from both female (FIG. 20, Left panel) and male (FIG. 20, Right panel) mice in three study groups. ** P value < 0.0089 based on Welch’s T-test.

[0092]

[0072] FIG. 21 provides data from a study in male and female mice in a FLEx.DUX4.CRE murine model of FSHD, showing effects of myostatin-selective inhibitor treatment on fatigue and exercise performance as measured by treadmill distance and treadmill time to exhaustion. FIG. 21, Left panel shows treadmill distance (Running Distance, in meters) and treadmill time to exhaustion (Running Time, in minutes) for female mice in the three study groups, and FIG. 21, Right panel shows the same for male mice. *P<0.05 based on Welch’s t-test.

[0093]

[0073] FIG. 22 provides data from a study in male and female 8-10-week-old FLExDUX4.CRE mice, showing a significant increase in grip strength in female mice treated with muSRK-015P (FIG. 22, Left panel) and a trend toward increase in grip strength in male mice treated with muSRK-015P (FIG. 22, Right panel) relative to wild-type CRE control mice (CRE WT). * P value = 0.0171 based on Welch’s T test.

[0094]

[0074] FIG. 23 provides data from male and female mice in a study (8-10-week-old FLExDUX4.CRE and wild-type mice) in a murine model of FSHD showing serum exposures of the antibody muSRK-015P. Serum exposures of the antibody are consistent on Days 7 and at end of study (“Term”) (FIG. 23, Left panel).

[0095] Circulating total latent myostatin levels are increased in muSRK-015P-treated mice, indicating target engagement (FIG. 23, Right panel). Data are shown as means (± SD): ug / mL for muSRK-015P levels; ng / mL for total latent myostatin; SD, standard deviation. These results are consistent with those from mice in the previous studies.

[0096]

[0075] FIG. 24 provides data from a study in male and female 8-10-week-old (FIG. 24) FLExDUX4.CRE FSHD model mice, showing average creatine / creatinine (Cr / Crn) ratios in FLExDUX4 FSHD mice treated with muSRK-015P or isotype control compared to wild-type CRE control mice (CRE WT). Treatment with muSRK-015P resulted in decreased creatine / creatinine (Cr / Crn) ratios compared to IgG control-treated FLExDUX4 FSHD mice. ** p value = 0.0016 and * p-value = 0.03 based on t-test.

[0097]

[0076] FIG. 25 provides pooled data from a study in 8-10-week-old male and female mice in a FLExDUX4.CRE murine model of FSHD, showing effects of myostatin-selective inhibitor treatment on terminal body weight (in grams). Shown are pooled body weights for male and female mice combined at study end in three study groupsAttorney Docket No. 15094.0067-00304

[0098]

[0077] FIG. 26 provides pooled data from a study in 8-10-week-old male and female mice in a FLExDUX4.CRE murine model of FSHD, showing effects of myostatin-selective inhibitor treatment on muscle weight (Gastroc and TA). FIG.26, Left panel shows pooled TA muscle weights for male and female mice combined at study end in three study groups. FIG. 26, Right panel shows Gastroc muscle weights for male and female mice combined at study end in three study groups. TA **** P value < 0.0001, and Gastroc * P value = 0.0150 based on Welch’s T-test.

[0099]

[0078] FIG. 27 provides pooled data from a study in male and female mice in a FLExDUX4.CRE murine model of FSHD, showing effects of myostatin-selective inhibitor treatment on grip strength and on fatigue and exercise performance as measured by treadmill distance and treadmill time to exhaustion. FIG. 27, Left panel provides pooled data from male and female mice combined, showing a significant increase in grip strength in mice treated with muSRK-015P relative to isotype control FLExDUX4.CRE mice. Grip strength * P value = 0.0157 based on Welch’s T test. FIG. 27, Center panel shows treadmill distance (Running Distance, in meters) and FIG.27, Right panel shows treadmill time to exhaustion (Running Time, in minutes) for pooled male and female mice combined in the three study groups, and the same for male mice. Running distance ***P 0.0005, Running time **P 0.0069 based on Welch’s t-test.

[0100]

[0079] FIG. 28 provides pooled data from a study in a male and female mice in a FLExDUX4.CRE murine model of FSHD showing effects of myostatin-selective inhibitor treatment on TA force generation. FIG.28, Left panel shows pooled TA max force male and female mice combined, while FIG.28, Right panel shows pooled TA specific force in male and female mice combined. Animals treated with muSRK-015P slightly increased TA force as determined by: measured max force; and max force normalized to muscle weight, known as “specific force”. P values based on 2-way ANOVA.

[0101]

[0080] FIG. 29 provides pooled data from a study in male and female mice in a FLExDUX4.CRE murine model of FSHD, showing pooled effects of myostatin-selective inhibitor treatment on max force (force at 150 Hz stimulation) for TA (FIG. 29, Left panel) and Gastroc muscles (FIG. 29, Right panel) from male and female mice combined in three study groups. ** P value 0.0013 based on Welch’s T-test.

[0102] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0103] Definitions

[0104]

[0081] In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure in its entirety and as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.

[0105]

[0082] Adjunct therapy. The terms “adjunct therapy” and “add-on” therapy are used interchangeably herein and are intended to refer to a therapeutic regimen in which a second agent (used as an adjunct therapy) is administered to a subject who is on, has received, or to be treated with, a first agent (e.g., backgroundAttorney Docket No. 15094.0067-00304

[0106] therapy). The terms “in conjunction with” and “complementary to” are used interchangeably herein and intended to refer to therapies used together, whether concurrent or partially overlapping in time.

[0107]

[0083] Administer / administratioir. The terms “administer”, “administering” or “administration” include any method or act of delivery of a pharmacological agent (e.g., medicament) to an intended subject, e.g., patient. The pharmacological agent can be biologic, such as an antibody or an antigen-binding fragment thereof (e.g., a pharmaceutical composition comprising such an antibody or antigen-binding fragment, peptide agents, such as hormones and modified or synthetic analogs thereof, or low molecular weight agents (i.e., structurally-defined small molecules or chemical entities), into a subject’s system or to a particular region in or on a subject (systemic and local administration, respectively).

[0108]

[0084] Antibody. As used herein the term “antibody” encompasses full-length immunoglobulins, antigenbinding portion(s) / fragment(s) thereof, and any variants thereof that retain the ability to bind a target antigen. Antibodies include human antibodies and humanized antibodies.

[0109]

[0085] Anti-fibrotic agent. As used herein, the term antifibrotic or anti-fibrotic agent refers to any pharmacological agent capable of partially or completely resolving existing fibrosis in a fibrotic tissue, slowing / retarding the process of fibrogenesis, delaying the onset of fibrosis, or otherwise reducing the amount of ECM deposition, which is typically measured by collagen synthesis and / or accumulation. Examples of antifibrotic agents include inhibitors of the TGFp signaling pathway.

[0110]

[0086] Background therapy. In the context of add-on or adjunct therapy, the term “background therapy” refers to approved treatment available to a patient or patient population, for a particular indication, or a condition associated with the indication, which the patient has received or is receiving prior to a second therapy to be added on as adjunct therapy for the same indication (e.g., prior to adjunct administration of a myostatin pathway inhibitor). Typically, background therapies are standard-on-care for the condition. In muscular dystrophy such as DMD, SOC typically includes anti-inflammatory agents such as corticosteroids / glucocorticoids.

[0111]

[0087] Baseline-. The terms “baseline” and “baseline measurement” refer to measurement of a particular metric in a subject or a population of subjects before an action occurs to modify that metric, e.g., at the beginning of a study (prior to dosing or pre-treatment). For example, in studying a treatment for a muscle disease or disorder, a baseline or baseline measurement may include measurement of muscle mass size (e.g., mass, volume), muscle strength (e.g., force generation), muscle tone, motor function, respiratory function, vocalization function, assessment of dysphagia, assessment of bulbar function, among other measurements.

[0112]

[0088] Combination therapy. As used herein, “combination therapy” refers to a therapeutic regimen involving administration of two or more active agents (e.g., two or more pharmacological agents) intended to treat a particular indication and / or conditions associated therewith. 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 each other and complementary to each other. In the context of the present disclosure, combination therapy for treating muscular dystrophiesAttorney Docket No. 15094.0067-00304

[0113] such as DMD include two or more agents selected from: i) myostatin pathway inhibitors, such as myostatin inhibitors and ActRII antagonists; ii) Dystrophin upregulators; and, iii) antifibrotic agents, such as TGFpl inhibitors.

[0114]

[0089] The term “decrease” or “reduce”, as used herein, in the context of a disease symptom refers to a statistically significant decrease in such level. 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%, or below the level of detection for 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 may achieve a level accepted as within the normal range for an individual without such disorder.

[0115]

[0090] Duchenne muscular dystrophy (DMD)-. Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration and weakness due to the alterations of the muscle protein dystrophin which provides structural integrity to the muscle. DMD is one of four conditions known as dystrophinopathies. The other three diseases that belong to this category are Becker Muscular dystrophy (BMD, a mild form of DMD); an intermediate clinical presentation between DMD and BMD; and DMD-associated dilated cardiomyopathy (heart-disease) with little or no clinical skeletal, or voluntary, muscle disease. DMD symptom onset is in early childhood, usually between ages 2 and 3. The disease primarily affects boys, but in rare cases it can affect girls. In Europe and North America, the prevalence of DMD is approximately 6 per 100,000 individuals.

[0116]

[0091] Dystrophin-. Dystrophin is a rod-shaped cytoplasmic protein, and a vital part of a protein complex that connects the cytoskeleton of a muscle fiber to the surrounding extracellular matrix through the cell membrane. Dystrophin is coded for by the DMD gene - the largest known human gene, covering 2.4 megabases at locus Xp21. The 79-exon muscle transcript codes for a protein of 3685 amino acid residues (MW 427 kDa). Dystrophin deficiency has been definitively established as one of the root causes of the general class of myopathies collectively referred to as muscular dystrophy. The deletions of one or several exons of the dystrophin DMD gene cause Duchenne and Becker muscular dystrophies.

[0117]

[0092] Dystrophin upregulator. As used herein, the term Dystrophin upregulator (or Dystrophin corrector) refers to any pharmacological agent capable of increasing Dystrophin levels in a subject. Examples of Dystrophin upregulators include splice modifying agents such as exon-skipping agents and gene therapy. Irrespective of the modalities, the aim is to increase the amount of functional Dystrophin protein in patients, which is either missing or significantly reduced in patients. The goal of exon skipping is to change the splicing pattern so that an out-of-frame, DMD-type mutation becomes an in-frame, BMD-type mutation. For example, skipping exon 51 of the dystrophin gene could restore the “reading frame” in patients who have specific out-of-frame deletions in some dystrophin exons.

[0118]

[0093] In some embodiments, the Dystrophin upregulator is an RNAi-based agent. In some embodiments, the RNAi agent is a small interference RNA (siRNA). In some embodiments, the RNAi agent is a microRNA (miRNA). In some embodiments, the Dystrophin upregulator is an antisenseAttorney Docket No. 15094.0067-00304

[0119] oligonucleotide agent. In some embodiments, the antisense oligonucleotide agent is a stabilized antisense oligonucleotide agent, wherein optionally, the stabilized antisense oligonucleotide agent is a Morpholino (phosphorodiamidate morpholino oligomer (PMO)).

[0120]

[0094] In some embodiments, the dystrophin upregulator is a splice modifier, wherein optionally the splice modifier is an exon-skipping agent, wherein further optionally, the exon-skipping agent is selected from: Exon 51 -skipping agents, Exon 53-skipping agents, Exon 45-skipping agents, Exon 44-skipping agents, Exon 50-skipping agents, Exon 52-skipping agents, Exon 43-skipping agents, Exon 55-skipping agents, and Exon 8-skipping agents. In preferred embodiments, the exon-skipping agent is selected from Exon 51 -skipping agents, Exon 53-skipping agents and Exon 45-skipping agents.

[0121]

[0095] In some embodiments, the dystrophin upregulator is selected from: casimersen (e.g., Amondys 45), eteplirsen (e.g., Exondys 51), viltolarsen (e.g., Viltepso), and golodirsen (e.g., Vyondys 53) in carrying out the invention.

[0122]

[0096] An alternative approach to increasing Dystrophin expression is through gene therapy. Thus, in some embodiments, the Dystrophin upregulator according to this disclosure is a gene therapy. In some embodiments, the gene therapy is a gene replacement therapy. In some embodiments, the gene replacement therapy aims to provide a gene that generates a truncated form of Dystrophin. In some embodiments, the gene therapy is a gene correction therapy, in which disease-associated mutation or mutations are repaired or “corrected” such that increased levels of functional Dystrophin can be produced. In some embodiments, the functional Dystrophin is a truncated form of Dystrophin protein. The gene correction therapy may be achieved by CRISPR-based techniques.

[0123]

[0097] Effective amount. As used herein, the terms "effective amount" and "effective dose" refer to any amount or dose of a compound or composition that is sufficient to fulfill its intended purpose(s), i.e., a desired biological or medicinal response in a tissue or subject at an acceptable benefit / risk ratio. For example, in certain embodiments of the present invention, the intended purpose may be to inhibit activation of myostatin in vivo, to achieve a clinically meaningful outcome associated with the myostatin inhibition.

[0124]

[0098] In some embodiments, an effective amount is an amount that, when administered according to a particular regimen, produces a positive clinical outcome with a reasonably acceptable level of adverse effects (e.g., toxicity), such that the adverse effects, if present, are tolerable enough fora patient to continue with the therapeutic regimen, and the benefit of the therapy overweighs risk of toxicity. Those of ordinary skill in the art will appreciate that in some embodiments of the invention, a unit dosage may be considered to contain an effective amount if it contains an amount appropriate for administration in the context of a dosage regimen correlated with a positive outcome.

[0125]

[0099] A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular pharmaceutical agent, a therapeutically effective amount (and / or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, on combination with other pharmaceutical agents. In some embodiments, the specific therapeutically effective amount (and / or unit dose) for any particular patient may depend upon a variety ofAttorney Docket No. 15094.0067-00304

[0126] factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and / or rate of excretion or metabolism of the specific pharmaceutical agent employed; the duration of the treatment; and like factors as is well known in the medical arts.

[0127]

[0100] An “effective amount” as used herein may also refer to the amount of each active agent required to confer a therapeutic effect on the subject, either alone or in combination with one or more other active agents. For example, an effective amount refers to the amount of a myostatin inhibitor, e.g., an antibody, or antigen-binding fragment thereof, of the present disclosure which is sufficient to achieve a biological effect, e.g., an increase in muscle mass or muscle fiber diameter, a switch in muscle fiber type, an increase in the amount of force generated by the muscle, an increase in mass and / or function of a muscle tissue in the subject; an increase in the metabolic rate of the subject; an increase in insulin sensitivity of the subject; an increase in a level of brown adipose tissue in the subject; an increase in a level of beige adipose tissue in the subject; a decrease in a level of white adipose tissue in the subject; a decrease in a level of visceral adipose tissue in the subject; a decrease in ratio of adipose-to-muscle tissue in the subject; an increase in glucose uptake by a brown adipose tissue, a beige adipose tissue, or a muscle tissue in the subject; a decrease in glucose uptake by a white adipose tissue or a liver tissue; a decrease in muscle catabolism of protein and / or muscle release of amino acids in the subject; an increase in insulin dependent glycemic control in the subject; a decrease in intramuscular fat infiltration in the subject; or a clinically significant outcome, e.g., partial and complete reversal of insulin resistance, overweight, or glycemic control, or the prevention of muscle loss or atrophy in the subject; and / or prevention of developing a metabolic disease in the subject.

[0128]

[0101] Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.

[0129]

[0102] Efficacy. The term “efficacy” refers to a measurable biological or medicinal response in a subject as the result of administering a therapy in an amount that is sufficient to fulfill its intended purpose(s). For example, in certain embodiments of the present invention, the efficacy of a myostatin pathway inhibitor may refer to any desired clinically meaningful outcome that is associated with use of the inhibitor.

[0130]

[0103] Exon-skipping agent: The goal of exon skipping is to change the splicing pattern so that an out-of-frame, DMD-type mutation becomes an in-frame, BMD-type mutation. For example, skipping exon 51 of the dystrophin gene could restore the “reading frame” in patients who have specific out-of-frame deletions in some dystrophin exons. Thus, the term exon-skipping agent refers to a type of Dystrophin upregulator capable of promoting favorable splicing that results in increased levels of functional Dystrophin.Attorney Docket No. 15094.0067-00304

[0131]

[0104] In some embodiments, an exon-skipping agent is an RNAi-based agent. In some embodiments, the RNAi agent is a small interference RNA (siRNA). In some embodiments, the RNAi agent is a microRNA (miRNA). In some embodiments, the exon-skipping agent is an antisense oligonucleotide agent. In some embodiments, the antisense oligonucleotide agent is a stabilized antisense oligonucleotide agent, wherein optionally, the stabilized antisense oligonucleotide agent is a Morpholino (phosphorodiamidate morpholino oligomer (PMO)).

[0132]

[0105] Examples of exon-skipping agents include but are not limited to: Exon 51 -skipping agents, Exon 53-skipping agents, Exon 45-skipping agents, Exon 44-skipping agents, Exon 50-skipping agents, Exon 52-skipping agents, Exon 43-skipping agents, Exon 55-skipping agents, and Exon 8-skipping agents. In preferred embodiments, the exon-skipping agent is selected from Exon 51 -skipping agents, Exon 53-skipping agents and Exon 45-skipping agents.

[0133]

[0106] In some embodiments, the exon-skipping agent is selected from: casimersen (e.g., Amondys 45), eteplirsen (e.g., Exondys 51), viltolarsen (e.g., Viltepso), and golodirsen (e.g., Vyondys 53) in carrying out the invention.

[0134]

[0107] Fc variant. The term “Fc variant” refers to an antibody (immunoglobulins) comprising one or more mutations within the Fc region thereof, typically having the same CDR sequences as the parental (reference) antibody from which it is derived. Fc variants can be generated to have altered (e.g., increased) affinities to the FcR, such as FcRn. In some embodiments, altered affinities to FcRn result in altered biological activities in vivo, such as a longer serum half-life of the Fc variant, as compared to its parental antibody without the Fc mutation(s).

[0135]

[0108] Human antibody / humanized antibody. The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences and fragments thereof. The human antibodies of the disclosure may include 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), for example in the CDRs and in particular CDR3. The term “humanized antibody”, 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.

[0136]

[0109] The term “increase” in the context, e.g., of a disease in which a symptom can be a loss of function or loss of mass, e.g., muscle mass associated with a disease, refers to a statistically significant increase in such level. 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 level of detection forthe 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 accepted as within the range of normal for an individual without such disorder which can also be referred to as a normalization of a level.Attorney Docket No. 15094.0067-00304

[0137] In certain embodiments, the increase is the normalization of the level of a sign or symptom of a disease, an increase in the difference between the subject level of a sign of the disease and the normal level of the sign for the disease. In certain embodiments, the methods include an increase in the mass and / or function of the muscle tissue after treatment of a subject with an antibody that specifically binds pro / latent myostatin. In certain embodiments, the methods include an increase in a level of pro-myostatin in a target muscle, as compared to a control level of pro-myostatin.

[0138]

[0110] Inhibitor of the myostatin signaling pathway: The terms “inhibitor of the myostatin signaling pathway” and “myostatin pathway inhibitor” are used interchangeably herein and encompass any agent that decreases (reduces) or suppresses the activity of the myostatin signaling pathway, irrespective of the mechanism of action. Decreased or suppressed activity of the myostatin signaling pathway may be a result of, for example, a reduced degree of activity, shorter duration of activity, reduced availability of one or more components of the signaling pathway, etc.

[0139]

[0111] Ligand trap: As used herein, the term “ligand trap” refers to a molecule or class of molecules comprising a ligand-binding fragment / moiety that serves as a “trap” to sequester ligands (such as growth factors), so as to prevent the ligand from binding to and activating endogenous receptors. Ligand traps may be derived from the naturally-occurring receptors of the ligand by incorporating the ligand-binding portion thereof. Examples of myostatin ligand traps include, without limitation, soluble receptor-based ligand traps, follistatin-based ligand traps, etc. Myostatin ligand traps may not be selective for myostatin but may also bind and inhibit additional ligands such as GDF11 and Activin A.

[0140]

[0112] Mature myostatin: The term “mature myostatin” refers to the dimeric growth factor, which is also known as GDF8 or GDF-8, and is released from the latent myostatin complex. Mature myostatin is the soluble and biologically active ligand capable of binding to and activating its receptors. Unless explicitly stated otherwise, the term “mature myostatin” refers to a fully processed, biologically active form of myostatin. A wildtype sequence of human mature myostatin has the following amino acid sequence: DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGS AGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS (SEQ ID NO: 982). In some cases, mature myostatin may contain one or more mutations, which may exhibit altered structure / function or stability.

[0141]

[0113] Metabolic disorder: The term “metabolic disorder” may also be referred to as metabolic disease or metabolic condition and encompasses any conditions involving dysregulation of the body’s metabolic function, resulting in perturbation of the normal physiological state of homeostasis due to an alteration in metabolism (anabolism and / or catabolism). Metabolic disorders may be inherited or acquired. Non-limiting examples of metabolic disorders include obesity / o verweight, type 2 diabetes mellitus, type 2 diabetes mellitus associated with obesity, and metabolic syndrome.

[0142]

[0114] Muscle force: Muscle strength can be determined by the force it generates. Experimentally, muscle force is typically assessed by measuring maximum force generated at a given frequency of stimulation. Muscle performance in a subject or in an animal model is assessed by measuring the muscle force. The term “muscle force” or “force” as used herein refers to the measurement of feree generated byAttorney Docket No. 15094.0067-00304

[0143] a muscle. The terms “maximum force,” “max force” and “max (muscle) force” refer to the maximum force generated by a muscle in response to a stimulation (e.g. maximum force generated by a muscle in response to electrical stimulation at a given frequency). The term “specific force” refers to muscle force normalized to muscle weight of a specific muscle (e.g. tibialis anterior or gastroc muscle). Muscle force may be normalized to body weight (and expressed, e.g. maximum force normalized to body weight as “Max / BW”). Muscle force may be normalized to muscle length of a specific muscle (e.g. tibialis anterior or gastroc muscle). In some embodiments, muscle force generation is measured in a mouse model (e.g., a FLExDUX4 mouse model or a D2.mdx mouse model) and expressed as a function of electrical stimulation frequency. In some embodiments, muscle force is measured for the plantarflexor muscle group. In some embodiments, the maximum force is determined at 150 Hz stimulation. In some embodiments, maximum force is determined at day 28 following weekly administration of the myostatin-selective inhibitor (e.g. a myostatin-selective inhibitor antibody at 20 mg / kg). In some embodiments, treatment of a FLExDUX4 mouse model with a myostatin-selective inhibitor increases muscle force (e.g. as measured by maximum force generated at a given frequency) as compared to a control. In some embodiments, treatment of a D2.mdx mouse model with a myostatin-selective inhibitor increases muscle force (e.g. as measured by maximum force generated at a given frequency) as compared to a control. In some embodiments, muscle strength is determined by cross-sectional area (CSA) of a muscle or muscles. In some embodiments, treatment of a D2.mdx mouse model with a myostatin-selective inhibitor increases muscle force or strength as measured by an increase in CSA of a specific muscle or muscles as compared to a control. In some embodiments, the control is measured prior to treatment with the myostatin-selective inhibitor. In some embodiments, the control is a mouse model that has received isotype matched control IgG, optionally wherein the isotype matched control IgG is a murine lgG1. In some embodiments, the control is a mouse model treated with a control / comparator antibody.

[0144]

[0115] Muscular dystrophy. Muscular dystrophies refer to a class of muscle disorders that include several categories: dystrophinopathies, limb girdle muscular dystrophies, congenital muscular dystrophies, distal muscular dystrophies, myofibrillar myopathy, as well as other types of muscular dystrophies. Dystrophinopathies include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, and DMD-associated dilated cardiomyopathy.

[0145]

[0116] Myostatin: In the context of the present disclosure, unless explicitly defined otherwise, the term “myostatin” can refer to any forms of the myostatin protein, such as pro-myostatin, latent myostatin and mature myostatin, each of which exists as dimers in vivo.

[0146]

[0117] Myostatin inhibitor. As used herein, the term “myostatin inhibitor” refers to any agent that inhibits one or more forms of myostatin (e.g., pro-myostatin, latent myostatin and / or mature myostatin) and encompasses both selective inhibitors of myostatin and non-selective inhibitors of myostatin. Selective inhibitors of myostatin (i.e., myostatin-selective inhibitors) are substantially specific to and potent towards myostatin over other structurally related members of the TGFp superfamily at given concentrations. 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 greater affinity as compared to another member of the TGFp superfamily (e.g., GDF11 or Activin A). Examples of myostatin-selective inhibitors known in the art includeAttorney Docket No. 15094.0067-00304

[0147] apitegromab, trevogrumab, and GYM329. By contrast, non-selective inhibitors of myostatin inhibit myostatin, as well as one or more additional members of the TGFp superfamily, such as GDF11 , Activin A, Activin B, BMPs, etc. at given concentrations. Most of the myostatin inhibitors in literature fall within the non-selective category. The term myostatin inhibitor encompasses any molecular modalities such as large molecules (biologies, such as antibodies and engineered protein constructs) and small molecules (such as structurally defined low molecular weight chemical entities), as well as nucleic acid-based molecules, such as ASOs and siRNAs, which reduce myostatin expression. A myostatin inhibitor may be an anti-myostatin antibody, or antigen binding fragment thereof, that binds pro- and / or latent myostatin and / or mature myostatin. In some embodiments, the myostatin inhibitor may be an anti-pro / latent myostatin antibody, or antigen binding fragment thereof, that preferentially (e.g., selectively) binds pro- and / or latent myostatin over mature myostatin. In various embodiments, the myostatin inhibitor may 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 other embodiments, the myostatin inhibitor refers to a gene therapy.

[0148]

[0118] Myostatin pathway inhibitor. The terms “myostatin pathway inhibitor” and “inhibitor of the myostatin signaling pathway” are used interchangeably herein and encompass any agent that decreases (reduces) or suppresses the activity of the myostatin signaling pathway, irrespective of the mechanism of action. Decreased or suppressed activity of the myostatin signaling pathway may be a result of, for example, a reduced degree of activity, shorter duration of activity, reduced availability of one or more components of the signaling pathway, etc. Such inhibitors may be selective to myostatin perse, or may be non-selective such that they also inhibit at least one additional growth factor, such as GDF11 and Activin A. Such agents may reduce myostatin-related signaling by acting upstream of myostatin, on myostatin, or downstream of myostatin via a member of the myostatin-induced signaling cascade to alter a signaling function of the myostatin pathway. In some embodiments, myostatin pathway inhibitors encompass inhibitors that act on molecules upstream of myostatin, e.g., to prevent activation of proteases that cleave pro- and / or latent-myostatin to its active form. In some embodiments, myostatin pathway inhibitors encompass myostatin inhibitors that act on myostatin to prevent its activation or to prevent its interaction with receptors, including myostatin-selective inhibitors. In some embodiments, myostatin pathway inhibitors encompass antibodies and other inhibitors that act on myostatin receptors (e.g., ActRII receptor) or downstream to prevent one or more effects of the myostatin signaling cascade. In some embodiments, the myostatin pathway inhibitors include, but are not limited, antibodies or antigen-binding fragments thereof, small molecules, receptor traps, adnectins, affibodies, DARPins, Anticalins, Avimers, Versabodies, receptor inhibitors (such as a receptor antibody or a receptor kinase inhibitor), or gene therapies.

[0149]

[0119] Myostatin-selective inhibitor. The term “myostatin-selective inhibitor” refers to an inhibitor of the myostatin signaling pathway (i.e., myostatin pathway inhibitor) which is selective to myostatin, but not GDF11 , Activin A, or other TGFp family members. In some embodiments, a myostatin inhibitor is selective if it binds to myostatin with a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1 ,000-fold, or greater affinity toward as compared to another member of the TGFp superfamily (e.g., GDF11 or Activin A). In preferred embodiments, a selective myostatin inhibitor exhibits no detectable binding or potencyAttorney Docket No. 15094.0067-00304

[0150] towards other TGFp family members. In some embodiments, myostatin-selective inhibitors are neutralizing antibodies that bind mature myostatin thereby inhibiting its activity. In some embodiments, myostatin-selective inhibitors are antibodies that bind pro- and / or latent myostatin, thereby inhibiting the activation step of myostatin. In some embodiments, the myostatin-selective inhibitor is Ab2, apitegromab, trevogrumab, GYM329, or a variant of the any one of the foregoing. In some embodiments, the myostatin-eslective inhibitor is selected from the antibodies and antigen-binding fragments disclosed in WO 2024 / 138076.

[0151]

[0120] Pro / latent myostatin-. As used herein, the term “pro / latent myostatin” refers to pro-myostatin, latent myostatin, or both (i.e., pro-forms or precursors of myostatin), but excludes mature myostatin. The pro-and latent forms of myostatin remain associated with a prodomain (e.g., LAP) and are inactive in that they are incapable of binding to the cellular myostatin receptors.

[0152]

[0121] In human, proGDF8 has the following amino acid sequence:

[0153] NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLETAPNISKDVIRQLLPKAPPLRELIDQYDV QRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETP TTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLA VTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKAN YCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS

[0154] (SEQ ID NO: 52), where the furin cleavage site is shown in bold, and the growth factor domain is shown underlined.

[0155]

[0122] “Specific” and “specificity” in the context 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) refer to the selective reactivity of the interaction. The phrase “specifically binds to” and analogous phrases, in the context of antibodies, refer to the ability of antibodies (or antigenically reactive fragments thereof) to bind specifically to an antigen (ora fragment thereof) and not bind specifically to other entities. Specific binding is understood as a preference for binding a certain antigen, epitope, receptor ligand, or binding partner with, 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 can also refer to binding pairs based on binding kinetics such as Kon, Kotf, and KD. For example, a ligand can be understood to bind specifically to its target site if it has a Koff of 10-2sec1or less, 10-3sec1or less, 10_4sec1or less, 10-5sec1or less, or 10-6sec1or less; and / or a KD of 10-8M or less, 10-9M or less, 10-10M or less, or 10-11M or less, or 10-12M or less, e.g., as measured by ELISA and Octet. It is understood that various proteins can share common epitopes or other binding sites (e.g., kinase reactive sites). In certain embodiments, binding sites may bind more than one ligand, but still can be considered to have specificity based on binding preference as compared to a non-specific antigen and / or by having certain binding kinetic parameters. Methods of selecting appropriate non-specific controls are within the ability of those of skill in the art. Binding assays are typically performed under physiological conditions. In some embodiments, an antibody may also “selectively” (i.e., “preferentially”) bind a target antigen if it binds that target with a comparatively greater strength than the strength of binding shown to other antigens, e.g., a 2-fold, 5-fold,Attorney Docket No. 15094.0067-00304

[0156] 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or greater comparative affinity for a target antigen (e.g., pro / latent myostatin) than for a non-target antigen (e.g., GDF11 , Activin A, or other TGFp superfamily members). In preferred embodiments, a selective myostatin inhibitor exhibits no detectable binding or potency towards other TGFp family members.

[0157]

[0123] Subject. As used herein, the term “subject” is a target to whom the therapy or therapies described herein may be administered. In a clinical context, the terms “subject” (e.g., human subjects) and “patient” may be used interchangeably. In some embodiments, a subject is a vertebrate, in particular a mammal, in need of treatment, e.g., companion animals (e.g., dogs, cats and the like), farm animals (e.g., cows, pigs, horses, sheep, goats, poultry and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like). In some embodiments, the subject is a human who will benefit from or in need of treatment. In some embodiments, a subject is a human subject. In the context of the present disclosure, the subject suffers from a muscular dystrophy, such as DMD.

[0158]

[0124] Treat / treating / treatment: Treatment of a condition or disease in a subject refers to the act of providing a therapeutic regimen aimed to alleviate, prevent, or improve a medical condition. Thus, the terms treating or treatment do not necessarily require a complete treatment or prevention of the 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%.

[0159] Muscular dystrophies

[0160]

[0125] The present disclosure relates to improved therapeutic methods for treating muscular dystrophies. More specifically, various embodiments of combination therapies for the treatment of muscular dystrophies are provided herein.

[0161]

[0126] Muscular dystrophies include several categories: dystrophinopathies, limb girdle muscular dystrophies, congenital muscular dystrophies, distal muscular dystrophies, myofibrillar myopathy, as well as other types of muscular dystrophies. Each of these categories is further described below.

[0162]

[0127] Dystrophinopathies include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, and DMD-associated dilated cardiomyopathy.

[0163]

[0128] DMD is the most common childhood form of muscular dystrophy. Because the underlining genetic mutation is associated with the X chromosome, DMD primarily affects boys, although girls who carry the defective gene may show some symptoms. DMD results from an absence of the muscle protein dystrophin. DMD usually becomes apparent during the toddler years, sometimes soon after an affected child begins to walk. Progressive weakness and muscle wasting (a decrease in muscle strength and size) caused by degenerating muscle fibers begins in the upper legs and pelvis before spreading into the upper arms. Other symptoms include: Loss of some reflexes; A waddling gait; Frequent falls and clumsiness (especially when running); Difficulty when getting up from a sitting position or when climbing stairs; Changes to overall posture; Impaired breathing; and, Heart problems (cardiomyopathy).

[0164]

[0129] Many children with DMD are unable to run or jump. The calf muscles, and less commonly, muscles in the buttocks, shoulders, and arms, may be enlarged by an accumulation of fat and connective tissue,Attorney Docket No. 15094.0067-00304

[0165] causing them to look larger and healthierthan they actually are (called pseudohypertrophy). As the disease progresses, the muscles in the diaphragm that assist in breathing and coughing may weaken. Individuals may experience breathing difficulties, respiratory infections, and swallowing problems. Bone thinning and scoliosis (curving of the spine) are common. Some children have cognitive and behavioral impairments.

[0166]

[0130] In recent years, the availability of DMD therapies have extended the life expectancy and improved the quality of life significantly for children with DMD. Many people with DMD now survive into their 20s or 30s.

[0167]

[0131] In addition to muscle defects, there have been multiple metabolic findings in Duchenne muscular dystrophy research, which include mitochondrial impairment, abnormal lipid metabolism (e.g., lipid buildup in muscle), abnormalities in macronutrient uptake and availability, glycolysis, glycogen storage and utilization, fat oxidation, the creatine phosphagen system and the purine nucleotide cycle.

[0168]

[0132] Becker muscular dystrophy is less severe than but closely related to DMD. The disorder usually appears around age 11 but may occur as late as age 25, and people with Becker MD usually live into middle age or later. The rate of muscle atrophy and weakness varies greatly. Many people maintain their ability to walk until they are in their mid-30s or later, while others are unable to walk past their teens. Muscle weakness is typically noticed first in the upper arms and shoulders, upper legs, and pelvis. Cognitive and behavioral impairments and heart problems are not as common or severe as in DMD, but they do occur. Early symptoms of Becker MD include: walking on one’s toes, frequent falls, and difficulty rising from the floor.

[0169]

[0133] Limb-girdle muscular dystrophy (LGMD) refers to more than 20 inherited conditions marked by progressive loss of muscle and the symmetrical weakening of voluntary muscles, primarily those in the shoulders and around the hips. At least five forms of autosomal dominant limb-girdle MD (known as Type 1) and 17 forms of autosomal recessive limb-girdle MD (known as Type 2) have been identified. Some autosomal recessive forms of the disorder are caused by a deficiency of any of fourdystrophin-glycoprotein complex proteins called the sarcoglycans. Deficiencies in dystroglycan, classically associated with congenital muscular dystrophies, may also cause LGMD. LGMD, as defined by the European Neuromuscular Centre in 2018, are named by the following system: LGMD, recessive or dominant inheritance (R or D), order of discovery (number), affected protein. Examples of LGMSs include the following: LGMD D1 DNAJB6-related; LGMD D2 TNP03-related; LGMD D3 HNRNPDL-related; LGMD D4 calpain3-related; LGMD D5 collagen 6-related; LGMD R1 calpain3-related (Calpainopathy); LGMD R2 dysferlin-related; LGMD R3 a-sarcoglycan-related; LGMD R4 p-sarcoglycan-related; LGMD R5 y-sarcoglycan-related; LGMD R6 6-sarcoglycan-related; LGMD R7 telethonin-related; LGMD R8 TRIM 32-related; LGMD R9 FKRP-related; LGMD R10 titin-related; LGMD R11 POMT1 -related; LGMD R12 anoctamin5-related; LGMD R13 Fukutin-related; LGMD R14 POMT2-related; LGMD R15 POMGnTI-related; LGMD R16 a-dystroglycan-related; LGMD R17 plectin-related; LGMD R18 TRAPPC11 -related; LGMD R19 GMPPB-related; LGMD R20 ISPD-related; LGMD R21 POGLUT1 -related; LGMD R22 collagen 6-related; LGMD R23 laminin a2-related; and, LGMD R24 POMGNT2-related.Attorney Docket No. 15094.0067-00304

[0170]

[0134] Congenital muscular dystrophy refers to a group of muscular dystrophies that are either present at birth or become evident before age 2. The degree and progression of muscle weakness and degeneration vary with the type of disorder. Weakness may be first noted when children do not meet developmental milestones related to motor function and muscle control. Muscle degeneration in congenital muscular dystrophy is restricted primarily to skeletal muscle. Most people with this type of MD are unable to sit or stand without support, and some may never learn to walk. Congenital muscular dystrophies include the following: LAMA2-related (merosin deficient) congenital muscular dystrophy (Emery-Dreifuss muscular dystrophy); Collagen Vl-related muscular dystrophy (Bethlem myopathy, Ullrich congenital muscular dystrophy); a-Dystroglycanopathies (Walker-Warburg syndrome, muscle-eye-brain disease); and, Laminopathies. In some embodiments, the congenital muscular dystrophy is Merosin-negative disorders, in which the protein merosin (found in the connective tissue that surrounds muscle fibers) is missing. In some embodiments, the congenital muscular dystrophy Merosin-positive disorders, in which merosin is present but other necessary proteins are missing.

[0171]

[0135] Distal muscular dystrophy, also known as distal myopathy, refers to any muscle disease that primarily affect the distal muscles (those farthest away from the shoulders and hips) in the forearms, hands, lower legs, and feet. Distal MDs are typically less severe, progress more slowly, and involve fewer muscles than other forms of MD, although they can spread to other muscles. Distal MD can affect the heart and respiratory muscles, and individuals with distal MD may eventually need a ventilator. They may not be able to perform fine hand movement and may have difficulty extending the fingers. Walking and climbing stairs may become difficult and some people may be unable to hop or stand on their heels. Examples include: Late adult-onset type 1 ; Late adult-onset type 2a; Late adult-onset type 2b; Early adult-onset type 1 ; Early adult-onset type 2; and, Early adult-onset type 3.

[0172]

[0136] Myofibrillar myopathies are diseases that share histological similarities in affected muscle and include: Desminopathy; Myotilinopathy; Zaspopathy; Filaminopathy; and, Bag3opathy.

[0173]

[0137] Other muscular dystrophies include: Myotonic dystrophy; Facioscapulohumeral muscular dystrophy (FSHD); Emery-Dreifuss muscular dystrophy (EDMD); and, Oculopharyngeal muscular dystrophy (OPMD).

[0174]

[0138] In some embodiments, the muscular dystrophy to be treated according to the present disclosure is Facioscapulohumeral muscular dystrophy (FSHD). FSHD is a genetic muscle disorder in which the muscles of the face, shoulder blades, and upper arms are among the most affected. FSHD initially affects muscles of the face (facio), shoulders (scapulo), and upper arms (humera) with progressive weakness. Also known as Landouzy-Dejerine disease, this is a relatively common form of MD and is characterized as an autosomal dominant disorder. Most people with FSHD have a normal life span, but some become severely disabled. Typically, weakness first and most seriously affects the face, shoulders, and upper arms, but the disease usually also causes weakness in other muscles. In around 90% of FSHD patients, symptoms usually begin before age 20, with weakness and atrophy of the muscles around the eyes and mouth, shoulders, abdominal muscles, upper arms, and lower legs, usually with asymmetric involvement.

[0175]

[0139] In some embodiments, the myotonic dystrophy is Myotonic dystrophy (DM1), also known as Steinert's disease or dystrophia myotonica. In some embodiments, the myotonic dystrophy is myotonicAttorney Docket No. 15094.0067-00304

[0176] dystrophy type 2 (DM2), which similar to the classic form, but usually affects proximal muscles more significantly.

[0177] General features of DMD

[0178]

[0140] In some embodiments, myostatin inhibitors and related methods disclosed herein are used in the treatment of DMD. The pathophysiology of DMD is complex and can manifest in many ways, which can include: muscle defects (e.g., motor function impairment), bone defects (e.g., fragility, fractures, and osteoporosis), chronic inflammation, muscle fibrosis, skin abnormality, and metabolic dysregulation. The objective of the combination therapies disclosed herein is to target multiple facets of the disease with the use of two or more therapeutic agents to address the condition, in order to enhance overall therapeutic benefit, as compared to monotherapy of any single agent.

[0179]

[0141] Muscle impairment:

[0180]

[0142] DMD is characterized by weakness and wasting (atrophy) of the muscles of the pelvic area followed by the involvement of the shoulder muscles. As the disease progresses, muscle weakness and atrophy spread to affect the trunk and forearms and gradually progress to involve additional muscles of the body. DMD can affect not only ambulation but also respiratory function, bulbar function and cardiac function.

[0181]

[0143] Bone fragility:

[0182]

[0144] Duchenne muscular dystrophy is associated with an increased risk of bone fragility due to the adverse effects of prolonged glucocorticoid therapy and progressive muscle weakness on bone strength. DMD patients frequently experience bone pain and loss of ambulation due to fractures. Glucocorticoid-related bone morbidity is common, which can lead to osteoporosis.

[0183]

[0145] Suh et al. recently demonstrated that transgenic overexpression of follistatin, an endogenous inhibitor of myostatin, GDF11 , and activins, substantially enhanced muscle mass but induced spontaneous tibial fractures due to a reduction in bone mineral density, implying that inhibition of GDF11 may have adverse effects on bone (Proc. Natl Acad. Sci. USA 117, 4910-4920 (2020)). These findings suggest that those who are at risk of bone fractures or bone disorders in particular should avoid non-selective myostatin inhibitors that also inhibit GDF11. Advantageously, in carrying out various embodiments of the present disclosure, myostatin inhibitors that do not inhibit GDF11 may be selected to treat these patient populations.

[0184]

[0146] Chronic inflammation:

[0185]

[0147] In DMD, the prolonged activation of the innate immune response leads to excessive inflammation resulting in chronic inflammation, which can exacerbate muscle damage. Inflammatory cytokines can activate a variety of signaling pathways, such as the nuclear factor (NF)-KB, Janus-activated kinase / signal transducer and activator of transcription (JAK / STAT), and p38 mitogen-activated protein kinase (MAPK) pathways, resulting in imbalance between protein synthesis and degradation in the skeletal muscle, which favors muscle breakdown, eventually leading to skeletal muscle atrophy. Thus, chronic inflammation can lead to muscle atrophy and / or bone loss (e.g., reduced bone density), and this process may at least in part be mediated by myostatin signaling.

[0186]

[0148] Indeed, role of myostatin in chronic inflammation is implicated in inflammatory conditions in various tissues, including IPF (lung fibrosis; see: Ito et al. Respir Investig 2023 61(4):371-378), CKD (kidneyAttorney Docket No. 15094.0067-00304

[0187] fibrosis; see: Zhang et al. 2011. FASEB J. 25(5):1653-1663)), as well as autoinflammatory disorders such as rheumatoid arthritis (see, for example: Dankbar et al,. Ann Rheum Dis. 2011. 70(Suppl 2):A1-A94), inflammatory bowel disease (IBD; reviewed in: Front. Immunol., 13 July 2021 Sec. Autoimmune and Autoinflammatory Disorders, Volume 12-2021), including Crohn’s disease (CD) and ulcerative colitis (UC), as well as metabolic disorders such as obesity and type 2 diabetes. Patients suffering from one or more of these conditions often experience muscle atrophy and also impaired bone homeostasis.

[0188]

[0149] Based on these observations, Applicant reasoned that myostatin inhibition may mitigate bone destruction and muscle atrophy associated with chronic inflammation in dystrophic muscle. Accordingly, the invention encompasses combination therapy that comprises a myostatin inhibitor, preferably a myostatin-selective inhibitor in the treatment of muscular dystrophy such as DMD.

[0189]

[0150] Fibrosis:

[0190]

[0151] Chronic exposures to an inflammatory environment drive fibrogenesis in the affected tissue. In DMD, fibrosis is a hallmark of the pathogenesis in which muscle tissues become replaced with fat / lipids and accumulate extracellular matrix depositions, particularly collagens. Over time, the chronic nature of the injury to the muscle tissue induces even more fibrosis, which results in matrix remodeling and greater stiffness of the ECM, causing muscle impairment. Indeed, Applicant previously demonstrated that increased stiffness of the matrix / substrate lowers the threshold for TGFpl activation, indicating that dysregulation in the ECM renders the tissue even more susceptible to exacerbation of fibrosis.

[0191]

[0152] As reviewed by Mogharerhabed and Czubryt (Am J Physiol - cell physiol., 325(5): C1155-C1386), the extracellular matrix (ECM) is primarily comprised of collagens that typically represent more than 80% of the ECM volume, providing tissue strength and resilience yet maintaining flexibility during muscle contraction. The most common collagen in muscle is fibrillar type 1 , levels of which correlate with muscle stiffness. As alluded to above, physical damage or stress can induce tissue fibrosis, resulting in increased ECM deposition, particularly collagen. Patients with DMD exhibit markedly increased levels of muscle collagen content, and fibrosis is known to occur in both cardiac and skeletal muscle. The degree of collagen cross-linking is also important in determining muscle stiffness, and increased collagen cross-linking has been observed in dystrophic patients. Structural remodeling of the ECM can therefore affect muscle functionality. Mesenchymal cells contribute to fibrosis in both the heart and skeletal muscle. In the myocardium, activation of resident fibroblasts by physical damage, stress, or inflammation results in conversion to myofibroblasts which drive the fibrotic process and impair cardiac contraction and relaxation. In skeletal muscle, damage to muscle fibers leads to satellite cell activation - muscle stem cells located between myofibers that, upon stimulation, can convert to myoblasts that subsequently form myotubes to facilitate muscle repair. However, in patients with DMD, increased inflammation negatively impacts this process, promoting fibrotic remodeling and fiber replacement, degrading muscle function. Mesenchymal fibroadipogenic progenitor cells (FAPs) that are located within the interstitial spaces of skeletal muscles can become activated to form adipocytes that result in fat accumulation, or alternatively to form fibroblasts that produce ECM and contribute to fibrosis.

[0192]

[0153] A number of factors contribute to fibrogenesis, but among them the TGFp pathway has been long recognized as a key driver of fibrosis. Historically, many groups attempted to develop inhibitors of the TGFpAttorney Docket No. 15094.0067-00304

[0193] signaling pathway without success. Applicant previously demonstrated that specifically targeting the ECM-associated pool of latent TGFpl , while sparing immune cell-associated TGFpl , using a monoclonal antibody that selectively binds and inhibits activation of LTBP-bound TGFpl is sufficient to achieve antifibrotic effects in multiple preclinical models (see, for example: WO 2020 / 160291 ; see also: Jackson et al., Sci. Signal. 17, eadn6052 (2024)). The rationale for this approach was to address the ECM deposition driving the fibrosis, while avoiding unwanted immune stimulation caused by TGFpl inhibition associated with immunosuppressive cells, such as regulatory T cells, polarized macrophages and MDSCs, which could further aggravate inflammation in fibrotic tissues. Because muscular dystrophies such as DMD are associated with muscle fibrosis which accompanies inflammation of the affected tissue, Applicant reasoned that preferred antifibrotic agents which can be used in accordance with the disclosure are TGFpl inhibitors that do not trigger unwanted immune stimulation to aggravate inflammation.

[0194]

[0154] Skin abnormality / wound:

[0195]

[0155] Clinical observations reported in literature suggest that patients with DMD are prone to developing skin abnormalities, such as wounds and impaired skin integrity, although the exact causal link between these manifestations and the disease-causing genetic mutations in the dystrophin gene is not well understood. Skin breakdown frequently seen in DMD patients includes, but is not limited to, rashes, nonhealing surgical wounds, burns, and pressure injuries. It has been reported that prolonged, systemic use of anti-inflammatory such as corticosteroids, can significantly impair wound healing. Furthermore, chronic inflammation plays a major role in defective wound healing and in the development of chronic wounds. Thus, multiple factors may contribute to the skin abnormalities often observed in DMD patients. Notably, Activin A is known to play a positive role in the process of wound healing. Many non-selective inhibitors of myostatin known in the art also inhibit Activin A, including, for example, ActRII antagonists such as bimagrumab, ligand traps, anti-myostatin Adnectins, and most of the neutralizing antibodies that bind mature myostatin. Use of these non-selective inhibitors should be cautioned due to its potential interference with wound healing in dystrophic patients, particularly those receiving corticosteroid therapy. Accordingly, preferred myostatin inhibitors to carry out the combination therapies disclosed herein are myostatin-selective inhibitors.

[0196]

[0156] Metabolic dysregulation:

[0197]

[0157] Dystrophic patients often suffer from metabolic dysregulation. Metabolic impairment has been reported in several tissues such as the skeletal and cardiac muscles, liver and brain. Skeletal muscle plays an important role in glucose metabolism and is a major participant in different signaling pathways. Therefore, its damage may lead to different metabolic disruptions. Two of the most important metabolic alterations in muscular dystrophies may be insulin resistance and obesity. In recent years, myostatin has garnered attention as a potential therapeutic target for metabolic disorders. Many, if not most, of the myostatin inhibitors in development are non-selective inhibitors that also block other similar ligands, such as GDF11. However, caution should be exercised in employing a non-selective inhibitor of myostatin because in addition to detrimental effects of GDF11 inhibition on enhancing muscle mass, active GDF11 may also provide metabolic benefits. This suggests that it is advantageous to select myostatin inhibitors which are capable of retaining GDF11 activities in the context of metabolic health.Attorney Docket No. 15094.0067-00304

[0198]

[0158] In some aspects, the present disclosure provides myostatin inhibitors for use in treating a muscular dystrophy, such as DMD. In some embodiments, the myostatin inhibitor is a myostatin-selective inhibitor (e.g., apitegromab). In some embodiments, the myostatin inhibitor is administered to a patient with DMD.

[0199]

[0159] In some embodiments, the myostatin inhibitor is administered to a patient with BMD. In some embodiments, the myostatin inhibitor is administered to the patient as a monotherapy. In some embodiments, the myostatin inhibitor is a myostatin-selective inhibitor. In some embodiments, the myostatin inhibitor is a myostatin-selective inhibitor selected from: apitegromab, GYM329, trevogrumab, SRK-439, or a variant thereof. In some embodiments, the myostatin-selective inhibitor is apitegromab.

[0200] General features of FSHD

[0201]

[0160] In some embodiments, myostatin inhibitors and related methods disclosed herein are used in the treatment of FSHD.

[0202]

[0161] FSHD is an inherited neuromuscular disorder that causes weakness most prominently of the muscles in the face, shoulder blades, and upper arms. It often progresses to cause widespread muscle weakness, and it can also cause loss of hearing.

[0203]

[0162] The region of human chromosomes that causes FSHD contains a section with multiple identical units of DNA called D4Z4 repeats. The number of D4Z4 repeat units in the general population varies from 11 to 100. Each repeat contains a copy of a gene called DUX4, which encodes a transcription factor involved in fetal development, but in adulthood, the DNA in this region is normally “condensed,” or packed tightly together, preventing the expression of the DUX4 gene. As a result, no protein is made from it once fetal development is completed.

[0204]

[0163] In approximately 95% of patients with FSHD, the D4Z4 allele is contracted, meaning that multiple D4Z4 units are lost. This leads to the decondensation of the chromatin, causing the reactivation of the DUX4 gene, allowing aberrant production of the DUX4 protein. Synthesis of DUX4 transcripts and protein is toxic in muscle cells. In most patients with FSHD, one D4Z4 allele (a variant form of a gene) is contracted to between 1 and 10 repeat units and the other D4Z4 allele has the normal number, a condition termed FSHD1.

[0205]

[0164] There is an inverse relationship between the onset and clinical severity of FSHD, and the size of the pathogenic (contracted) D4Z4 repeat units. Individuals with one to three repeat D4Z4 units are typically at the severe end of the disease spectrum. With four to 10 repeat units, the clinical variation ranges from asymptomatic to severely affected. An allele with 10 to 11 D4Z4 repeats is considered borderline. The current view among FSHD researchers is that elevation of DUX4 protein causes the symptoms of FSHD.

[0206]

[0165] In less than 5% of people with FSHD, the D4Z4 region is of normal length, which is a condition termed FSHD2. In many individuals with FSHD2, mutations in a different gene, called SMCHD1 , cause the DNA in the vicinity of the D4Z4 repeats to be spread out, allowing the DUX4 gene to be read and protein to be produced.

[0207]

[0166] FSHD1 is inherited in an autosomal dominant pattern, meaning it takes only one mutation (from one parent) to cause the disorder. This altered piece of DNA also can occur spontaneously in a child as he or she develops in the womb, which accounts for 10% to 30% of FSHD1 cases. The inheritance of FSHD2 isAttorney Docket No. 15094.0067-00304

[0208] not fully established and in most cases may require mutations in SMCHD1 on chromosome 18. Approximately 60% of FSHD2 cases appear to be sporadic.

[0209]

[0167] In some embodiments, the therapeutic use according to the present disclosure is directed to treating a subject having FSHD1 or FSHD2. In some embodiments, the subject has FSHD1 or FSHD2 confirmed by a genetic test and / or has symptoms consistent with FSHD according to the judgment of the subject’s physician. In some embodiments, the subject has FSHD with a severity of FSHD determined according to the Ricci Clinical Severity Scale with a score of > 2.5 and < 4 (Ricci et al. Ann. Neurol., 45, 751-757, 1999; Ricci et al., J Neurol. 8(263):1204-1214, 2016; see Table 1 below). In some embodiments, the subject with FSHD has a clinical severity of FSHD as indicated by a Ricci Clinical Severity Scale score of 0.5 to 4. In some embodiments, the subject with FSHD has a clinical severity of FSHD as indicated by a Ricci Clinical Severity Scale score of 1.5 to 3.0. In some embodiments, the subject with FSHD has a clinical severity of FSHD as indicated by a Ricci Clinical Severity Scale score of less than 2.5. In some embodiments, the subject with FSHD has a clinical severity of FSHD as indicated by a Ricci Clinical Severity Scale score of 1.5 to less than 2.5. In some embodiments, the subject with FSHD has a clinical severity of FSHD as indicated by a Ricci Clinical Severity Scale score of 1.5 to 2.4. In some embodiments, the subject with FSHD1 or FSHD2 has a baseline 10-meter walk / run test (10MWRT) time of less than or equal to 5 seconds. In some embodiments, the subject with FSHD has a clinical severity of FSHD as indicated by a Ricci score of 1 .5 to 3.0 (e.g., a Ricci Clinical Severity Scale score of less than 2.5, e.g., 1.5 to 2.4) and a baseline 10-meter walk / run test (10MWRT) time of less than or equal to 5 seconds.

[0210] Table 1 : Ricci Clinical Severity Scale for FSHD.

[0211] > >

[0212] < <

[0213] >

[0214] >

[0215] <

[0216]

[0217] *Muscle strength as evaluated by Manual Muscle Testing (MMT).

[0218]

[0168] In some embodiments, administration of a myostatin-selective inhibitor (e.g., apitegromab) achieves an increase in lean muscle volume (LMV) in the subject with FSHD. In some embodiments, administration of a myostatin-selective inhibitor (e.g., apitegromab) achieves one or more of the following effects: an increase in lean muscle volume (LMV) from baseline (e.g., as measured by a full body MRIAttorney Docket No. 15094.0067-00304

[0219] scan) at 24 weeks of treatment, an increase in lean muscle volume (LMV) from baseline (e.g., as measured by full body MRI) at 52 weeks of treatment.

[0220]

[0169] In some embodiments, administration of a myostatin-selective inhibitor (e.g., apitegromab) achieves an improvement in motor function in the subject with FSHD as assessed by a motor function test. In some embodiments, administration of a myostatin-selective inhibitor (e.g., apitegromab) achieves an improvement in motor function, e.g., an increase in motor function score, as assessed by quantitative muscle testing (QMT), reachable workspace (RWS), 10-meter walk / run test (10MWRT), timed up and go (TUG), manual muscle testing (MMT), maximal voluntary isometric contraction testing (MVICT), and / or 5* sit-to-stand (5* STS) tests. In some embodiments, this improvement is achieved relative to baseline after 12 weeks, 24 weeks, 36 weeks, 48 weeks, or 52 weeks of treatment.

[0221]

[0170] In some embodiments, administration of a myostatin-selective inhibitor (e.g., apitegromab) achieves an improvement in a patient-reported outcome (PRO) and / or a clinician-reported outcome (CRO) in the subject with FSHD. In some embodiments, the improvement in the patient-reported outcome (PRO) and / or a clinician-reported outcome (CRO) is indicated by, e.g., an increase in scoring, based on one or more of the following: FSHD - health index (FSHD-HI), FSHD - composite outcome measure (FSHD-COM), Patient’s Global Impression of Change (PGI-C), Patient-Reported Outcomes Measurement Information System (PROMIS), Quality of Life in Neurological Disorders (NeuroQoL), FSHD Rasch-Built Overall Disability Scale (FSHD-RODS), and Activity Limitations (ACTIVLIM) Questionnaire. In some embodiments, this improvement is achieved relative to the baseline score of the corresponding test in the subject after 12 weeks, 24 weeks, 36 weeks, 48 weeks or 52 weeks of treatment.

[0222]

[0171] In some embodiments, administration of a myostatin-selective inhibitor (e.g., apitegromab) achieves an improvement in one or more biomarkers in the subject with FSHD. In some embodiments, administration of a myostatin-selective inhibitor (e.g., apitegromab) achieves an improvement (e.g., a reduction) in the expression level of one or more biomarkers affected by DUX4 expression selected from the group consisting of: methyl-CpG-binding protein 3-like 2 (MBD3L2), tripartite motif protein 43 (TRIM43), PRAME family member 1 (PRAMEF1), Zinc finger and SCAN domain-containing 4 (ZSCAN4), K-homology domain-containing 1 like (KHDC1L), Leucine twenty homeobox (LEUTX), whey acidic protein four-disulfide core 3 (WFDC3), I IvB-like (ILVBL; Bacterial Acetolactate Synthase-Like), Solute carrier (SLC) family 15 member 2 (SLC15A2; PepT2), and sorbitol dehydrogenase (SORD). In some embodiments, administration of a myostatin-selective inhibitor (e.g., apitegromab) achieves an improvement (e.g., a reduction) in the expression level of one or more genes selected from the group consisting of: ZSCAN4, LEUTX, MBD3L2, TRIM43, PRAMEF1 , and KHDC1L. In some embodiments, administration of a myostatin-selective inhibitor (e.g., apitegromab) achieves an improvement (e.g., a reduction) in the expression level of one or more genes selected from the group consisting of: MBD3L2, TRIM43, PRAMEF1 , and KHDC1L. In some embodiments, this improvement is achieved relative to the baseline level of the corresponding gene expression in the subject after 12 weeks, 24 weeks, 36 weeks, 48 weeks or 52 weeks of treatment.

[0223]

[0172] In some embodiments, administration of a myostatin-selective inhibitor (e.g., apitegromab) achieves an improvement in the subject’s muscle quality, including one or more of a decrease in fattyAttorney Docket No. 15094.0067-00304

[0224] tissue replacement or infiltration of muscle, a decrease in muscle damage, including edema or inflammation, and / or an increase in muscle volume (e.g. a decrease in muscle atrophy or an increase in muscle hypertrophy), in the muscle in the subject with FSHD as detected by MRI or ultrasound. In some embodiments, this improvement is achieved relative to baseline after 12 weeks, 24 weeks, 36 weeks, 48 weeks or 52 weeks of treatment.

[0225]

[0173] In some embodiments, administration of a myostatin-selective inhibitor (e.g., apitegromab) achieves an improvement (e.g., a reduction) in creatine kinase. In some embodiments, administration of a myostatin-selective inhibitor (e.g., apitegromab) achieves a decrease in one or more of the following: serum creatine kinase (e.g. creatine kinase MM and MB isoforms), serum carbonic anhydrase III, serum troponin I type 2, serum interleukin 6, muscle expression of chemokine (C-X-C motif) ligand 13 (CXCL13), and / or DL / X4-related microRNA signatures (e.g., muscle expression of miR-31-5p and miR-206 and / or serum levels of miR-206). In some embodiments, this improvement is achieved relative to baseline after 12 weeks, 24 weeks, 36 weeks, 48 weeks or 52 weeks of treatment.

[0226]

[0174] In some embodiments, administration of a myostatin-selective inhibitor (e.g., apitegromab) achieves an improvement in the muscle in the subject with FSHD as detected by MRI or ultrasound. In some embodiments, administration of a myostatin-selective inhibitor (e.g., apitegromab) achieves an improvement in fatty tissue replacement or infiltration of muscle, muscle damage, including edema or inflammation, and / or muscle volume (atrophy or hypertrophy), in the muscle in the subject with FSHD as detected by MRI or ultrasound. In some embodiments, the administration of the myostatin-selective inhibitor reduces fatty tissue replacement of or infiltration of muscle as detected by MRI or ultrasound. In some embodiments, the administration of the myostatin-selective inhibitor reduces infiltration of muscle by fatty tissue as detected by MRI or ultrasound. In some embodiments, the administration of the myostatin-selective inhibitor reduces muscle damage (e.g., edema, inflammation) as detected by MRI or ultrasound. In some embodiments, administration of the myostatin-selective inhibitor reduces muscle atrophy as detected by MRI or ultrasound. In some embodiments, administration of the myostatin-selective inhibitor increases muscle volume as detected by MRI or ultrasound. In some embodiments, administration of the myostatin-selective inhibitor increases lean muscle volume as detected by MRI or ultrasound. In some embodiments, administration of a myostatin-selective inhibitor (e.g., apitegromab) achieves an increase in total lean muscle volume as detected by MRI or ultrasound. In some embodiments, this improvement is achieved relative to baseline after 12 weeks, 24 weeks, 36 weeks, 48 weeks or 52 weeks of treatment.

[0227]

[0175] In some aspects the present disclosure provides a myostatin inhibitor for use in the treatment of facioscapulohumeral muscular dystrophy (FSHD) in a patient in need thereof, wherein the treatment comprises administration of a myostatin inhibitor to the patient in an amount effective to treat FSHD, wherein the patient has a Ricci Clinical Severity Scale score of 1.5 to 3.0 at baseline (at screening). In some embodiments, the subject with FSHD has a clinical severity of FSHD as indicated by a Ricci Clinical Severity Scale score of less than 2.5. In some embodiments, the subject with FSHD has a clinical severity of FSHD as indicated by a Ricci Clinical Severity Scale score of 1.5 to less than 2.5. In some embodiments, the subject with FSHD has a clinical severity of FSHD as indicated by a Ricci Clinical Severity Scale score of 1.5 to 2.4. In some aspects, the present disclosure provides a myostatin inhibitorAttorney Docket No. 15094.0067-00304

[0228] for use in the treatment of facioscapulohumeral muscular dystrophy (FSHD) in a patient in need thereof, wherein the treatment comprises administration of a myostatin inhibitor to the patient in an amount effective to treat FSHD, wherein the patient has a 10-meter walk / run test (10MWRT) time of less than or equal to 5 seconds at baseline (at screening). In some aspects, the present disclosure provides a myostatin inhibitor for use in the treatment of FSHD in a patient in need thereof, wherein the treatment comprises administration of a myostatin inhibitor to the patient in an amount effective to treat FSHD, wherein the patient has a timed-up-and-go (TUG) time of 20 seconds or less (e.g., 16 seconds or less, 14 seconds or less, 12 seconds or less) at baseline (at screening). In some embodiments, the amount effective to treat FSHD is an amount sufficient to delay disease progression.

[0229]

[0176] In some embodiments, the patient is at least 16 years of age at initiation of the administration of the myostatin inhibitor. In some embodiments, the patient is 16-65 years of age (e.g., 16-60 years of age) at initiation of the administration of the myostatin inhibitor. In some embodiments, the patient is between the age of 18-60 years at initiation of the administration of the myostatin inhibitor. In some embodiments, the patient has been genetically diagnosed with FSHD1 or FSHD2. In some embodiments, the patient has been genetically diagnosed with FSHD1 or FSHD2 and is an ambulatory adult.

[0230]

[0177] In some embodiments, the patient has later-onset FSHD. In some embodiments, the patient has mild-to-moderate FSHD. In some embodiments, the patient has moderate FSHD. In some embodiments, the patient has mild FSHD. In some embodiments, a patient with mild FSHD exhibits one or more of the following symptoms: facial weakness, mild scapular involvement without limitation of arm abduction, moderate involvement of scapular and arm muscles or both (arm abduction >60° and strength >3 in arm muscles, without involvement of pelvic and leg muscles), severe scapular involvement (arm abduction <60°on at least one side); strength <3 in at least one muscular district of the arms without involvement of pelvic and leg muscles, tibioperoneal weakness without weakness of pelvic and proximal leg muscles, and / or mild weakness of pelvic and proximal leg muscles or both (strength >4 in all these muscles and with ability to stand up from a chair without support), where muscle strength values are as evaluated by Manual Muscle Testing (MMT). In some embodiments, a patient with moderate FSHD exhibits one or more of the following symptoms: moderate weakness of pelvic and proximal leg muscles or both (strength >3 in all these muscles and with ability to stand up from a chair with monolateral support), and / or severe weakness of pelvic and proximal leg muscles or both (strength <3 in at least one of these muscles and with ability to stand up from a chair with double support and ability to walk unaided). However, because of the high degree of heterogeneity among FSHD patients, in some embodiments, some patients with FSHD will not present symptoms according to these groupings and might be considered more or less severe based on the severity and type of the affected muscles. In some embodiments, a patient may be considered to have more severe FSHD than indicated by these typical groupings because of the leg and / or pelvic girdle being more affected than other muscle types. In some embodiments, a patient may be considered to have milder FSHD than indicated by these typical groupings because of the facioscapulohumeral muscles being more affected than the leg and / or pelvic girdle muscles. In some embodiments, muscle strength values are as evaluated by Manual Muscle Testing (MMT).

[0231]

[0178] In some embodiments, the myostatin inhibitor is a myostatin-selective inhibitor. In some embodiments, the myostatin-selective inhibitor is selected from: an antibody that selectively bindsAttorney Docket No. 15094.0067-00304

[0232] myostatin but does not bind Activin A or GDF11 ; an antibody that selectively binds pro / latent myostatin thereby inhibiting myostatin activation; or a nucleic acid-based agent that blocks expression of endogenous myostatin. In some embodiments, the myostatin-selective inhibitor is selected from apitegromab, GYM329, trevogrumab, SRK-439, or a variant thereof. In some embodiments, the myostatin-selective inhibitor is apitegromab. In some embodiments, the apitegromab is administered to the patient at a dose of 2-20 mg / kg. In some embodiments, the apitegromab is administered to the patient at a dose of 10 mg / kg. In some embodiments, the apitegromab is administered to the patient once every four weeks or once a month. In some embodiments, the myostatin inhibitor is administered to the patient intravenously. In some embodiments, the apitegromab is administered to the patient intravenously at a dose of 10 mg / kg and at a frequency of once every four weeks or once a month.

[0233]

[0179] In some embodiments, the present disclosure provides myostatin-selective inhibitors (e.g., apitegromab) for treatment of FSHD in specific patient populations, such as those exhibiting certain characteristics of disease or biomarkers indicating they are likely to be responsive to a myostatin-selective inhibitor (e.g., apitegromab) treatment. Thus, baseline features such as age, level of muscle weakness (e.g., Ricci scores), or biomarkers detectable in samples (e.g., D4Z4 repeat numbers) collected from a patient prior to receiving a myostatin-selective inhibitor treatment may inform the likelihood of clinical response, which may allow for selection of patient(s) likely to benefit from a myostatin-selective inhibitor treatment for FSHD.

[0234]

[0180] It is recognized herein that FSHD myocytes comprise sporadic DUX4 expression (e.g., mixed pattern of DUX4-expressing and DUX4-non-expressing groups of cells), and that FSHD is the result of a toxic gain-of-function from de-repression of the DUX4 gene. The data presented herein demonstrate the beneficial effect of myostatin-selective inhibition on non-affected myocytes, increasing muscle mass and function, thereby improving the quality of muscle, and reducing cell death and macrophage. Without being bound by theory, use of myostatin-selective inhibition (e.g., apitegromab) may promote DUX4-non-expressing myocytes in the muscles of the patient and also help prevent expanded atrophy in affected muscle, e.g., in muscles of FSHD patients having milder (e.g., mild-to-moderate, later-onset) FSHD. Thus, the present disclosure provides a rationale for choosing monotherapy with a myostatin-selective inhibitor (e.g., apitegromab) for the treatment of FSHD in a patient with milder (mild-to-moderate, later-onset) FSHD.

[0235]

[0181] In some embodiments, this recognition helps in selecting those patients with FSHD who are likely to benefit from a myostatin-selective inhibitor (e.g., apitegromab) treatment, particularly in context of a monotherapy. Accordingly, the present disclosure provides therapeutic use of myostatin-selective inhibitors (e.g., apitegromab) for the treatment of FSHD, particularly in a patient with mild-to-moderate or later onset FSHD. In some embodiments, the patient with mild-to-moderate or later-onset FSHD has a clinical severity of FSHD are indicated by a Ricci Clinical Severity Scale score of 0.5 to 4. In some embodiments, the patient has mild FSHD and a Ricci Clinical Severity Scale score of 0.5 to 3.0. In some embodiments, the patient has moderate FSHD and a Ricci Clinical Severity Scale score of 3.5 to 4. In some embodiments, the patient with mild-to-moderate or later-onset FSHD has a clinical severity of FSHD as indicated by a Ricci Clinical Severity Scale score of 1.5 to 3.0. In some embodiments, the patient with mild-to-moderate or later-onset FSHD has a clinical severity of FSHD as indicated by a Ricci Clinical Severity Scale score of less than 2.5. In some embodiments, the patient with mild-to-moderate or later-onset FSHD has a clinical severity ofAttorney Docket No. 15094.0067-00304

[0236] FSHD as indicated by a Ricci Clinical Severity Scale score of 1.5 to less than 2.5. In some embodiments, the patient with mild-to-moderate or later-onset FSHD has a clinical severity of FSHD as indicated by a Ricci Clinical Severity Scale score of 1.5 to 2.4. In some embodiments, the patient with mild-to-moderate or later-onset FSHD has about 4 to about 10 D4Z4 repeats. In some embodiments, the patient has FSHD2 and about 11 to about 20 D4Z4 repeats, along with a mutation in a chromatin modifier gene (e.g. a mutation in SMCHD1). In some embodiments, a patient with mild-to-moderate FSHD is treated with a myostatin-selective inhibitor as a monotherapy. In some embodiments, a patient with mild FSHD is treated with a myostatin-selective inhibitor as a monotherapy. In some embodiments, a patient with moderate FSHD is treated with a myostatin-selective inhibitor as a monotherapy. In some embodiments, a patient with later-onset FSHD is treated with a myostatin-selective inhibitor as a monotherapy. In some embodiments, the myostatin-selective inhibitor is apitegromab.

[0237]

[0182] In some embodiments, the patient is further treated with a DUX4 inhibitor or is on a DUX4 inhibitor therapy. In some embodiments, the DUX4 inhibitor is an siRNA that blocks DUX4 expression. In some embodiments, the DUX4 inhibitor is delpacibart braxlosiran (del-brax). In some embodiments, the DUX4 inhibitor is a TfR1-binding Fab conjugated to an siRNA against DUX4 (e.g., DYNE-302). In some embodiments, the myostatin inhibitor and the DUX4 inhibitor are engineered into a single molecule construct.

[0238]

[0183] In some embodiments, the treatment of FSHD with a myostatin-selective inhibitor (e.g., apitegromab) further comprises a standardized exercise regimen consistent with standard-of-care guidelines. In some embodiments, the standardized exercise regimen is a defined home exercise regimen tailored to the patient’s baseline function and adjusted as needed for safety and tolerability over the course of treatment in consultation with a physical therapist with neuromuscular disease expertise. In some embodiments, the standardized exercise regimen comprises a combination of moderate strength training and aerobic exercise. In some embodiments, the standardized exercise regimen is appropriate to the patient’s level of function at baseline and implemented with adjustment for safety and tolerability as needed.

[0239]

[0184] In some embodiments, the treatment increases total lean muscle volume (LMV) in the patient as compared to baseline. In some embodiments, the total LMV is measured by a whole-body Magnetic Resonance Imaging (MRI) scan. In some embodiments, the treatment increases total lean muscle volume as detected by an MRI or ultrasound. In some embodiments, the treatment improves motor function in the patient as compared to baseline. In some embodiments, the motor function is measured by a quantitative myometry test, a 10-meter walk / run test (10WMRT), a Timed Up and Go test, a 5x sit-to-stand test (5x STS), and / or an exercise questionnaire.

[0240]

[0185] In some embodiments, the treatment increases exercise capacity and / or endurance in the patient as compared to baseline. In some embodiments, the increase in exercise capacity and / or endurance is measured by a graded treadmill test, e.g., as measured by time and / or distance to exhaustion. In some embodiments, the treatment increases exercise capacity in the patient. In some embodiments, the treatment increases running capacity in the patient. In some embodiments, the treatment increases endurance in the patient.Attorney Docket No. 15094.0067-00304

[0241]

[0186] In some embodiments, the treatment increases muscle function in the patient as compared to baseline. In some embodiments, the treatment increases a max force level of a muscle in the patient. In some embodiments, the treatment increases a max force level of a tibialis anterior (TA) muscle in the patient. In some embodiments, the treatment increases muscle mass in the patient as compared to baseline. In some embodiments, the treatment increases the muscle mass of a gastrocnemius muscle in the patient. In some embodiments, the treatment increases grip strength in the patient.

[0242]

[0187] In some embodiments, the treatment decreases the creatine / creatinine (Cr / Crn) ratio in the patient compared to baseline. In some embodiments, the treatment decreases the Cr / Crn ratio in the patient compared to a patient receiving a control (e.g., a placebo, vehicle, or isotype-matched control). In some embodiments, the decrease in Cr / Crn ratio in the patient is achieved by 14 days, 28 days, 42 days, or 56 days after initiating the treatment. In some embodiments, the Cr / Crn ratio is determined using a serum creatine level and a serum creatinine level.

[0243]

[0188] In some embodiments, the treatment reduces inflammation in the patient as compared to baseline. In some embodiments, the treatment reduces pro-inflammatory macrophage infiltration in muscle in the patient.

[0244]

[0189] FSHD can be categorized by the timing of disease onset: adult-onset (or later-onset) and early-onset (childhood onset or infantile-onset). In some embodiments, a patient with early-onset FSHD is one who becomes symptomatic for FSHD at earlier than about 10 years old. In some embodiments, a patient with early-onset FSHD is one who becomes symptomatic before the age of 10 years old for shoulder-girdle weakness and / or less than about 5 years old for facial weakness. In some embodiments, a patient with later-onset FSHD is one who becomes symptomatic for FSHD during adolescence to adulthood (e.g., at about 15 to 30 years old). In some embodiments, a patient with later-onset FSHD is one who becomes symptomatic after 30 years of age. In some embodiments, a patient with later-onset FSHD is one who becomes symptomatic before 15 years of age but who does not otherwise fall into the early-onset category. In some embodiments, a patient with later-onset FSHD (e.g., one who presents with symptoms of FSHD later in life, e.g., at about 30-40 years of age) has less severe symptoms than patients who present with symptoms earlier in life.

[0245]

[0190] In some embodiments, the therapeutic use according to the present disclosure is directed to treating a subject having adult-onset FSHD. The adult-onset (which includes FSHD that begins in adolescence) is far more common. In eitherthe adult or infantile onset type of FSHD, facial weakness can start in childhood. Occasionally, other FSHD symptoms appear in early childhood.

[0246]

[0191] In some embodiments, the therapeutic use according to the present disclosure is directed to treating a subject having infantile-onset FSHD. Infantile-onset FSHD generally runs a more pronounced course with regard to muscle weakness, and sometimes also affects hearing and vision. It is characterized by facial weakness appearing before the age of 5 and / or shoulder and arm weakness appearing before the age of 10. Infantile-onset FSHD tends to progress more rapidly, often requiring subjects with infantile-onset FSHD to use a mobility device sooner after the onset of the disorder than subjects with adult-onset FSHD. FSHD may be inherited through either the father or the mother, or it may occur without a family history. The most probable cause of FSHD is a genetic flaw (mutation) that leads to inappropriate expression of the doubleAttorney Docket No. 15094.0067-00304

[0247] homeobox protein 4 gene (DUX4) on chromosome 4, in the 4q35 region. The segment is not part of any particular gene, but it nevertheless seems to interfere with the correct processing of genetic material.

[0248]

[0192] According to the present disclosure, a myostatin inhibitor is used in the treatment of FSHD. Inhibition of the myostatin signaling pathway is aimed to enhance muscle mass and function by favoring synthesis over muscle breakdown. In preferred embodiments, the myostatin inhibitor is an inhibitor that spares the function of the most closely related growth factor, GDF11. In particularly preferred embodiments, the myostatin inhibitor is selective to myostatin, i.e., a myostatin-selective inhibitor, which does not inhibit GDF11 or Activin A. In some embodiments, the myostatin-selective inhibitor comprises GYM329 or apitegromab.

[0249]

[0193] In some embodiments, such myostatin inhibitor is used in conjunction with a DUX4-directed therapy in the treatment of FSHD in a subject. Examples of DUX4-directed therapy include agents that inhibit or reduce expression or activity of DUX4, including agents that inhibit DUX4 mRNA and agents that activate CpG methylation.

[0250]

[0194] In some embodiments, the subject is further treated with additional therapy, such as anabolic stimulators, p2-adrenergic agonists (p2-agonists), creatine, and / or anti-inflammatory agents (e.g., glucocorticoids).

[0251]

[0195] In some aspects, a method of treating muscular dystrophy (e.g., FSHD, DMD, BMD) is provided, the method comprising treatment with a selective myostatin inhibitor, wherein the treatment improves muscle quality. In some embodiments, the improvement in muscle quality comprises an increase in maximum force generated by a muscle. In some embodiments, the improvement in muscle quality comprises an increase in the specific force generated by a muscle. In some embodiments, the muscle is tibialis anterior (TA). In some embodiments, the muscle is gastrocnemius (Gastroc).

[0252]

[0196] In preclinical models, muscle function or performance can be evaluated by assessing exercise capacity. Measures of exercise capacity can include, for example, measures of running capacity, such as distance to exhaustion and / or time to exhaustion. In some aspects, a method of treating a muscular dystrophy (e.g. FSHD, DMD, BMD) is provided, the method comprising treatment with a selective myostatin inhibitor, wherein the treatment improves or increases exercise capacity and / or endurance. In some embodiments, the increase in exercise capacity and / or endurance is an increase in exercise capacity. In some embodiments, the increase in exercise capacity comprises an increase in running capacity. In some embodiments, the increase in exercise capacity and / or endurance is an increase in endurance. In some aspects, a method of increasing exercise capacity and / or endurance in a subject with a muscular dystrophy (e.g. FSHD, DMD, BMD) is provided, the method comprising treatment with an amount of the selective myostatin inhibitor effective to increase exercise capacity and / or endurance. In some embodiments, the increase in exercise capacity and / or endurance is an increase in exercise capacity. In some embodiments, the increase in exercise capacity comprises an increase in running capacity. In some embodiments, the increase in exercise capacity and / or endurance is an increase endurance. In some aspects, a method of improving or increasing endurance in a subject with a muscular dystrophy (e.g. FSHD, DMD, BMD) is provided, the method comprising treatment with an amount of the selective myostatin inhibitor effective to improve or increase endurance and / or running capacity. In some embodiments, the improvement orAttorney Docket No. 15094.0067-00304

[0253] increase in endurance and / or running capacity is measured during a graded treadmill test, e.g. as measured by time and / or distance to exhaustion. In some embodiments, the treatment with selective myostatin inhibitor results in improvement or increase in endurance and / or running capacity compared to a control. In some embodiments, the subject with muscular dystrophy has FSHD. In some embodiments, the subject with muscular dystrophy has DMD.

[0254]

[0197] In some embodiments, treatment of a FLExDUX4 mouse model with a myostatin-selective inhibitor increases endurance or running capacity as compared to a control. In some embodiments, the treatment with selective myostatin inhibitor results in improvement or increase in endurance and / or running capacity in a murine model of FSHD (e.g. FLExDUX4 mouse model) compared to control. In some embodiments, the control is a wild-type counterpart to the FLExDUX4 mouse model. In some embodiments, the control is a FLExDUX4 mouse treated with control isotype Ig. In some embodiments, treatment with the selective myostatin inhibitor improves or increases endurance as measured by running capacity during a graded treadmill test, e.g. as measured by time and / or distance to exhaustion.

[0255]

[0198] In some embodiments, the selective myostatin inhibitor improves or increases time to exhaustion during a graded treadmill test, compared to a wild-type control, e.g. to at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the time to exhaustion for a wild-type control. In some embodiments, the selective myostatin inhibitor improves or increases time to exhaustion during a graded treadmill test to at least 90% of the time to exhaustion for a wild-type control. In some embodiments, the selective myostatin inhibitor improves or increases time to exhaustion during a graded treadmill test, compared to an isotype Ig treated control, e.g. at least a 10%, at least a 15%, at least a 20%, at least 25%, or at least 30% increase in time to exhaustion relative to that for an isotype Ig treated control. In some embodiments, the selective myostatin inhibitor improves or increases time to exhaustion during a graded treadmill test to at least a 20% increase in time to exhaustion relative to that for an isotype Ig treated control.

[0256]

[0199] In some embodiments, the selective myostatin inhibitor improves or increases distance to exhaustion during a graded treadmill test, compared to a wild-type control, e.g. to at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the distance to exhaustion for a wild-type control. In some embodiments, the selective myostatin inhibitor improves or increases distance to exhaustion during a graded treadmill test to at least 80% of the distance to exhaustion for a wild-type control. In some embodiments, the selective myostatin inhibitor improves or increases distance to exhaustion during a graded treadmill test, compared to an isotype Ig treated control, e.g. at least a 25%, at least a 30%, at least a 35%, at least a 40%, or at least a 45% increase in distance to exhaustion relative to that for an isotype Ig treated control. In some embodiments, the selective myostatin inhibitor improves or increases distance to exhaustion during a graded treadmill test to at least a 35% increase in distance to exhaustion relative to that for an isotype Ig treated control.

[0257]

[0200] In some embodiments, treatment of a FLExDUX4 mouse model with a myostatin-selective inhibitor increases grip strength. In some embodiments, the treatment increases grip strength as measured by forelimb and / or hindlimb peak force as compared to a control. In some embodiments, the treatment increases grip strength as measured by duration on wire hang, hanging bar, box hang or similar suspension latency test) as compared to a control.Attorney Docket No. 15094.0067-00304

[0258]

[0201] In some aspects, a method of treating a muscular dystrophy (e.g., FSHD, DMD, BMD) is provided, the method comprising administering to a subject in need thereof a selective myostatin inhibitor, wherein the treatment reduces macrophage infiltration of muscle (e.g., is administered in an amount sufficient to reduce macrophage infiltration). In some aspects, a method of reducing macrophage infiltration of muscle in a subject with a muscular dystrophy (e.g. FSHD, DMD, BMD) is provided, the method comprising administering to the subject in need thereof an amount of a selective myostatin inhibitor (e.g., apitegromab) effective to reduce macrophage infiltration of muscle. In some embodiments, the reduction in macrophage infiltration of muscle is measured by a histological test on a biopsy sample from the subject. In some embodiments, the reduction in macrophage infiltration of muscle is measured by one or more of the following histological tests in a muscle biopsy sample: staining of F4 / 80, staining of CD68, staining of CD86 and / or CD80, staining of CD163 and / or CD206. In some embodiments, the treatment results in a reduction of macrophage infiltration of muscle as compared to a control (e.g., a placebo, vehicle, or isotype-matched control). In some embodiments, the treatment results in a reduction of macrophage infiltration of muscle as compared to a baseline level measured prior to the subject receiving the selective myostatin inhibitor. In some embodiments, the muscular dystrophy is FSHD. In some embodiments, the muscular dystrophy is DMD. In some embodiments, the muscular dystrophy is BMD.

[0259]

[0202] Non-invasive in vivo imaging techniques may be applied in a variety of suitable methods for purposes of: diagnosing patients; selecting or identifying patients who are likely to benefit from selective myostatin inhibitor therapy; and / or monitoring patients for therapeutic response upon treatment. Any cells with a known cell-surface marker may be detected / localized by virtue of employing an antibody or similar molecules that specifically bind to the cell marker. Typically, cells to be detected by the use of such techniques are immune cells, such as cytotoxic T lymphocytes, regulatory T cells, MDSCs, tumor-associated macrophages, NK cells, dendritic cells, and neutrophils. Antibodies or engineered antibody-like molecules that recognize such markers can be coupled to a detection moiety.

[0260]

[0203] In some embodiments, an effective amount of the selective myostatin inhibitor may normalize, as compared to control, the levels of multiple inflammatory serum biomarkers as assessed following the start of the therapy, at, for example, 24 or at 52 weeks. In some embodiments, normalizing the levels of multiple inflammatory serum biomarkers comprises lowering abnormally high levels to a normal range. In some embodiments, normalizing the levels of multiple inflammatory serum biomarkers comprises increasing abnormally low levels to a normal range. In some embodiments, the normal range for the levels of multiple inflammatory serum biomarkers is based on levels of the inflammatory serum biomarkers in a subject that does not have FSHD. In some embodiments, inflammatory biomarkers may be used to assess severity of FSHD, select patients for treatment, and / or monitor disease progression or treatment response. Blood biomarkers may include inflammatory markers, such as CRP, TNF, IL-8, and CXCL10.

[0261]

[0204] Non-limiting examples of suitable immune cell markers include monocyte markers, macrophage markers (e.g., M1 and / or M2 macrophage markers), CTL markers, suppressive immune cell markers, MDSC markers (e.g., markers for G- and / or M-MDSCs), including but are not limited to: CD8, CD3, CD4, CD11 b, CD163, CD206, CD68, CD14, CD15, CD66, CD34, CD25, and CD47. M1 macrophages typically express cell surface HLA-DR, CD68 and CD86, while M2 macrophages typically express cell surface HLA-DR, CD68, CD163 and CD206.Attorney Docket No. 15094.0067-00304

[0262]

[0205] In some embodiments, the present disclosure provides a method of treating FSHD in a subject in need thereof, comprising administering to the subject a selective myostatin inhibitor in an amount effective to reduce macrophage infiltration of muscle as compared to a control (e.g., a placebo, vehicle, or isotype-matched control). In some embodiments, treatment results in a reduction of macrophage infiltration of muscle as compared to a baseline measured prior to the subject receiving the selective myostatin inhibitor.

[0263]

[0206] In some embodiments, the present disclosure provides a method of treating DMD in a subject in need thereof, comprising administering to the subject a selective myostatin inhibitor in an amount effective to reduce macrophage infiltration of muscle as compared to a control (e.g., a placebo, vehicle, or isotype-matched control). In some embodiments, treatment results in a reduction of macrophage infiltration of muscle as compared to a baseline measured prior to the subject receiving the selective myostatin inhibitor.

[0264]

[0207] In some embodiments, the present disclosure provides a method of treating BMD in a subject in need thereof, comprising administering to the subject a selective myostatin inhibitor in an amount effective to reduce macrophage infiltration of muscle as compared to a control (e.g., a placebo, vehicle, or isotype-matched control). In some embodiments, treatment results in a reduction of macrophage infiltration of muscle as compared to a baseline measured prior to the subject receiving the selective myostatin inhibitor.

[0265]

[0208] In some aspects, a method of treating a muscular dystrophy (e.g., FSHD, DMD, BMD) is provided, the method comprising administering to a subject in need thereof a selective myostatin inhibitor, wherein the treatment increases satellite cell number in muscle (e.g., wherein the treatment is in an amount sufficient to increase satellite cell numbers). In some aspects, a method of increasing satellite cell number in muscle in a subject with a muscular dystrophy (e.g. FSHD, DMD, BMD) is provided, the method comprising administering to a subject in need thereof an amount of the selective myostatin inhibitor effective to increase satellite cell number in muscle. In some embodiments, the increase in satellite cell number in muscle is measured by a histological test on a biopsy, e.g. as measured by staining of Pax7 in a sample from a muscle biopsy. In some embodiments, the treatment results in an increase in satellite cell number in muscle as compared to a control (e.g., a placebo, vehicle, or isotype-matched control). In some embodiments, the treatment results in an increase in satellite cell number in muscle as compared to a baseline level measured prior to the subject receiving the selective myostatin inhibitor. In some embodiments, the muscular dystrophy is FSHD. In some embodiments, the muscular dystrophy is DMD. In some embodiments, the muscular dystrophy is BMD.

[0266]

[0209] In some embodiments, the present disclosure provides a method for treating FSHD in a subject in need thereof, comprising administering to the subject a selective myostatin inhibitor in an amount effective to increase a satellite cell number in a muscle as compared to control (e.g., a placebo, vehicle, or isotype-matched control). In some embodiments, the treatment results in an increase in satellite cell number in muscle as compared to a baseline level measured prior to the subject receiving the selective myostatin inhibitor.

[0267]

[0210] In some embodiments, the present disclosure provides a method of treating FSHD in a subject in need thereof, comprising administering to the subject a selective myostatin inhibitor in an amount effective to reduce apoptosis of skeletal muscle cells as compared to a control (e.g., a placebo, vehicle, or isotype-matched control). In some embodiments, treatment results in a reduction of apoptosis of skeletal muscleAttorney Docket No. 15094.0067-00304

[0268] cells as compared to a baseline measured prior to the subject receiving the selective myostatin inhibitor. In some embodiments, the reduction in apoptosis in skeletal muscle cells is measured by TUNEL (TdT-mediated dUTP Nick-End Labeling) method for detection of apoptotic cells. In some embodiments, the method of treating FSHD in a subject in need thereof, comprises administering to the subject a selective myostatin inhibitor in an amount effective to reduce apoptosis of skeletal muscle cells, as measured by TUNEL (TdT-mediated dUTP Nick-End Labeling) method for detection of apoptotic cells.

[0269]

[0211] In some aspects, a method of treating a muscular dystrophy (e.g., FSHD, DMD, BMD) is provided, the method comprising administering to a subject in need thereof a selective myostatin inhibitor, wherein the treatment increases grip strength (e.g., maximum grip strength, dynamic grip endurance, etc), in one or more muscles (e.g., wherein the treatment is in an amount sufficient to increase grip strength). In some aspects, a method of increasing grip strength in a muscle in a subject with a muscular dystrophy (e.g. FSHD, DMD, BMD) is provided, the method comprising administering to a subject in need thereof an amount of the selective myostatin inhibitor effective to increase grip strength. In some embodiments, the increase in grip strength in muscle is measured by hand grip strength, e.g. as assessed by measuring hand grip strength by dynamometry (Rooks et al. J Am Geriatr Soc. 2017;65(9):1988-1995). In some embodiments, the treatment results in an increase in grip strength in muscle as compared to a control (e.g., a placebo, vehicle, or isotype-matched control). In some embodiments, the treatment results in an increase in grip strength in muscle as compared to a baseline level measured prior to the subject receiving the selective myostatin inhibitor. In some embodiments, the muscular dystrophy is FSHD. In some embodiments, the muscular dystrophy is DMD. In some embodiments, the muscular dystrophy is BMD.

[0270]

[0212] In some embodiments, the present disclosure provides a method for treating DMD in a subject in need thereof, comprising administering to the subject a selective myostatin inhibitor in an amount effective to increase a satellite cell number in a muscle as compared to control (e.g., a placebo, vehicle, or isotype-matched control). In some embodiments, the treatment results in an increase in satellite cell number in muscle as compared to a baseline level measured prior to the subject receiving the selective myostatin inhibitor.

[0271]

[0213] In some embodiments, the present disclosure provides a method for treating BMD in a subject in need thereof, comprising administering to the subject a selective myostatin inhibitor in an amount effective to increase a satellite cell number in a muscle as compared to control (e.g., a placebo, vehicle, or isotype-matched control). In some embodiments, the treatment results in an increase in satellite cell number in muscle as compared to a baseline level measured prior to the subject receiving the selective myostatin inhibitor.

[0272]

[0214] In some embodiments, the selective myostatin inhibitor comprises a pro / latent myostatin selective inhibitor. In some embodiments, the selective myostatin inhibitor comprises SRK-015, SRK-439, or a MST1032 variant as described in PCT / JP2015 / 006323.

[0273] Combination therapy for DMD

[0274]

[0215] The invention encompasses pharmaceutical compositions and related methods used as combination therapies for treating subjects who may benefit from myostatin inhibition and at least one other therapy for the treatment of muscular dystrophies in vivo. In any of these embodiments, such subjects may receiveAttorney Docket No. 15094.0067-00304

[0275] combination therapies that include a first composition comprising at least one myostatin pathway inhibitor (e.g., a myostatin inhibitor, e.g., a myostatin-selective inhibitor) in conjunction with a second composition comprising at least one additional therapeutic 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 discrete 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 therapies may be administered in conjunction with each other. The phrase “in conjunction with,” in the context of combination therapies, means that therapeutic effects of a first therapy overlaps temporarily and / or spatially with therapeutic effects of a second therapy in the subject receiving the combination therapy. Thus, the combination therapies may be formulated as a single formulation for concurrent administration, or as separate formulations, for simultaneous or sequential administration of the therapies. The term “concurrent administration”, as used herein, may include coformulated 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 therapeutic regimens in which therapeutics are administered at separate times. In some embodiments, a subject receives concurrent administration of two or more therapies when subject is on one or more first therapeutic(s) at the beginning of the treatment regimen and one or more second therapeutic(s) is / are added to the regimen at a specific point during the treatment.

[0276]

[0216] In some embodiments, combination therapies produce synergistic effects in the treatment of a disease. The term “synergistic” refers to effects that are greater than additive effects (e.g., greater efficacy) of each monotherapy in aggregate. In some embodiments, combination therapies produce additive effects in the treatment of a disease. The invention encompasses pharmaceutical compositions and related methods used as combination therapies for treating subjects who may benefit from myostatin inhibition and Dystrophin upregulation in vivo.

[0277]

[0217] Accordingly, the present disclosure provides combination therapy for treating a muscular dystrophy, such as DMD. According to the invention, the combination therapy comprises at least two of the following pharmacological agents: i) a myostatin inhibitor, preferably a myostatin-selective inhibitor; ii) a Dystrophin upregulator; and, iii) an anti-fibrotic agent. Patients who receive the combination therapy may be further treated with background therapy (e.g., SOC), such as anti-inflammatory (e.g., corticosteroids). Combination therapies according to the present disclosure are not limited to particular modalities of therapeutic agents.

[0278]

[0218] In some embodiments, the combination therapy for treating a muscular dystrophy such as DMD is comprised of a myostatin inhibitor and a Dystrophin upregulator. Optionally, the combination therapy further comprises an anti-inflammatory, wherein further optionally the anti-inflammatory is a corticosteroid.

[0279]

[0219] In some embodiments, the combination therapy for treating a muscular dystrophy such as DMD is comprised of a myostatin inhibitor and an anti-fibrotic agent. Optionally, the combination therapy further comprises an anti-inflammatory, wherein further optionally the anti-inflammatory is a corticosteroid.Attorney Docket No. 15094.0067-00304

[0280]

[0220] In some embodiments, the combination therapy for treating a muscular dystrophy such as DMD is comprised of a Dystrophin upregulatorand an anti-fibrotic agent. Optionally, the combination therapy further comprises an anti-inflammatory, wherein further optionally the anti-inflammatory is a corticosteroid.

[0281]

[0221] In some embodiments, the combination therapy for treating a muscular dystrophy such as DMD is comprised of a myostatin inhibitor, a Dystrophin upregulator, and an anti-fibrotic agent. Optionally, the combination therapy further comprises an anti-inflammatory, wherein further optionally the antiinflammatory is a corticosteroid.

[0282]

[0222] Each of the components of combination therapies contemplated herein is discussed in more detail below.

[0283] Myostatin inhibitors

[0284]

[0223] According to the present disclosure, combination therapy for the treatment of muscular dystrophies can include an agent aimed at inhibiting myostatin signaling. Such agents can include inhibitors of myostatin and ActRII receptor antagonists. Most of such agents known in the art for inhibiting myostatin signaling are non-selective inhibitors of myostatin characterized by their ability of block signaling of not only myostatin but also other structurally similar growth factors, such as GDF11 and Activin A. In preferred embodiments, the myostatin inhibitors suitable for carrying out the present invention are selective inhibitors of myostatin (i.e., “myostatin-selective inhibitors”). As used herein, the term myostatin-selective inhibitor refers to an agent capable of inhibiting myostatin (also known as GDF8 or GDF-8) but not other structurally similar growth factors, specifically, GDF11 and Activin A, at a dose effective in vivo and are not limited to a particular modality.

[0285]

[0224] In some embodiments, the myostatin-selective inhibitor is an antibody or antibody-based construct that comprises an antigen-binding fragment. Examples of such myostatin-selective inhibitors include: neutralizing antibodies that specifically bind mature myostatin but do not bind GDF11 or Activins; anti-myostatin Adnectins engineered to selectively bind myostatin; and antibodies and antigen-binding fragments that selectively bind latent complex of myostatin and capable of inhibiting activation of mature myostatin from the latent form; and, nucleic acid-based inhibitors of myostatin expression. In some embodiments, the myostatin-selective inhibitor is an antibody comprising a heavy chain comprising an N-terminal glutamine and / or a light chain comprising an N-terminal glutamine. In some embodiments, the myostatin-selective inhibitor is an antibody comprising a heavy chain comprising an N-terminal pyroglutamate and / or a light chain comprising an N-terminal pyroglutamate.

[0286]

[0225] The inhibitor of the myostatin signaling pathway (e.g., “myostatin inhibitors”) may be any agent capable of suppressing myostatin signaling. In some embodiments, the inhibitors ofthe myostatin signaling pathway useful for carrying out the present invention may be myostatin-selective inhibitors. In some embodiments, the inhibitors ofthe myostatin signaling pathway useful for carrying out the present invention may be myostatin non-selective inhibitors. In various embodiments, inhibitors of the myostatin signaling pathway useful for carrying out the present invention may include low molecular weight compounds (i.e., small molecules) that inhibit the pathway, as well as biologies, such as antibodies or antigen-binding fragments thereof, and engineered protein constructs that comprise ligand-binding domains (such as soluble receptor ligand traps, follistatin-based engineered constructs, and adnectins). In someAttorney Docket No. 15094.0067-00304

[0287] embodiments, the myostatin pathway inhibitors are antibodies (e.g., antigen-binding fragments thereof or engineered constructs that comprise such fragments) that bind mature myostatin (also known as GDF-8 or GDF8). Such antibodies are typically referred to as neutralizing antibodies because they bind the growth factor thereby blocking its ability to bind endogenous receptors for activating the signaling pathway. In some embodiments, a myostatin pathway inhibitor binds a myostatin receptor thereby interfering with ligand binding. These include antibodies that bind the extracellular portion(s) of the receptor. Non-limiting examples of such antibodies include bimagrumab (BYM338) which binds ActRIIB. In preferred embodiments, the inhibitors of the myostatin signaling pathway useful for carrying out the invention are myostatin-selective inhibitors. In some embodiments, the myostatin-selective inhibitors are neutralizing antibodies that selectively bind mature myostatin (but not other growth factors such as GDF11 or Activin A). One example of such myostatin-selective antibody istrevogrumab, also known as REGN1033. In some embodiments, the myostatin-selective inhibitors are antibodies or antigen-binding fragments thereof that bind the pro / latent myostatin complex, thereby inhibiting activation (e.g., release) of mature myostatin. Such antibodies or antigen-binding fragments are referred to as “activation inhibitors” of myostatin. Nonlimiting examples of myostatin-selective activation inhibitors include apitegromab (sequences shown in Tables 2 and 3 below, also known as SRK-015; described in WO 2017 / 049011 , the content of which is incorporated by reference) and variants thereof, SRK-439 and variants thereof (such as those described in WO 2024 / 138076, the content of which is incorporated by reference), GYM329 (RO7204239) and variants thereof, and a MST1032 variant as described in PCT / JP2015 / 006323, the content of which is incorporated by reference. In some embodiments, SRK-439 comprises a heavy chain variable region sequence comprising SEQ ID NO: 402 and a light chain variable region sequence comprising SEQ ID NO: 412. In some embodiments, SRK-439 comprises a heavy chain sequence of SEQ ID NO: 503 and a light chain sequence of 504.

[0288] Table 2.

[0289]

[0290] Table 3.

[0291]

[0292] Attorney Docket No. 15094.0067-00304

[0293]

[0294]

[0226] In some embodiments, the anti-pro / latent myostatin antibody or antigen-binding portion thereof comprises the heavy chain variable region, light chain variable region, and / or full chain sequences from Table 3, wherein the N-terminal glutamine of the heavy chain variable region sequence is a pyroglutamateAttorney Docket No. 15094.0067-00304

[0295] and / or the N-terminal glutamine of the light chain variable region sequence is a pyroglutamate. In some embodiments, the term “SRK-015” or “apitegromab” encompasses antibody sequences where the N-terminal glutamine of the heavy chain variable region sequence is a pyroglutamate and / or the N-terminal glutamine of the light chain variable region sequence is a pyroglutamate.

[0296]

[0227] In some embodiments, the myostatin pathway inhibitor is a non-selective myostatin inhibitor, e.g., 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-ActRllb antibody (e.g., bimagrumab); a neutralizing antibody that binds mature myostatin (e.g., stamulumab (MYO-029), domagrozumab (PF-06252616), landogrozumab (LY2495655), AMG-745 / PINTA-745 (Myostatin peptibody), RG6206 (an antimyostatin adnectin, which is a single-strand fusion protein containing domains of fibronectin), BMS-986089 (an anti-myostatin Adnectin® also known as taldefgrobep alfa). In some embodiments, the myostatin pathway inhibitor is a receptor antagonist (e.g., Alk4 / 5 inhibitors).

[0297]

[0228] In various embodiments, the myostatin pathway inhibitor comprises stamulumab, trevogrumab, LY2495655, AMG 745, bimagrumab, BIIB-110, domagrozumab (PF-06252616), apitegromab, GYM329, taldefgrobep alfa (also known as BMS-986089), or efmitermant 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 International Publication No. WO2016 / 098357, herein incorporated by reference. In some embodiments, the myostatin pathway inhibitor comprises a MST1032 variant as disclosed in International Patent Application No. PCT / JP2015 / 006323, the content of which is hereby incorporated in its entirety. In some embodiments, the myostatin pathway inhibitor comprises an antibody or antigen-binding fragment comprising additional sequences found in, e.g., in International Patent Publication Nos. WO 2018 / 129395; WO 2017 / 218592; WO 2017 / 120523; WO 2017 / 049011 ; WO 2016 / 073853; WO 2014 / 182676, or WO 2024 / 138076, the contents of which are hereby incorporated by reference in their entirety.

[0298]

[0229] A “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 sequences of MS1032LQ01-SG1 , MS1032L006-SG1 , MS1032LO11-SG1 , MS1032LO18-SG1 , MS1032LO19-SG1 , MS1032LO21-SG1 , MS1032LO25-SG1 , and sequences provided in Table 2a, Table 11a, Table 11 b, or Table 13 of PCT / JP2015 / 006323. In certain embodiments, a MST1032 variant comprises an antibody or antigen-binding fragment comprising a heavy chain variable domain comprising three CDR sequences of HCDR1 , HCDR2, and HCDR3, and a light chain variable domain comprising three CDR sequences of LCDR1 , LCDR2, and LCDR3, wherein the heavy chain CDRs comprise the amino acid sequences of: X1X2DIS (HCDR1); IISYAGSTYYASWAKG (HCDR2; SEQ ID NO: 18); GVPAYSX3GGDL (HCDR3; SEQ ID NO: 19), respectively; and the light chain CDRs comprise 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 -X9 is any amino acid residue. In certain embodiments, 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; X9 is L or R. In certain embodiments, a MST1032 variant comprises the six CDR sequences of SEQ ID NOs: 18, 66, 67, 68-70; SEQ ID Nos: 119, 18, 120, 68, 121 , 122; or SEQ IDAttorney Docket No. 15094.0067-00304

[0299] Nos: 123, 18, 120, 124, 121 , 122. In certain embodiments, a MST1032 variant comprises a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 125-128. In certain embodiments, a MST1032 variant comprises a light chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 129, 126, 127 and 130. In certain embodiments, a MST1032 variant comprises a set of sixCDRs (e.g., from the same MST1032 variant antibody) or a paired VH / VL (e.g., from the same MST1032 variant antibody) from those listed in the tables below. In certain embodiments, an antibody or antigen-binding fragment of the disclosure comprises one or more of the sequences from T ables 4-9.

[0300] Table 4.

[0301]

[0302] Table 5.

[0303]

[0304] Table 6.

[0305]

[0306] Table 7.

[0307]

[0308] Attorney Docket No. 15094.0067-00304

[0309] Table 8.

[0310]

[0311] Table 9.

[0312]

[0313]

[0230] In some embodiments, a myostatin antibody or antigen binding fragment thereof may include or may be engineered to include a mutation or modification that causes an extended half-life of the antibody. In some embodiments, such mutations or modifications may be within the Fc domain of the antibodies (e.g., Fc-modified antibodies), e.g., to promote circulating half-life or other PK properties. In some embodiments, the mutation is the 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 herein incorporated by reference).

[0314]

[0231] In some embodiments, an anti-pro / latent myostatin antibody, or antigen binding fragment thereof, binds specifically to latent myostatin. In some embodiments, an anti-pro / latent myostatin antibody, or antigen binding fragment thereof, binds specifically to both latent myostatin and pro-myostatin. In preferred embodiments, the anti-pro / latent myostatin antibody, or antigen binding fragment thereof, that binds specifically to pro-myostatin and / or latent myostatin does not bind mature myostatin. In preferred embodiments, the anti-pro / latent myostatin antibody, or antigen binding fragment thereof, that binds specifically to pro-myostatin and / or latent myostatin does not bind pro / latent GDF11 or mature GDF11 .Attorney Docket No. 15094.0067-00304

[0315] Anti-Pro / Latent Myostatin Antibodies and Antigen-Binding Fragments Thereof

[0316]

[0232] In some embodiments, antibodies, or antigen binding fragments thereof, described herein are capable of binding to a pro / latent-myostatin, thereby inhibiting the proteolytic activation of pro / latent-myostatin into mature myostatin. In some instances, antibodies, or antigen binding fragments thereof, described herein can inhibit the proteolytic activation of pro / latent-myostatin by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher. In some instances, antibodies described herein can inhibit the proteolytic cleavage of pro-myostatin by a proprotein convertase (e.g., furin) by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher. In some instances, antibodies, or antigen binding fragments thereof, described herein can inhibit the proteolytic cleavage of pro-myostatin or latent myostatin by a tolloid protease (e.g., mTLL2) by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher.

[0317]

[0233] In some embodiments, antibodies, or antigen binding fragments thereof, described herein are capable of binding to a pro / latent-myostatin, thereby inhibiting myostatin activity. In some instances, the antibodies, or antigen binding fragments thereof, described herein can inhibit myostatin signaling by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher. 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 WO 2016 / 073853, the entire contents of which are expressly incorporated herein by reference. However, it should be appreciated that additional methods may be used for measuring myostatin signaling activity.

[0318]

[0234] It should be appreciated that the extent of proteolytic cleavage of myostatin, e.g., by a proprotein convertase and / or a tolloid protease, can be measured and / or quantified using any suitable method. In some embodiments, the extent of proteolytic cleavage of myostatin is measured and / or quantified using an enzyme-linked immunosorbent assay (ELISA). For example, an ELISA may be used to measure the level of released growth factor (e.g., mature myostatin). As another example, an antibody, or antigen binding fragment thereof, that specifically binds to pro- myostatin, latent myostatin and / or mature myostatin can be used in an ELISA to measure the level of a specific form of myostatin (e.g., pro / latent / mature-myostatin), to quantify the extent of proteolytic cleavage of myostatin. In some embodiments, the extent of proteolytic cleavage of myostatin is measured and / or quantified using immunoprecipitation followed by SDS-PAGE or mass spectrometry of tryptic peptides, fluorescence anisotropy-based techniques, FRET assays, hydrogen-deuterium-exchange mass spectrometry, and / or NMR spectroscopy.

[0319]

[0235] Anti-pro / latent-myostatin antibodies, or antigen binding fragments thereof, suitable for use in the methods of the present invention include those described in International Patent Application Nos. PCT / US15 / 59468, PCT / US16 / 52014, and PCT / US2023 / 085574. The entire contents of each of the foregoing applications are incorporated herein by reference in their entireties.

[0320]

[0236] According to the present disclosure, an antibody or antigen-binding fragment that binds human pro / latent myostatin may be used to carry out various embodiments disclosed herein. In some embodiments, such antibody and antigen-binding fragment binds an epitope within the prodomain, wherein the epitope comprises one or more amino acid residues F147, Q149, L151 , Y186, S168, K170, K205,Attorney Docket No. 15094.0067-00304

[0321] and / or L207, as numbered according to SEQ ID NO: 52 (Dagbay et al. J Biol Chem. 2020 Apr 17;295(16):5404-5418).

[0322]

[0237] In some embodiments, anti-pro / latent-myostatin antibodies, or antigen binding fragments thereof, of the present disclosure and the nucleic acid molecules of the present disclosure that encode the antibodies, or antigen binding fragments thereof, include the CDR amino acid sequences shown in Tables 10 to 12.

[0323] Table 10.

[0324]

[0325]

[0238] In Table 10, the single sequences of CDRH3 and CDRL3 reflect Kabat and IMGT.

[0326] Table 11.

[0327]

[0328] Attorney Docket No. 15094.0067-00304

[0329]

[0330] Attorney Docket No. 15094.0067-00304

[0331]

[0332] Attorney Docket No. 15094.0067-00304

[0333]

[0334] Table 12.

[0335]

[0336] Attorney Docket No. 15094.0067-00304

[0337]

[0338] Attorney Docket No. 15094.0067-00304

[0339]

[0340] Attorney Docket No. 15094.0067-00304

[0341]

[0342] Attorney Docket No. 15094.0067-00304

[0343]

[0344]

[0239] In some embodiments, anti-pro / latent-myostatin antibody, or antigen-binding portion thereof, of the disclosure include any antibody, or antigen binding fragment thereof, that includes a CDRH1 , CDRH2, CDRH3, CDRL1 , CDRL2, or CDRL3, or combinations thereof, as provided for any one of the antibodies shown in Tables 10 to 12. In some embodiments, anti-pro / latent-myostatin antibodies, or antigen-binding portions thereof, comprise the CDRH1 , CDRH2, CDRH3, CDRL1 , CDRL2, and CDRL3 of any one of the antibodies shown in Tables 10 to 12. The disclosure also includes any nucleic acid sequence that encodes a molecule comprising a CDRH1 , CDRH2, CDRH3, CDRL1 , CDRL2, or CDRL3 as provided for any one of the antibodies shown in Tables 10 to 12. Antibody heavy and light chain CDR3 domains may play a particularly important role in the binding specificity / affin ity of an antibody for an antigen. Accordingly, the anti-pro / latent myostatin antibodies, or antigen-binding portions thereof, of the disclosure, or the nucleic acid molecules thereof, may include at least the heavy and / or light chain CDR3s of antibodies as shown in Tables 10 to 12.

[0345]

[0240] Aspects of the disclosure relate to a monoclonal antibody, or antigen binding fragment, that binds to pro / latent-myostatin protein and that comprises six complementarity determining regions (CDRs): CDRH1 , CDRH2, CDRH3, CDRL1 , CDRL2, and CDRL3.

[0346]

[0241] 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.

[0347]

[0242] In some embodiments (e.g., as for anti-pro / latent-myostatin antibody Ab1 , shown in Tables 10 and 11 , or an antigen-binding portion thereof), CDRH1 comprises a sequence as set forth in SEQ ID NO: 1 or 2, CDRH2 comprises a sequence as set forth in SEQ ID NO: 4 or 5, CDRH3 comprises a sequence as set forth in SEQ ID NO: 10, CDRL1 comprises a sequence as set forth in SEQ ID NO: 12, or 13, CDRL2 comprises a sequence as set forth in SEQ ID NO: 60 or 61 , and CDRL3 comprises a sequence as set forth in SEQ ID NO: 65, and the antibody, or an antigen-binding portion thereof, binds to pro / latent-myostatin.

[0348]

[0243] In some embodiments (e.g., as for anti-pro / latent-myostatin antibody Ab2, shown in Tables 10 and 11 , or an antigen-binding portion thereof), CDRH1 comprises a sequence as set forth in SEQ ID NO: 1 or 2, CDRH2 comprises a sequence as set forth in SEQ ID NO: 4 or 5, CDRH3 comprises a sequence as set forth in SEQ ID NO: 66, CDRL1 comprises a sequence as set forth in SEQ ID NO: 12, or 13, CDRL2Attorney Docket No. 15094.0067-00304

[0349] comprises a sequence as set forth in SEQ ID NO: 60 or 61 , and CDRL3 comprises a sequence as set forth in SEQ ID NO: 67, and the antibody, or an antigen-binding portion thereof, binds to pro / latent-myostatin.

[0350]

[0244] In some embodiments (e.g., as for anti-pro / latent-myostatin antibody Ab3, shown in Tables 10 and 11 , or an antigen-binding portion thereof), CDRH1 comprises a sequence as set forth in SEQ ID NO: 1 or 3, CDRH2 comprises a sequence as set forth in SEQ ID NO: 6 or 7, CDRH3 comprises a sequence as set forth in SEQ ID NO: 11 , CDRL1 comprises a sequence as set forth in SEQ ID NO: 14, or 15, CDRL2 comprises a sequence as set forth in SEQ ID NO: 131 or 64, and CDRL3 comprises a sequence as set forth in SEQ ID NO: 23, and the antibody, or an antigen-binding portion thereof, binds to pro / latent-myostatin.

[0351]

[0245] In some embodiments (e.g., as for anti-pro / latent-myostatin antibody Ab5, shown in Tables 10 and I I , or an antigen-binding portion thereof), CDRH1 comprises a sequence as set forth in SEQ ID NO: 1 or 3, CDRH2 comprises a sequence as set forth in SEQ ID NO: 8 or 9, CDRH3 comprises a sequence as set forth in SEQ ID NO: 11 , CDRL1 comprises a sequence as set forth in SEQ ID NO: 16, or 59, CDRL2 comprises a sequence as set forth in SEQ ID NO: 131 or 64, and CDRL3 comprises a sequence as set forth in SEQ ID NO: 23, and the antibody, or an antigen-binding portion thereof, binds to pro / latent-myostatin.

[0352]

[0246] In some examples, any of the anti-pro / latent-myostatin antibodies, or antigen-binding portions thereof, of the 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 antibodies may include one or more CDR sequences as shown in Tables 10 to 12 (SEQ ID NOs: 1-16, 18, 23, 59-61 , 64-72, 76, 77, 81 , 82, 86, 87, 91 , 92, 96, 97, 101 , 102, 106, 107, I I I , 112, and 119-124) containing up to 5, 4, 3, 2, or 1 amino acid residue variations as compared to the corresponding CDR region in any one of the SEQ ID NOs: shown in Tables 10 to 12 (1-16, 18, 23, 59-61 , 64-72, 76, 77, 81 , 82, 86, 87, 91 , 92, 96, 97, 101 , 102, 106, 107, 111 , 112, and 119-124).

[0353]

[0247] In some embodiments, an anti-pro / latent myostatin antibody or antigen-binding portion thereof described herein comprises three heavy chain complementarity determining regions (HCDRs) comprising 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 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.

[0354]

[0248] In some embodiments, an anti-pro / latent myostatin antibody or antigen-binding portion thereof described herein comprises three heavy chain complementarity determining regions (HCDRs) comprising 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 amino acid sequences of SEQ ID NO: 13 (LCDR1), SDN (LCDR2), and SEQ ID NO: 65 (LCDR3), as defined by the IMGT numbering system.Attorney Docket No. 15094.0067-00304

[0355]

[0249] In various embodiments, the anti-pro / latent myostatin antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 25 or an amino acid sequence that is at least 98% or 99% identical thereto, and / or a light chain variable region comprising an amino acid sequence of SEQ ID NO: 31 or an amino acid sequence that is at least 98% or 99% identical thereto. In some embodiments, the anti-pro / latent myostatin antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 25 and / or a light chain variable region comprising an amino acid sequence of SEQ ID NO: 31. In some embodiments, the anti-pro / latent myostatin antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 25 and / or a light chain variable region comprising an amino acid sequence of SEQ ID NO: 31 , wherein the N-terminal glutamine of the amino acid sequence of SEQ ID NO: 25 is a pyroglutamate and / or the N-terminal glutamine of the amino acid sequence of SEQ ID NO: 31 is a pyroglutamate.

[0356]

[0250] In various embodiments, the anti-pro / latent myostatin antibody or antigen-binding portion thereof comprises a heavy chain region comprising an amino acid sequence of SEQ ID NO: 50 or an amino acid sequence that is at least 99% identical thereto, and / or a light chain region comprising an amino acid sequence of SEQ ID NO: 51 or an amino acid sequence that is at least 99% identical thereto. In various embodiments, the anti-pro / latent myostatin antibody or antigen-binding portion thereof comprises a heavy chain region comprising an amino acid sequence of SEQ ID NO: 50, and / or a light chain region comprising an amino acid sequence of SEQ ID NO: 51.

[0357]

[0251] In some embodiments, anti-pro / latent-myostatin antibodies, or antigen-binding portions thereof, of the 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 disclosure include any antibody that includes the heavy chain variable and light chain variable pairs 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).

[0358]

[0252] In some embodiments, anti-pro / latent-myostatin antibodies, or antigen-binding portions thereof, which can be used to carry out the combination therapy according to the present disclosure are selected from the antibodies disclosed in WO 2024 / 138076. These include the following (adopted from WO 2024 / 138076, Table 3):

[0359] Table 13.

[0360]

[0361] Attorney Docket No. 15094.0067-00304

[0362]

[0363] Attorney Docket No. 15094.0067-00304

[0364]

[0365] Attorney Docket No. 15094.0067-00304

[0366]

[0367] Attorney Docket No. 15094.0067-00304

[0368]

[0369] Table 14.

[0370]

[0371] Attorney Docket No. 15094.0067-00304

[0372]

[0373]

[0253] Aspects of the disclosure provide anti-pro / latent-myostatin antibodies, or antigen-binding portions thereof, having a heavy chain variable and / or a light chain variable amino acid sequence homologous to any of those described herein. In some embodiments, the anti-pro / latent-myostatin antibody, or antigenbinding portions thereof, comprises a heavy chain variable sequence or a light chain variable sequence that is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the heavy chain variable sequence of any of SEQ ID NOs: 24-29, 73, 78, 83, 88, 93, 98, 103, 108, 113, 125-128 or a light chain variable sequence 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, the homologous heavy chain variable and / or a light chain variable amino acid sequences do not vary within any of the CDR sequences provided herein. For example, in some embodiments, the degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) may occur within a heavy chain variable and / or a light chain variable sequence excluding any of the CDR sequences provided herein.

[0374]

[0254] 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 can be performed with the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between 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.

[0375]

[0255] In some embodiments, conservative mutations can be introduced into the CDRs or framework sequences at positions where the residues are not likely to be involved in interacting with pro / latent- myostatin as determined based on the crystal structure. As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst 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.

[0376]

[0256] In some embodiments, the antibodies, or antigen binding fragments thereof, provided herein comprise mutations that confer desirable properties to the antibodies, or antigen binding fragments thereof. For example, to avoid potential complications due to Fab-arm exchange, which is known to occur withAttorney Docket No. 15094.0067-00304

[0377] native lgG4 mAbs, the antibodies, or antigen binding fragments thereof, provided herein may comprise a stabilizing ‘Adair’ mutation (Angal S., et al., “A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (lgG4) antibody,” Mol Immunol 30, 105-108; 1993), where serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline resulting in an lgG1 -like (CPPCP (SEQ ID NO: 58)) hinge sequence. Accordingly, any of the antibodies may include a stabilizing ‘Adair’ mutation or the amino acid sequence CPPCP (SEQ ID NO: 58).

[0378]

[0257] Anti-pro / latent-myostatin antibodies, or antigen-binding portions thereof, of this disclosure may optionally comprise antibody constant regions or parts thereof. For example, a VL domain may be attached at its C-terminal end to a light chain constant domain like CK or CA. Similarly, a VH domain or portion thereof may be attached to all or part of a heavy chain like IgA, IgD, IgE, IgG, and IgM, and any isotype subclass. Antibodies may include suitable constant regions (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Therefore, antibodies within the scope of this may disclosure include VH and VL domains, or an antigen binding portion thereof, combined with any suitable constant regions.

[0379]

[0258] In certain embodiments, the VH and / or VL domains may be reverted to germline sequence, e.g., the FR of these domains are mutated using conventional molecular biology techniques to match those produced by the germline cells. For example, the VH and / or VL domains may be reverted to germline sequence of lgHV3-30 (SEQ ID NO: 36) and / or lgLV1-44 (SEQ ID NO: 37), respectively. It should be appreciated that any of the VH and / or VL domains may be reverted to any suitable germline sequence. In other embodiments, the FR sequences remain diverged from the consensus germline sequences.

[0380]

[0259] lgHV3-30

[0381]

[0260] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 36)

[0382]

[0261] lgLV1-44

[0383]

[0262] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSG SKSGTSASLAISGLQSEDEADYYCAAWDDSLNG (SEQ ID NO: 37)

[0384]

[0263] In some embodiments, anti-pro / latent-myostatin antibodies or antigen binding fragments may or may not include the framework region of the antibodies shown in SEQ ID NOs: 24-35. In some embodiments, anti-pro-latent-myostatin antibodies are murine antibodies and include murine framework region sequences.

[0385]

[0264] In some embodiments, an anti-pro / latent-myostatin antibodies, or antigen binding fragments thereof, can bind to pro / latent-myostatin with relatively high affinity, e.g., with a KD less than 10-8M, 10-9M, 1O-10M, 10-11M or lower. For example, anti-pro / latent-myostatin antibodies, or antigen binding fragments thereof, can bind to pro / latent-myostatin with an affinity between 5 pM and 500 nM, e.g., between 50 pM and 100 nM, e.g., between 500 pM and 50 nM. The invention also includes antibodies or antigen binding fragmentsAttorney Docket No. 15094.0067-00304

[0386] that compete with any of the antibodies described herein for binding to pro / l ate nt- myostatin and that have an affinity of 50 nM or lower (e.g., 20 nM or lower, 10 nM or lower, 500 pM or lower, 50 pM or lower, or 5 pM or lower). The affinity and binding kinetics of the anti-pro / latent-myostatin antibody can be tested using any suitable method including but not limited to biosensor technology (e.g., OCTET or BIACORE). When such binding profiles are measured with the use of OCTET or BIACORE, the assay is performed typically in accordance with the manufacturer’s instructions, unless otherwise specified.

[0387]

[0265] In some embodiments, antibodies, or antigen binding fragments thereof, are disclosed herein that specifically bind pro / latent-myostatin. In some embodiments, any of the antibodies, or antigen binding fragments thereof, provided herein bind at or near a tolloid cleavage site or at or near a tolloid docking site of pro / latent-myostatin. In some embodiments, an antibody binds near a tolloid cleavage site or near a tolloid docking site if it binds within 15 or fewer amino acid residues of the tolloid cleavage site or tolloid docking site. In some embodiments, any of the antibodies, or antigen binding fragments thereof, provided herein bind within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acid residues of a tolloid cleavage site or tolloid docking site. In some embodiments, an antibody binds at or near a tolloid cleavage site of GDF8. For example, an antibody may bind an amino acid sequence as set forth in SEQ ID NO: 62 PKAPPLRELIDQYDVQRDDSSDGSLEDDDYHAT (SEQ ID NO: 62). In other embodiments, any of the antibodies, or antigen binding fragments thereof, provided herein bind at or near a proprotein convertase cleavage site or at or near a proprotein convertase docking site of pro / latent-myostatin. In some embodiments, an antibody binds near a proprotein convertase cleavage site or near a proprotein convertase docking site if it binds within 15 or fewer amino acid residues of the proprotein convertase cleavage site or proprotein convertase docking site. In some embodiments, any of the antibodies, or antigen binding fragments 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 a proprotein convertase cleavage site or proprotein convertase docking site. In some embodiments, an antibody binds at or near a proprotein convertase cleavage site of GDF8. For example, an antibody may bind an amino acid sequence as set forth in SEQ ID NO: 63 (GLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRC).

[0388]

[0266] In some embodiments, the myostatin-selective inhibitor according to the present disclosure is an antibody or antigen-binding fragment thereof, which is optionally a myostatin-selective inhibitor, that binds an epitope that includes 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, wherein optionally the variant is an Fc variant. In some embodiments, the antibody is not apitegromab or its variant.

[0389]

[0267] In some embodiments, the myostatin-selective inhibitor according to the present disclosure is an antibody or antigen-binding fragment thereof, which is optionally a myostatin-selective inhibitor, that binds 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 proGDF8 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 antibody binds an epitope that includes 10 or more, 9 or more, 8 or more,Attorney Docket No. 15094.0067-00304

[0390] 7 or more, 6 or more, 5 or more, 4 or more, or 3 or more of the amino acid residues shown above. In some embodiments, the antibody is apitegromab or a variant thereof, wherein optionally the variant is an Fc variant. In some embodiments, the antibody is not apitegromab or its variant.

[0391]

[0268] In one example, the anti-pro / latent-myostatin antibodies, or antigen binding fragments thereof, described herein specifically bind pro / latent-myostatin as compared to other forms of Myostatin and / or other members of the TGFp family of growth factors. Members of the TGFp family of growth factors include, 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, TGFpl , TGFp2, and TGFp3 protein. Such antibodies, or antigen binding fragments thereof, may bind pro / latent-myostatin at a much higher affinity as compared to other members of the TGFp family of growth factors (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1 ,000-fold higher). In some embodiments, such antibodies, or antigen binding fragments thereof, may bind pro / latent-myostatin with an affinity of at least 1000-fold higher as compared to other members of the TGFp family of growth factors. In some embodiments, antibodies, or antigen binding fragments thereof, provided herein may bind to pro / latent-myostatin at a much higher affinity as compared to one or more forms of GDF11 or mature myostatin (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1 ,000-fold higher). In some embodiments, antibodies, or antigen binding fragments thereof, provided herein may bind to pro / latent-myostatin with an affinity of at least 1 ,000-fold higher as compared to one or more forms of GDF11 (e.g., proGDF11 , latent GDF11 or mature GDF11) or mature myostatin. Alternatively, or in addition, antibodies, or antigen binding fragments thereof, may exhibit a much higher inhibitory activity against proteolytic cleavage of pro / latent-myostatin (e.g., by a proprotein convertase or tolloid protease) as compared with other members of the TGFp family, such as pro / latent GDF11 (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1 ,000-fold higher). In another embodiment, the antibodies, or antigen binding fragments thereof, disclosed herein do not bind to GDF11. This avoids potential toxicity issues associated with antibodies that cross-react with both myostatin and GDF11 . An example of one such potential toxicity relates to impaired 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 content of which is hereby incorporated in its entirety.

[0392]

[0269] In some embodiments, antibodies bind an antigen but cannot effectively eliminate the antigen from the plasma. Thus, in some embodiments, the concentration of the antigen in the plasma may be increased by reducing the clearance of the antigen. However, in some embodiments, antibodies (e.g., sweeping antibodies) provided herein have an affinity to an antigen that is sensitive to pH. Such pH sensitive antibodies may bind to the antigen in plasma at neutral pH and dissociate from the antigen in an acidic endosome, thus reducing antibody-mediated antigen accumulation and / or promoting antigen clearance from the plasma.

[0393]

[0270] Aspects of the disclosure relate to sweeping antibodies. As used herein “sweeping antibodies” or antigen-binding fragments thereof refer to antibodies, or antigen-binding fragments thereof, having both pH-sensitive antigen binding and at least a threshold level of binding to cell surface neonatal Fc receptorAttorney Docket No. 15094.0067-00304

[0394] (FcRn) at neutral or physiological pH. In some embodiments, sweeping antibodies, or an antigen-binding portion thereof, bind to the neonatal Fc receptor FcRn at neutral pH. For example, sweeping antibodies may bind to the FcRn at a pH ranging from 7.0 to 7.6. In some embodiments, sweeping antibodies, or an antigen-binding portion thereof, can bind to an antigen at an antigen binding site and bind to a cellular FcRn via an Fc portion of the antibody. In some embodiments, sweeping antibodies, or an antigen-binding portion thereof, may then be internalized, releasing antigen in an acidic endosome, which may be degraded. In some embodiments, a sweeping antibody, or an antigen-binding portion thereof, no longer bound to the antigen, may then be released (e.g., by exocytosis) by the cell back into the serum.

[0395]

[0271] In some embodiments, FcRn in the vascular endothelia (e.g., of a subject) extends the half-life of a sweeping antibody, or an antigen-binding portion thereof. In some embodiments, vascular endothelial cells internalize sweeping antibodies, or antigen-binding portions thereof, which in some embodiments are bound to an antigen such as myostatin (e.g., pro-myostatin, latent myostatin or primed myostatin). In some embodiments, a sweeping antibody, or an antigen-binding portion thereof, is recycled back into the bloodstream. In some embodiments, a sweeping antibody, or an antigen-binding portion thereof, has an increased half-life (e.g., in the serum of a subject) as compared to its conventional counterpart. In some embodiments, a conventional counterpart of a sweeping antibody refers the antibody, or an antigen-binding portion thereof, from which the sweeping antibody, or an antigen-binding portion thereof, was derived (e.g., prior to engineering the Fc portion of the conventional antibody to bind FcRn with greater affinity at pH 7). In some embodiments, a sweeping antibody, or an antigen-binding portion thereof, has a half-life in the serum of a subject that is at least 1%, 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 100%, 150%, 200% or 250% longer as compared to its conventional counterpart.

[0396]

[0272] In some embodiments, an Fc portion of a sweeping antibody binds FcRn. In some embodiments, the Fc portion of a sweeping antibody binds to FcRn at a pH of 7.4 with a KD ranging from 10-3M to 10-8M. In some embodiments, a sweeping antibody binds to FcRn at a pH of 7.4 with a KD ranging from 10-3M to 10'7M, from 10-3M to 10-6M, from 10-3M to 10-5M, from 10-3M to 10-4M, from 10-4M to 10-8M, from 10-4M to 10-7M, from 104M to 10-6M, from 104M to 10-5M, from 105M to 10-8M, from 105M to 10-7M, from 10-5M to 10-6M, from 10-6M to 10-8M, from 10-6M to 10-7M, or from 10-7M to 10-8M. In some embodiments, FcRn binds to the CH2-CH3 hinge region of a 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 from FcyRs. In some embodiments, the amino acid residues AA of a sweeping antibody Fc region are required for binding to FcRn. In some embodiments, the amino acid residues AA of a sweeping antibody Fc region affect binding to FcRn.

[0397]

[0273] In some embodiments, any of the antibodies, or antigen binding fragments thereof, provided herein are engineered to bind FcRn with greater affinity. In some embodiments, any of the antibodies, or antigen binding fragments thereof, provided herein are engineered to bind FcRn with greater affinity at pH 7.4. In some embodiments, the affinity of antibodies, or antigen binding fragments thereof, to FcRn is increased to extend their pharmacokinetic (PK) properties as compared to their conventional counterparts. For example, in some embodiments, sweeping antibodies elicit less adverse reactions due to their efficacy at lower doses. In some embodiments, sweeping antibodies, or an antigen-binding portion thereof, areAttorney Docket No. 15094.0067-00304

[0398] administered less frequently. In some embodiments, transcytosis of sweeping antibodies, or an antigenbinding portion thereof, to certain tissue types are increased. In some embodiments, sweeping antibodies, or antigen-binding portions thereof, enhance efficiency of trans-placental delivery. In some embodiments, sweeping antibodies, or antigen-binding portions thereof, are less costly to produce.

[0399]

[0274] In some embodiments, any of the antibodies, or antigen binding fragments thereof, provided herein are engineered to bind FcRn with lower affinity. In some embodiments, any of the antibodies, or antigen binding fragments thereof, provided herein are engineered to bind FcRn with lower affinity at pH 7.4. In some embodiments, the affinity of sweeping antibodies, or an antigen-binding portion thereof, to FcRn is decreased to shorten their pharmacokinetic (PK) properties as compared to their conventional counterparts. For example, in some embodiments, sweeping antibodies, or an antigen-binding portion thereof, are more rapidly cleared for imaging and / or radioimmunotherapy. In some embodiments, sweeping antibodies, or an antigen-binding portion thereof, promote clearance of endogenous pathogenic antibodies as a treatment for autoimmune diseases. In some embodiments, sweeping antibodies, or antigen-binding portions thereof, reduce the risk of adverse pregnancy outcome, which may be caused by trans-placental transport of material fetus-specific antibodies.

[0400]

[0275] In some embodiments, sweeping antibodies, or an antigen-binding portion thereof, have decreased affinity to an antigen at low pH as compared to a neutral or physiological pH (e.g., pH 7.4). In some embodiments, sweeping antibodies, or an antigen-binding portion thereof, have a decreased affinity to an antigen at an acidic pH (e.g., a pH ranging from 5.5 to 6.5) as compared to a physiological pH (e.g., pH 7.4).

[0401]

[0276] It should be appreciated that any of the antibodies, or antigen binding fragments thereof, provided herein can be engineered to dissociate from the antigen depending on changes in pH (e.g., pH-sensitive antibodies). In some embodiments, sweeping antibodies, or an antigen-binding portion thereof, provided herein are engineered to bind antigen in a pH-dependent manner. In some embodiments, sweeping antibodies, or an antigen-binding portion thereof, provided herein are engineered to bind FcRn in a pH-dependent manner. In some embodiments, sweeping antibodies, or an antigen-binding portion thereof, provided herein are internalized by endocytosis. In some embodiments, sweeping antibodies, or an antigen-binding portion thereof, provided here are internalized by FcRn binding. In some embodiments, endocytosed sweeping antibodies, or antigen-binding portion thereof, release antigen in an endosome. In some embodiments, sweeping antibodies, or antigen-binding portions thereof, are recycled back to the cell surface. In some embodiments, sweeping antibodies remain attached to cells. In some embodiments, endocytosed sweeping antibodies, or an antigen-binding portion thereof, are recycled back to the plasma. It should be appreciated that the Fc portion of any of the antibodies, or antigen binding fragments thereof, provided herein may be engineered to have different FcRn binding activity. In some embodiments, FcRn binding activity affects the clearance time of an antigen by a sweeping antibody. In some embodiments, sweeping antibodies may be long-acting or rapid-acting sweeping antibodies.

[0402]

[0277] In some embodiments, converting a conventional therapeutic antibody, or an antigen-binding portion thereof, into a sweeping antibody, or an antigen-binding portion thereof, reduces the efficacious dose. InAttorney Docket No. 15094.0067-00304

[0403] some embodiments, converting a conventional therapeutic antibody, or an antigen-binding portion thereof, into a sweeping antibody, or an antigen-binding portion thereof, reduces the efficacious 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 an antigen-binding portion thereof, into a sweeping antibody, or an antigen-binding portion thereof, reduces the efficacious 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.

[0404]

[0278] In some embodiments, selecting an appropriate dose of a sweeping antibody, or an antigen-binding portion thereof, for therapy may be performed empirically. In some embodiments, a high dose of a sweeping antibody, or an antigen-binding portion thereof, may saturate FcRn, resulting in antibodies which stabilize antigen in serum without being internalized. In some embodiments, a low dose of a sweeping antibody, or an antigen-binding portion thereof, may not be therapeutically effective. In some embodiments, sweeping antibodies, or antigen-binding portions thereof, are administered once a day, once a week, once every two weeks, once every three weeks, once every four weeks, once every 6 weeks, once every 8 weeks, once every 10 weeks, once every 12 weeks, once every 16 weeks, once every 20 weeks, or once every 24 weeks.

[0405]

[0279] In some embodiments, any of the antibodies, or antigen binding fragments thereof, provided herein may be modified or engineered to be sweeping antibodies. In some embodiments, any of the antibodies, or antigen binding fragments thereof, provided herein may be converted into 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 hereby incorporated by reference. It should be appreciated, however, that the methods for making sweeping antibodies, or an antigen-binding portion thereof, as provided herein are not meant to be limiting. Thus, additional methods for making sweeping antibodies, or an antigen-binding portion thereof, are within the scope of this disclosure.

[0406]

[0280] Some aspects of the disclosure are based on the recognition that the affinity (e.g., as expressed as KD) of any of the anti-pro / latent-myostatin antibodies, or antigen binding fragments thereof, provided herein are sensitive to changes in pH. In some embodiments, the antibodies, or antigen binding fragments thereof, provided herein have an increased KD of binding to pro / latent-myostatin at a relatively low pH (e.g., a pH ranging from 4.0-6.5) as compared to a relatively high pH (e.g., a pH ranging from 7.0-7.4). In some embodiments, the antibodies, or antigen binding fragments thereof, provided herein have a KD of binding to pro / latent-myostatin ranging from 10-3M, 10-4M, 10-5M, 10-6M, 10-7M, 10-8M when the pH is between 4.0 and 6.5. In some embodiments, the antibodies, or antigen binding fragments thereof, provided herein have a KD of binding to pro / latent-myostatin ranging from 10-6M, 10-7M, 10-8M, 10-9M, IO-10M, 10-11M when the pH is between 7.0 and 7.4. In some embodiments, the antibodies, or antigen binding fragments thereof, provided herein have a KD of binding to pro / latent-Myostatin that is at least 2-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, or at least 10000-fold greater at a pH between 4.0 and 6.5 as compared to a pH between 7.0 and 7.4.Attorney Docket No. 15094.0067-00304

[0407]

[0281] In some embodiments, pro / latent-myostatin antibodies, or antigen binding fragments thereof, are provided herein that specifically bind to the same epitope as an antibody described in Table 2a, 11a, 11 b, or 13 of International Patent Application Publication No. WO 2016 / 098357, which was published on June 23, 2016, and which is based on International Patent Application No. PCT / JP2015 / 006323, which was filed on December 18, 2015. In some embodiments, pro / latent-myostatin antibodies, or antigen binding fragments thereof, are provided herein that compete for binding with such an antibody.

[0408]

[0282] In some embodiments, 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, 11a, 11 b, or 13 of International Patent Application Publication No. WO 2016 / 098357, which was published on June 23, 2016, and which is based on International Patent Application No. PCT / JP2015 / 006323, which was filed on December 18, 2015. In some embodiments, pro / latent-myostatin antibodies, or antigen binding fragments thereof, provided herein do not compete or do not cross-compete for binding to the same epitope as an antibody described in Table 2a, 11a, 11b, or 13 of International Patent Application Publication No. WO 2016 / 098357, which was published on June 23, 2016, and which is based on International Patent Application No. PCT / JP2015 / 006323, which was filed on December 18, 2015. In some embodiments, 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 a VL pair described in Table 2a, 11a, 11 b, or 13 of International Patent Application Publication No. WO 2016 / 098357, which was published on June 23, 2016, and which is based on International Patent Application No. PCT / JP2015 / 006323, which was filed on December 18, 2015. In some embodiments, pro / latent-myostatin antibodies, or antigen binding fragments thereof, provided herein do not compete or do not cross-compete for binding to the same epitope as an antibody comprising a VH and a VL pair described in Table 2a, 11a, 11b, or 13 of International Patent Application Publication No. WO 2016 / 098357, which was published on June 23, 2016, and which is based on International Patent Application No. PCT / JP2015 / 006323, which was filed on December 18, 2015.

[0409] Antibodies, and Antigen-Binding Fragments, that Compete with Anti-pro / latent-Myostatin Antibodies, or Antigen Binding Fragments Thereof

[0410]

[0283] Aspects of the 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. The term “compete”, as used herein with regard 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 binding of the first antibody with its epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, can, but need not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of the other antibody with its epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to “cross-compete” with each other for binding of their respective epitope(s). Both competing and cross-competing antibodies are within the scope of this disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope,Attorney Docket No. 15094.0067-00304

[0411] or portion thereof), the skilled artisan would appreciate that such competing and / or cross-competing antibodies are encompassed and can be useful for the methods and / or compositions provided herein.

[0412]

[0284] Aspects of the disclosure relate to antibodies, or antigen-binding portions thereof, that compete or cross-compete with any of the antibodies, or antigen binding fragments thereof, provided herein. In some embodiments, an antibody, or an antigen-binding portion thereof, binds at or near the same epitope as any of the antibodies provided herein. In some embodiments, an antibody, or an antigen-binding portion thereof, binds near an epitope if it binds within 15 or fewer amino acid residues of the epitope. In some embodiments, any of the antibodies, or antigen binding fragments thereof, provided herein bind within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acid residues of an epitope that is bound by any of the antibodies, or antigen binding fragments thereof, provided herein. In preferred embodiments, such antibody or antigen-binding fragment cross-competes with apitegromab for binding to human pro / latent myostatin. In some embodiments, such antibody or antigen-binding fragment binds an epitope within the prodomain of human myostatin, wherein the epitope comprises one or more amino acid residues F147, Q149, L151 , Y186, S168, K170, K205, and / or L207, as numbered according to SEQ ID NO: 52 disclosed herein. In some embodiments, such antibody or antigen-binding fragment binds an epitope within the prodomain of human myostatin, wherein the epitope comprises 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 antibody or antigen-binding fragment comprises an HCDR3 paratope that is a variant of SEQ ID NO: 10, wherein the variant contains of up to two amino acid substitutions as compared to SEQ ID NO: 10. In some embodiments, such antibody or antigen-binding fragment comprises an HCDR3 sequence comprising a leucine at amino acid position 3 and a tryptophan at amino acid position 9, as numbered according to SEQ ID NO: 10. In some embodiments, such antibody or antigen-binding fragment comprises 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 according to SEQ ID NO: 10.

[0413]

[0285] In another embodiment, an antibody, or an antigen-binding portion thereof, competes or crosscompetes for binding to any of the antigens provided herein (e.g., pro / latent-myostatin) with an equilibrium dissociation constant, KD, between the antibody and the protein of less than 10-8M. In other embodiments, an antibody, or an antigen-binding portion thereof, competes or cross-competes for binding to any of the antigens provided herein with a KD in a range from 10-11M to 10-8M.

[0414]

[0286] Aspects of the disclosure relate to antibodies, or antigen-binding portions thereof, that compete for binding to pro / latent-myostatin with any of the antibodies, or antigen binding fragments thereof, provided herein. In some embodiments, the antibody, or an antigen-binding portion thereof, binds to pro / latent-myostatin at the same epitope as any of the antibodies, or antigen-binding portions thereof, provided herein. For example, in some embodiments any of the antibodies provided herein bind at or near a tolloid cleavage site or at or near a tolloid docking site of pro / latent-myostatin. In other embodiments, any of the antibodies, or antigen binding fragments thereof, provided herein bind at or near a proprotein convertase cleavage site or at or near a proprotein convertase docking site of pro / latent-myostatin. In another embodiment, an antibody, or an antigen-binding portion thereof, competes for binding to pro / latent-myostatin with anAttorney Docket No. 15094.0067-00304

[0415] equilibrium dissociation constant, KD, between the antibody, or antigen-binding portion thereof, and pro / latent-myostatin of less than 10-6M. In other embodiments, the antibody, or antigen-binding portion thereof, that competes with any of the antibodies, or antigen-binding portions thereof, provided herein binds to pro / latent-myostatin with a KD in ranging from 10-11M to 10-8M.

[0416]

[0287] Any of the antibodies, or antigen binding fragments thereof, provided herein can be characterized using any suitable methods. 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 epitopes on proteins, including solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. In an additional example, epitope mapping can be used to determine the sequence to which an antibody, or an antigen-binding portion thereof, binds. The epitope can be a linear epitope, i.e., contained in a single stretch of amino acids, ora conformational epitope formed by a three-dimensional interaction of amino acids that may not necessarily be contained in a single stretch (primary structure linear sequence). Peptides of varying lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used for binding assays with an antibody. In another example, the epitope to which the antibody, or an antigen-binding portion thereof, binds can be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody, or an antigen-binding portion thereof. According to the gene fragment expression assays, the open reading frame encoding the target antigen is fragmented either randomly or by specific genetic constructions and the reactivity of the expressed fragments of the antigen with the antibody to be tested is determined. The gene fragments may, for example, be produced by PCR and then transcribed and translated into protein in vitro, in the presence of radioactive amino acids. The binding of the antibody, or an antigen-binding portion thereof, to the radioactively labeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Certain 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 the test antibody, or an antigen-binding portion thereof, in simple binding assays. In an additional example, mutagenesis of an antigen binding domain, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and / or necessary for epitope binding. For example, domain swapping experiments can be performed using a mutant of a target antigen in which various fragments of the pro / latent-myostatin polypeptide have been replaced (swapped) with sequences from a closely related, but antigenically distinct protein, such as another member of the TGFp superfamily (e.g., GDF11). By assessing binding of the antibody, or antigen-binding portion thereof, to the mutant pro / latent-myostatin, the importance of the particular antigen fragment to antibody, or antigenbinding portion thereof, binding can be assessed.

[0417]

[0288] Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine whether an antibody, or an antigen-binding portion thereof, binds to the same epitopeAttorney Docket No. 15094.0067-00304

[0418] as the other antibodies, or antigen-binding portions thereof. Competition assays are well known to those of skill in the art.

[0419]

[0289] Any of the suitable methods, e.g., the epitope mapping methods as described herein, can be applied to determine whether an anti-pro / latent-myostatin antibody, or an antigen-binding portion thereof, binds one or more of the specific residues / segments in pro / latent-myostatin as described herein. Further, the interaction of the antibody, or an antigen-binding portion thereof, with one or more of those defined residues in pro / latent-myostatin can be determined by routine technology. For example, a crystal structure can be determined, and the distances between the residues 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, whether a specific residue in pro / latent-myostatin interacts with one or more residues in the antibody, or antigen-binding portion thereof, can be determined. Further, suitable methods, such as competition assays and target mutagenesis assays can be applied to determine the preferential binding of a candidate anti-pro / latent-myostatin antibody, or an antigen-binding portion thereof, to pro / latent-myostatin as compared to another target such as a mutant pro / latent-myostatin.

[0420]

[0290] According to the present disclosure, therapeutic effects of myostatin inhibition in patients with muscular dystrophy may include: enhanced muscle strength and motor function; improved metabolic health; prevention or lesser degree of bone loss or fractures, and / or alleviation of chronic inflammation.

[0421]

[0291] Accordingly, the present disclosure encompasses therapeutic use of myostatin inhibitors to treat chronic inflammation associated with muscular dystrophy, e.g., DMD. In some embodiments, a myostatin inhibitor is used in the treatment of chronic inflammation in a subject, wherein the treatment comprises administration of a myostatin inhibitor in an amount sufficient to alleviate the inflammation and associated dysregulation, such as muscle atrophy and bone metabolism. Chronic inflammation may include, without limitation, inflammation associated with muscle disorders (e.g., inflammatory myopathies, myositis), inflammation associated with diabetes, inflammation associated with diabetic retinopathy, inflammation associated with ocular myositis, inflammation associated with eye muscle weakness such as with myasthenia gravis, and inflammation associated with obesity. In some embodiments, inflammation associated with diabetes is diabetic nephropathy. In some embodiments, inflammation associated with diabetes is diabetic retinopathy. In preferred embodiments, the myostatin inhibitor is a myostatin-selective inhibitor (i.e., agents that selectively inhibit myostatin signaling, as opposed to agents that also inhibit additional growth factor signaling such as GDF11 and Activin A). Examples of myostatin-selective inhibitors include, without limitation, the antibodies and antigen-binding fragments of the novel myostatin inhibitors disclosed herein, apitegromab, trevogrumab, GYM329, and any variants thereof. In some embodiments, the myostatin selective inhibitor is an antibody or antigen binding fragment selective for pro / latent myostatin, e.g., any one of Ab101-141 or an antigen binding fragment thereof.

[0422]

[0292] The standard-of-care commonly prescribed to patients with muscular dystrophies (e.g., DMD and Becker’s) include anti-inflammatory drugs, such as corticosteroids. Corticosteroids are a class of steroid hormones released by the adrenal cortex, which includes glucocorticoids and mineralocorticoids, although the terms “corticosteroids” and “glucocorticoids” are commonly used interchangeably. Prolonged use of corticosteroids can lead to muscle atrophy and weakness. The mechanism underlining this effectAttorney Docket No. 15094.0067-00304

[0423] can be at least in part explained by the evidence that an excess of glucocorticoids directly stimulates myostatin production via interactions with a glucocorticoid response element in the myostatin promoter, thus inducing loss of muscle mass. According to the present disclosure, preferred myostatin inhibitors used in the treatment of muscular dystrophies in patients who are on a corticosteroid therapy (e.g., SOC background therapy) are capable of preventing glucocorticoid-induced muscle atrophy. Preferably, such myostatin inhibitors are myostatin-selective inhibitors that spare GDF11 and Activin A. Examples of myostatin-selective inhibitors that are capable of preventing glucocorticoid-induced muscle atrophy include SRK-015 (apitegromab) and the antibodies disclosed in WO 2024 / 138076 (PCT / US2023 / 085574), e.g., Abs101-141 as published.

[0424]

[0293] The lack of specificity observed in myostatin antagonists described elsewhere may pose a greater risk to dystrophic patient populations because of off-target effects. For instance, myostatin inhibitors that also bind to mature myostatin may block additional biological pathways in addition to the myostatin signaling pathway due to the high homology of the mature myostatin protein with other members of the TGFp superfamily (e.g., Activin A or GDF11). Both Activin A and GDF11 are widely expressed and are known to play diverse biological roles; therefore, interfering with the activities of these growth factors may inadvertently perturb normal biological processes. Such off-target effects may therefore potentially limit the population of patients who can safely undergo therapy due to unacceptable adverse-effects such as abnormal bleeding, wound healing, or reproductive problems caused by off-target antibody binding (Campbell, et al. Muscle Nerve (2016); David, L„ Blood 109, 1953-1961 (2007)).

[0425]

[0294] For example, Activin A is involved in both wound healing and reproductive biology, and inhibition of Activin A would therefore limit use in patients who have recently undergone surgery or injury, or in women of reproductive age. Such increased risk of adverse effects or toxicity may be particularly concerning where i) a patient population requires a long-term treatment (such as chronic conditions); and / or, ii) a patient population is or includes pediatric patients, who may be susceptible to such adverse effects and / or toxicity. Accordingly, the present disclosure provides improved myostatin inhibitors that target pro / latent myostatin specifically and with high potency, thereby providing potentially greater safety profiles. Notably, clinical observations reported in literature suggest that patients with DMD are prone to developing skin abnormalities, such as wounds and impaired skin integrity, although the exact causal link between these manifestations and the disease-causing genetic mutations in the dystrophin gene is not well understood. Skin breakdown frequently seen in DMD patients includes, but is not limited to, rashes, nonhealing surgical wounds, burns, and pressure injuries. Against this background, muscle-enhancing therapies that target Activin A may be detrimental due to the role Activin A plays in would healing. Thus, in preferred embodiments, myostatin inhibitors used in carrying out the invention do not inhibit Activin A at a dose effective to inhibit myostatin. Most preferably, the myostatin inhibitor is a myostatin-selective inhibitor. Dystrophin upregulators (correctors)

[0426]

[0295] According to the present disclosure, combination therapy for the treatment of muscular dystrophies can include an agent aimed to increase Dystrophin levels in patients. Such agents are referred to as Dystrophin correctors or upregulators. Examples of Dystrophin upregulators as used herein include splice modifying agents such as exon-skipping agents and gene therapy. Irrespective of the modalities, the aim is to increase the amount of functional Dystrophin protein in patients, which is either missing or significantlyAttorney Docket No. 15094.0067-00304

[0427] reduced in patients. The goal of exon skipping is to change the splicing pattern so that an out-of-frame, DMD-type mutation becomes an in-frame, BMD-type mutation. For example, skipping exon 51 of the dystrophin gene could restore the “reading frame” in patients who have specific out-of-frame deletions in some dystrophin exons.

[0428]

[0296] Currently, there are several FDA-approved exon skipping drugs and more are being tested in clinical trials. The approved drugs skip exons 51 , 53 and 45, which could treat up to 29% of all DMD patients. It is thought that up to 80% of DMD could potentially benefit from exon skipping if it is applied to other mutations.

[0429]

[0297] The combination therapy disclosed herein therefore can employ appropriate Dystrophin upregulators in conjunction with one or more additional therapies. In some embodiments, the Dystrophin upregulator is an RNAi-based agent. In some embodiments, the RNAi agent is a small interference RNA (siRNA). In some embodiments, the RNAi agent is a microRNA (miRNA). In some embodiments, the Dystrophin upregulator is an antisense oligonucleotide agent. In some embodiments, the antisense oligonucleotide agent is a stabilized antisense oligonucleotide agent, wherein optionally, the stabilized antisense oligonucleotide agent is a Morpholino (phosphorodiamidate morpholino oligomer (PMO)).

[0430]

[0298] In some embodiments, the dystrophin upregulator is a splice modifier, wherein optionally the splice modifier is an exon-skipping agent, wherein further optionally, the exon-skipping agent is selected from: Exon 51 -skipping agents, Exon 53-skipping agents, Exon 45-skipping agents, Exon 44-skipping agents, Exon 50-skipping agents, Exon 52-skipping agents, Exon 43-skipping agents, Exon 55-skipping agents, and Exon 8-skipping agents. In preferred embodiments, the exon-skipping agent is selected from Exon 51 -skipping agents, Exon 53-skipping agents and Exon 45-skipping agents.

[0431]

[0299] In some embodiments, the dystrophin upregulator is selected from: casimersen (e.g., Amondys 45), eteplirsen (e.g., Exondys 51), viltolarsen (e.g., Viltepso), and golodirsen (e.g., Vyondys 53) in carrying out the invention.

[0432]

[0300] Because exon skipping is available for certain mutations, it’s important to have genetic testing performed to determine if it’s right for particular patients with DMD.

[0433]

[0301] An alternative approach to increasing Dystrophin expression is through gene therapy. Thus, in some embodiments, the Dystrophin upregulator according to this disclosure is a gene therapy. In some embodiments, the gene therapy is a gene replacement therapy. In some embodiments, the gene replacement therapy aims to provide a gene that generates a truncated form of Dystrophin. In some embodiments, the gene therapy is a gene correction therapy, in which disease-associated mutation or mutations are repaired or “corrected” such that increased levels of functional Dystrophin can be produced. In some embodiments, the functional Dystrophin is a truncated form of Dystrophin protein. The gene correction therapy may be achieved by CRISPR-based techniques.

[0434] Anti-fibrotic agents

[0435]

[0302] Certain types of muscular dystrophies, DMD in particular, are associated with the manifestation of fibrosis in the affected muscle tissues, such as skeletal muscle and cardiac muscle. The role of fibrosis in the pathophysiology of muscular dystrophy is reviewed, for example, by Mogharerhabed and Czubryt (AmAttorney Docket No. 15094.0067-00304

[0436] J Physiol - cell physiol., 325(5): C1155-C1386). Thus, the present disclosure contemplates the use of an antifibrotic agent as part of combination therapy for the treatment of muscular dystrophy, such as DMD.

[0437]

[0303] Traditional approach to TGFp inhibition has been to block multiple isoforms of TGFp (i.e., TGFpl , TGFp2 and TGFp3, or at least two of the three isoforms), based at least on the assumption that it would be necessary to do so in order to achieve efficacy, given that all three isoforms signal through the same receptors. However, Applicant’s recent observations in murine fibrosis models suggest detrimental effects of TGFp3 inhibition in tissues with dysregulated ECM, raising the possibility that role of TGFp3 expands beyond homeostasis. Applicant previously found that TGFpl -selective inhibitors are capable of mitigating fibrosis in multiple preclinical models, including mouse liver fibrosis model where both the TGFpl and TGFp3 isoforms are expressed in fibrotic liver tissue, albeit in discrete cell types. Surprisingly, inhibition of TGFp3 promoted pro-fibrotic phenotypes. The exacerbation of fibrosis was observed when the TGFp3 inhibitor was used alone. Furthermore, when used in combination with a TGFpl -selective inhibitor, the TGFp3-selective inhibitor attenuated the anti-fibrotic effect of the TGFpl inhibitor, indicating that TGFp3 may counteract the anti-fibrotic effects of TGFpl and that inhibition of TGFp3 is in fact detrimental for the treatment of fibrosis. These observations point to isoform-selective inhibition of TGFpl as the preferred approach in treating fibrosis.

[0438]

[0304] In addition to the possible concerns of inhibiting TGFp3 addressed above, Takahashi et al., (Nat Metab. 2019, 1(2): 291-303) recently reported a beneficial role of TGFp2 in regulating metabolism. The authors identified TGFp2 as an exercise-induced adipokine, which stimulated glucose and fatty acid uptake in vitro, as well as tissue glucose uptake in vivo; which improved metabolism in obese mice; and, which reduced high fat diet-induced inflammation. Moreover, the authors observed that lactate, a metabolite released from muscle during exercise, stimulated TGFp2 expression in human adipocytes and that a lactate-lowering agent reduced circulating TGFp2 levels and reduced exercise-stimulated improvements in glucose tolerance. These observations suggest that therapeutic use of a TGFp inhibitor with inhibitory activity towards the TGFp2 isoform may be harmful, at least in the metabolic aspect. Because patients suffering from muscular dystrophy, such as DMD, are prone to metabolic dysfunction, they may be particularly susceptible to TGFp2 inhibition, further pointing to the advantage of TGFp inhibitors that do not target the TGFp2 isoform.

[0439]

[0305] Accordingly, the present disclosure contemplates the use of LTBP-selective inhibitors of TGFpl in the treatment of muscular dystrophy, such as DMD, in a subject. Preferred LTBP-selective inhibitors of TGFpl are the monoclonal antibodies and antigen-binding fragments thereof which are disclosed in WO 2020 / 160291 , as well as antibodies that compete antigen binding with one or more of the antibodies disclosed therein. In some embodiments, the LTBP-selective inhibitor of TGFpl is the human version of the monoclonal antibody referred to as LTBP-49247 (Jackson et al., Sci. Signal. 17, eadn6052 (2024). In some embodiments, the LTBP-selective inhibitor of TGFpl is used in combination with a myostatin inhibitor in the treatment of muscular dystrophy (e.g., DMD and BMD) in a subject. In some embodiments, the LTBP-selective inhibitor of TGFpl is used in combination with a Dystrophin upregulator in the treatment of muscular dystrophy (e.g., DMD and BMD) in a subject. In some embodiments, the LTBP-selective inhibitor of TGFpl is used in combination with a myostatin inhibitor and a Dystrophin upregulator in the treatment of muscular dystrophy (e.g., DMD and BMD) in a subject. In some embodiments, the LTBP-selective inhibitorAttorney Docket No. 15094.0067-00304

[0440] of TGFpl and a myostatin inhibitor are used in combination in the treatment of muscular dystrophy (e.g., DMD and BMD) in a subject, wherein optionally the subject has received a Dystrophin upregulator. In any of the above embodiments, a preferred myostatin inhibitor is a myostatin-selective inhibitor that does not inhibit GDF11 or Activin A at a dose effective to inhibit myostatin. Non-limiting examples of myostatin-selective inhibitors include: apitegromab, trevogrumab (REGN1033), GYM329 (RO7204239), the antibodies disclosed in WO 2024 / 138076 (e.g., Ab101-Ab141), and nucleic acid-based agents aimed to eliminate or reduce myostatin expression, such as RNAi agents and antisense oligonucleotides. In any of the embodiments, the subject may be on a SOC therapy, wherein the SOC therapy comprises a corticosteroid. In some embodiments, the subject is treated with the myostatin-selective inhibitor and the LTBP-selective inhibitor of TGFpl in lieu of the SOC such as corticosteroids. In some cases, the subject may receive a reduced dose of corticosteroids, as compared to typical recommended doses as SOC.

[0441] Patient selection

[0442]

[0306] According to the present disclosure, a subject who benefits from the combination therapy described herein is a patient who has been diagnosed with a muscular dystrophy selected from the listing below.

[0443]

[0307] Muscular dystrophies include several categories: dystrophinopathies, limb girdle muscular dystrophies, congenital muscular dystrophies, distal muscular dystrophies, myofibrillar myopathy, as well as other types of muscular dystrophies. Each of these categories is further described below.

[0444]

[0308] Dystrophinopathies include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, and DMD-associated dilated cardiomyopathy.

[0445]

[0309] DMD is the most common childhood form of muscular dystrophy. Because the underlining genetic mutation is associated with the X chromosome, DMD primarily affects boys, although girls who carry the defective gene may show some symptoms. DMD results from an absence of the muscle protein dystrophin. DMD usually becomes apparent during the toddler years, sometimes soon after an affected child begins to walk. Progressive weakness and muscle wasting (a decrease in muscle strength and size) caused by degenerating muscle fibers begins in the upper legs and pelvis before spreading into the upper arms. Other symptoms include: Loss of some reflexes; A waddling gait; Frequent falls and clumsiness (especially when running); Difficulty when getting up from a sitting position or when climbing stairs; Changes to overall posture; Impaired breathing; and, Heart problems (cardiomyopathy).

[0446]

[0310] Many children with DMD are unable to run or jump. The calf muscles, and less commonly, muscles in the buttocks, shoulders, and arms, may be enlarged by an accumulation of fat and connective tissue, causing them to look larger and healthierthan they actually are (called pseudohypertrophy). As the disease progresses, the muscles in the diaphragm that assist in breathing and coughing may weaken. Individuals may experience breathing difficulties, respiratory infections, and swallowing problems. Bone thinning and scoliosis (curving of the spine) are common. Some children have cognitive and behavioral impairments.Attorney Docket No. 15094.0067-00304

[0447]

[0311] In recent years, the availability of DMD therapies have extended the life expectancy and improved the quality of life significantly for children with DMD. Many people with DMD now survive into their 20s or 30s.

[0448]

[0312] In addition to muscle defects, there have been multiple metabolic findings in Duchenne muscular dystrophy research, which include mitochondrial impairment, abnormal lipid metabolism (e.g., lipid buildup in muscle), abnormalities in macronutrient uptake and availability, glycolysis, glycogen storage and utilization, fat oxidation, the creatine phophagen system and the purine nucleotide cycle.

[0449]

[0313] In some embodiments, the subject with DMD who is treated as disclosed herein is under the age of 10. In some embodiments, the subject with DMD is age 8 years or younger. In some embodiments, the subject with DMD is newborn (age 0) to 8 years old. In some embodiments, the subject with DMD is newborn to 7 years old. In some embodiments, the subject with DMD is 4 to 8 years old. In some embodiments, the subject with DMD is 4 to 7 years old. In some embodiments, the subject with DMD is 4 years old or older and less than 7 years old.

[0450]

[0314] In some embodiments, the subject with DMD is at least 4 years old and has stabilized motor function. In some embodiments, stabilized motor function is the maintenance of motor function as measured by NSAA, TTR, 6MWT, 10MWRT and / or 4-stair climb. In some embodiments, stabilized motor function is the maintenance of muscle size or strength (e.g., quadriceps size or strength).

[0451]

[0315] In some embodiments, the subject with DMD who is treated as disclosed herein has high motor function and are early in their disease progression. In some embodiments, the subject with DMD has high motor function as measured by NSAA and / or time to rise (TTR) at baseline. In some embodiments, the subject with DMD has high motor function as measured by North Star Ambulatory Assessment (NSAA) (e.g. NSAA of at least 17) at baseline. In some embodiments, the subject with DMD has high motor function as measured by TTR (e.g. TTR of between 4-8 seconds) at baseline.

[0452]

[0316] In some embodiments, the subject with DMD is age 0-7 years old and retains a threshold of motor function, such as baseline TTR of at least 17 and / or NSAA of between 4-8 seconds.

[0453]

[0317] Becker muscular dystrophy is less severe than but closely related to DMD. The disorder usually appears around age 11 but may occur as late as age 25, and people with Becker MD usually live into middle age or later. The rate of muscle atrophy and weakness varies greatly. Many people maintain their ability to walk until they are in their mid-30s or later, while others are unable to walk past their teens. Muscle weakness is typically noticed first in the upper arms and shoulders, upper legs, and pelvis. Cognitive and behavioral impairments and heart problems are not as common or severe as in DMD, but they do occur. Early symptoms of Becker MD include: walking on one’s toes, frequent falls, and difficulty rising from the floor.

[0454]

[0318] Limb-girdle muscular dystrophy (LGMD) refers to more than 20 inherited conditions marked by progressive loss of muscle and the symmetrical weakening of voluntary muscles, primarily those in the shoulders and around the hips. At least five forms of autosomal dominant limb-girdle MD (known as Type 1) and 17 forms of autosomal recessive limb-girdle MD (known as Type 2) have been identified. Some autosomal recessive forms of the disorder are caused by a deficiency of any of fourdystrophin-glycoproteinAttorney Docket No. 15094.0067-00304

[0455] complex proteins called the sarcoglycans. Deficiencies in dystroglycan, classically associated with congenital muscular dystrophies, may also cause LGMD. LGMD, as defined by the European Neuromuscular Centre in 2018, are named by the following system: LGMD, recessive or dominant inheritance (R or D), order of discovery (number), affected protein. Examples of LGMSs include the following: LGMD D1 DNAJB6-related; LGMD D2 TNP03-related; LGMD D3 HNRNPDL-related; LGMD D4 calpain3-related; LGMD D5 collagen 6-related; LGMD R1 calpain3-related (Calpainopathy); LGMD R2 dysferlin-related; LGMD R3 a-sarcoglycan-related; LGMD R4 p-sarcoglycan-related; LGMD R5 y-sarcoglycan-related; LGMD R6 6-sarcoglycan-related; LGMD R7 telethonin-related; LGMD R8 TRIM 32-related; LGMD R9 FKRP-related; LGMD R10 titin-related; LGMD R11 POMT1 -related; LGMD R12 anoctamin5-related; LGMD R13 Fukutin-related; LGMD R14 POMT2-related; LGMD R15 POMGnTI-related; LGMD R16 a-dystroglycan-related; LGMD R17 plectin-related; LGMD R18 TRAPPC11 -related; LGMD R19 GMPPB-related; LGMD R20 ISPD-related; LGMD R21 POGLUT1 -related; LGMD R22 collagen 6-related; LGMD R23 laminin a2-related; and, LGMD R24 POMGNT2-related.

[0456]

[0319] Congenital muscular dystrophy refers to a group of muscular dystrophies that are either present at birth or become evident before age 2. The degree and progression of muscle weakness and degeneration vary with the type of disorder. Weakness may be first noted when children do not meet developmental milestones related to motor function and muscle control. Muscle degeneration in congenital muscular dystrophy is restricted primarily to skeletal muscle. Most people with this type of MD are unable to sit or stand without support, and some may never learn to walk. Congenital muscular dystrophies include the following: LAMA2-related (merosin deficient) congenital muscular dystrophy (Emery-Dreifuss muscular dystrophy); Collagen Vl-related muscular dystrophy (Bethlem myopathy, Ullrich congenital muscular dystrophy); a-Dystroglycanopathies (Walker-Warburg syndrome, muscle-eye-brain disease); and, Laminopathies. In some embodiments, the congenital muscular dystrophy is Merosin-negative disorders, in which the protein merosin (found in the connective tissue that surrounds muscle fibers) is missing. In some embodiments, the congenital muscular dystrophy Merosin-positive disorders, in which merosin is present but other necessary proteins are missing.

[0457]

[0320] Distal muscular dystrophy, also known as distal myopathy, refers to any muscle disease that primarily affect the distal muscles (those farthest away from the shoulders and hips) in the forearms, hands, lower legs, and feet. Distal MDs are typically less severe, progress more slowly, and involve fewer muscles than other forms of MD, although they can spread to other muscles. Distal MD can affect the heart and respiratory muscles, and individuals with distal MD may eventually need a ventilator. They may not be able to perform fine hand movement and may have difficulty extending the fingers. Walking and climbing stairs may become difficult and some people may be unable to hop or stand on their heels. Examples include: Late adult-onset type 1 ; Late adult-onset type 2a; Late adult-onset type 2b; Early adult-onset type 1 ; Early adult-onset type 2; and, Early adult-onset type 3.

[0458]

[0321] Myofibrillar myopathies are diseases that share histological similarities in affected muscle and include: Desminopathy; Myotilinopathy; Zaspopathy; Filaminopathy; and, Bag3opathy.

[0459]

[0322] Other muscular dystrophies include: Myotonic dystrophy; Facioscapulohumeral muscular dystrophy (FSHD); Emery-Dreifuss muscular dystrophy (EDMD); and, Oculopharyngeal muscular dystrophy (OPMD).Attorney Docket No. 15094.0067-00304

[0460]

[0323] In some embodiments, the facioscapulohumeral muscular dystrophy (FSHD) initially affects muscles of the face (facio), shoulders (scapulo), and upper arms (humera) with progressive weakness. Also known as Landouzy-Dejerine disease, this is a relatively common form of MD and is characterized as an autosomal dominant disorder. Most people with FSHD have a normal life span, but some become severely disabled.

[0461]

[0324] In some embodiments, the myotonic dystrophy is Myotonic dystrophy (DM1), also known as Steinert's disease or dystrophia myotonica. In some embodiments, the myotonic dystrophy is myotonic dystrophy type 2 (DM2), which similar to the classic form, but usually affects proximal muscles more significantly.

[0462]

[0325] In some embodiments, the patient is on standard-of-care therapy prescribed for the muscular dystrophy. Examples of pharmacological SOC therapies include steroids, such as prednisone and deflazacort, and immunosuppressive drugs can help slow the rate of muscle deterioration and damage to muscle cells, but some carry side effects that can be especially troubling in children. There are also drugs available to treat the symptoms of MD, including myotonia (muscle spasms and weakness). Breathing symptoms may be treated with antibiotics. Non-pharmacological care or interventions may include: physical therapy, occupational therapy, speech therapy, moderate exercise / physical activity, deep breathing and / or coughing exercise, corrective surgery, support aids such as wheelchairs, splints, braces, spinal supports, and posture correction devises, and assisted ventilation.

[0463]

[0326] In some embodiments, the patient is naive to Dystrophin upregulator therapy.

[0464]

[0327] In some embodiments, the patient has received or is on a Dystrophin upregulator therapy, such as ASOs and gene therapy. In preferred embodiments, the patient has shown a clinical response to the upregulator therapy. The clinical response includes increased levels of Dystrophin protein.

[0465] NUMBERED EMBODIMENTS

[0466] 1. A myostatin-selective inhibitor for use in the treatment of a muscular dystrophy in a subject, wherein the treatment comprises administration of a myostatin-selective inhibitor to a subject suffering from a muscular dystrophy in an amount effective to treat the muscular dystrophy, wherein the subject is treated with a dystrophin upregulator.

[0467] 2. A dystrophin upregulator for use in the treatment of a muscular dystrophy in a subject, wherein the treatment comprises administration of a dystrophin upregulator to a subject suffering from a muscular dystrophy in an amount effective to treat the muscular dystrophy, wherein the subject is treated with a myostatin-selective inhibitor.

[0468] 3. A myostatin-selective inhibitor and a dystrophin upregulator for use in the treatment of a muscular dystrophy in a subject, wherein the treatment comprises administration of a myostatin-selective inhibitor and a dystrophin upregulator to a subject suffering from a muscular dystrophy in amounts effective to treat the muscular dystrophy.Attorney Docket No. 15094.0067-00304

[0469] 4. The myostatin-selective inhibitor for use according to any one of the preceding embodiments, wherein the treatment further comprises an anti-fibrotic agent, wherein optionally the anti-fibrotic agent is a TGFpl inhibitor.

[0470] 5. The myostatin-selective inhibitor for use according to any one of the preceding embodiments, wherein the myostatin-selective inhibitor is an antibody or antigen-binding fragment thereof that binds mature myostatin and inhibits receptor activation but does not bind GDF11.

[0471] 6. The myostatin-selective inhibitor for use according to any one of embodiments 1-4, wherein the myostatin-selective inhibitor is an antibody or antigen-binding fragment thereof that binds latent myostatin complex thereby inhibiting release of mature myostatin from the latent complex.

[0472] 7. The myostatin-selective inhibitor for use according to embodiment 1 , wherein the myostatin-selective inhibitor is an antibody or antigen-binding fragment thereof that specifically binds pro / latent myostatin.

[0473] 8. The myostatin-selective inhibitor for use according to embodiment 6 or embodiments 7, wherein the myostatin-selective inhibitor is apitegromab.

[0474] 9. The myostatin-selective inhibitor for use according to any one of embodiments 1-4, wherein the myostatin-selective inhibitor is an anti-myostatin-Adnectin comprising an antigen-binding fragment that selectively binds to mature myostatin or latent myostatin.

[0475] 10. The myostatin-selective inhibitor for use according to any one of embodiments 1-4, wherein the myostatin-selective inhibitor is a nucleic acid-based inhibitor of myostatin expression, wherein optionally the nucleic acid-based inhibitor of myostatin expression is an antisense oligonucleotide agent, an RNA interference (RNAi) agent, or a CRISPR agent.

[0476] 11. The myostatin-selective inhibitor or the Dystrophin upregulator for use according to any one of the preceding embodiments, wherein the Dystrophin upregulator is an RNAi agent, wherein optionally the RNAi agent is a small interference RNA (siRNA) or microRNA (miRNA).

[0477] 12. The myostatin-selective inhibitor or the Dystrophin upregulator for use according to any one of embodiments 1-10, wherein the Dystrophin upregulator is an antisense oligonucleotide agent, wherein optionally the antisense oligonucleotide agent is a Morpholino (phosphorodiamidate morpholino oligomer (PMO)).

[0478] 13. The myostatin-selective inhibitor or the Dystrophin upregulator for use according to any one of embodiments 1-10, wherein the dystrophin upregulator is a splice modifier, wherein optionally the splice modifier is an exon-skipping agent.

[0479] 14. The myostatin-selective inhibitor or the Dystrophin upregulator for use according to embodiment 13, wherein the exon-skipping agent is selected from: Exon 51 -skipping agents, Exon 53-skipping agents,Attorney Docket No. 15094.0067-00304

[0480] Exon 45-skipping agents, Exon 44-skipping agents, Exon 50-skipping agents, Exon 52-skipping agents, Exon 43-skipping agents, Exon 55-skipping agents, and Exon 8-skipping agents.

[0481] 15. The myostatin-selective inhibitor or the Dystrophin upregulator for use according to embodiment 13, wherein the exon skipping agent is selected from Exon 51 -skipping agents, Exon 53-skipping agents and Exon 45-skipping agents, wherein optionally the dystrophin upregulator is selected from: casimersen (e.g., Amondys 45), eteplirsen (e.g., Exondys 51), viltolarsen (e.g., Viltepso), golodirsen (e.g., Vyondys 53).

[0482] 16. The myostatin-selective inhibitor or the Dystrophin upregulator for use according to any one of embodiments 1 -10, wherein the dystrophin upregulator is a gene therapy, wherein optionally the gene therapy is a gene replacement therapy or a gene correction therapy, wherein further optionally the gene correction therapy comprises a CRISPR agent.

[0483] 17. A TGFpl inhibitor for use in the treatment of a muscular dystrophy in a subject, wherein the treatment comprises administration of a TGFpl inhibitor in conjunction with a myostatin inhibitor in amounts effective to treat the muscular dystrophy, wherein optionally the subject is treated with a dystrophin upregulator, and wherein further optionally the subject receives a reduced dose of a corticosteroid or discontinues a corticosteroid therapy.

[0484] 18. A TGFpl inhibitor and a myostatin inhibitor for use in the treatment of a muscular dystrophy in a subject, wherein the treatment comprises administration of a combination of a TGFpl inhibitor and a myostatin inhibitor to the subject in amounts effective to treat the muscular dystrophy, wherein optionally the subject is treated with a dystrophin upregulator, wherein further optionally the subject receives a reduced dose of a corticosteroid or discontinues a corticosteroid therapy.

[0485] 19. The TGFpl inhibitor and / or a myostatin inhibitor for use according to embodiment 17 or embodiment 18, wherein the TGFpl inhibitor is a TGFpl -selective inhibitor, wherein optionally the TGFpl -selective inhibitor specifically targets proTGFpl complex associated with the extracellular matrix, wherein further optionally the proTGFpl complex associated with the extracellular matrix is LTBP1-proTGFpl and LTBP3-proTGFp1.

[0486] 20. The myostatin-selective inhibitor, the Dystrophin upregulator, and / or the TGFpl inhibitor for use according to any one of the preceding embodiments, wherein the subject is on a glucocorticoid therapy, wherein optionally the glucocorticoid is selected from prednisone / prednisolone and deflazacort.

[0487] 21. A myostatin-selective inhibitor for use in the treatment of a muscular dystrophy in a subject in need thereof, wherein the treatment comprises administration of a myostatin-selective inhibitor to a subject suffering from a muscular dystrophy in an amount effective to treat the muscular dystrophy, and wherein the treatment increases exercise capacity and / or endurance of the subject, optionally wherein the increase in exercise capacity and / or endurance is measured by a graded treadmill test, e.g. as measured by time and / or distance to exhaustion.Attorney Docket No. 15094.0067-00304

[0488] 22. The myostatin-selective inhibitor for use according to embodiment 21 , wherein the myostatin-selective inhibitor is an antibody or antigen-binding fragment thereof that binds mature myostatin and inhibits receptor activation but does not bind GDF11.

[0489] 23. The myostatin-selective inhibitor for use according to embodiment 21 , wherein the myostatin-selective inhibitor is an antibody or antigen-binding fragment thereof that binds latent myostatin complex, thereby inhibiting release of mature myostatin from the latent complex.

[0490] 24. The myostatin-selective inhibitor for use according embodiment 21 , wherein the myostatin-selective inhibitor is an antibody or antigen-binding fragment thereof that specifically binds pro / latent myostatin.

[0491] 25. The myostatin-selective inhibitor for use according to embodiment 23 or embodiment 24, wherein the myostatin-selective inhibitor is apitegromab.

[0492] 26. The myostatin-selective inhibitor for use according to embodiment 21 , wherein the myostatin-selective inhibitor is an anti-myostatin-Adnectin comprising an antigen-binding fragment that selectively binds to mature myostatin or latent myostatin.

[0493] 27. The myostatin-selective inhibitor for use according to any one of embodiments 21-26, wherein the increase in exercise capacity and / or endurance is an increase in exercise capacity.

[0494] 28. The myostatin-selective inhibitor for use according to any one of embodiments 21-27, wherein the increase in exercise capacity comprises an increase in running capacity.

[0495] 29. The myostatin-selective inhibitor for use according to any one of embodiments 21-28, wherein the increase in exercise capacity and / or endurance is an increase in endurance.

[0496] 30. The myostatin-selective inhibitor for use according to any one of embodiments 21-29, wherein the treatment increases the max force of a muscle.

[0497] 31. The myostatin-selective inhibitor for use according to embodiment 30, wherein the muscle is tibialis anterior (TA).

[0498] 32. The myostatin-selective inhibitor for use according to any one of embodiments 21-31 , wherein the disorder is facioscapulohumeral muscular dystrophy (FSHD).

[0499] 33. A myostatin-selective inhibitor for increasing exercise capacity and / or endurance in a subject suffering from a muscular dystrophy, comprising administration of a myostatin-selective inhibitor to the subject in an amount effective to increase the exercise capacity and / or endurance of the subject.

[0500] 34. The myostatin-selective inhibitor according to embodiment 33, wherein the myostatin-selective inhibitor is an antibody or antigen-binding fragment thereof that binds mature myostatin and inhibits receptor activation but does not bind GDF11.Attorney Docket No. 15094.0067-00304

[0501] 35. The myostatin-selective inhibitor according to embodiment 33, wherein the myostatin-selective inhibitor is an antibody or antigen-binding fragment thereof that binds latent myostatin complex, thereby inhibiting release of mature myostatin from the latent complex.

[0502] 36. The myostatin-selective inhibitor according to embodiment 33, wherein the myostatin-selective inhibitor is an antibody or antigen-binding fragment thereof that specifically binds pro / latent myostatin.

[0503] 37. The myostatin-selective inhibitor according to embodiment 35 or embodiment 36, wherein the myostatin-selective inhibitor is apitegromab.

[0504] 38. The myostatin-selective inhibitor according to embodiment 33, wherein the myostatin-selective inhibitor is an anti-myostatin-Adnectin comprising an antigen-binding fragment that selectively binds to mature myostatin or latent myostatin.

[0505] 39. The myostatin-selective inhibitor according to any one of embodiments 33-38, wherein the increase in exercise capacity and / or endurance is an increase in exercise capacity.

[0506] 40. The myostatin-selective inhibitor according to any one of embodiments 33-39, wherein the increase in exercise capacity comprises an increase in running capacity.

[0507] 41. The myostatin-selective inhibitor according to any one of embodiments 33-40, wherein increase in exercise capacity and / or endurance is an increase in endurance.

[0508] 42. The myostatin-selective inhibitor according to any one of embodiments 33-41 , wherein the administration increases the max force of a muscle.

[0509] 43. The myostatin-selective inhibitor for use according to embodiment 42, wherein the muscle is tibialis anterior (TA).

[0510] 44. The myostatin-selective inhibitor according to any one of embodiments 33-43, wherein in the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

[0511] 45. A myostatin-selective inhibitor for use in the treatment of DMD in a subject diagnosed with DMD, wherein the treatment comprises administration of a myostatin-selective inhibitor in an amount effective to treat DMD, wherein the subject is a human subject under the age of 10 years old.

[0512] 46. The myostatin-selective inhibitor for use according to embodiment 45, wherein the subject is between the age of 0-9 years old.

[0513] 47. The myostatin-selective inhibitor for use according to embodiment 46, wherein optionally, the subject is between the age of 1-9, 2-9, 3-9, 4-9, 4-8, or 4-7 years old.

[0514] 48. The myostatin-selective inhibitor for use according to embodiment 46, wherein the subject is a human subject under the age of 8.Attorney Docket No. 15094.0067-00304

[0515] 49. The myostatin-selective inhibitor for use according to any one of the preceding embodiments, wherein the subject is on a dystrophin restorer (a dystrophin upregulator or a dystrophin corrector) therapy, wherein optionally the dystrophin restorer therapy comprises an exon-skipping agent / splice modifier or a gene therapy.

[0516] 50. The myostatin-selective inhibitor for use according to embodiment 49, wherein the dystrophin restorer therapy comprises an exon-skipping agent / splice modifier or a gene therapy.

[0517] 51. The myostatin-selective inhibitor for use according to embodiment 49, wherein the exon-skipping agent / splice modifier is a nucleic acid-based therapy, wherein optionally the nucleic acid-based therapy is an anti-sense oligonucleotide (ASO) therapy.

[0518] 52. The myostatin-selective inhibitor for use according to embodiment 51 , wherein the nucleic acid-based therapy is an anti-sense oligonucleotide (ASO) therapy.

[0519] 53. A myostatin-selective inhibitor for use in the treatment of DMD in a subject diagnosed with DMD, wherein the treatment comprises administration of a myostatin-selective inhibitor in an amount effective to treat DMD, wherein the subject has a dystrophin expression level sufficient to retain motor function.

[0520] 54. The myostatin-selective inhibitor for use according to embodiment 53, wherein the retention of motor function is as measured by North Star Ambulatory Assessment (NSAA) (e.g. NSAA of at least 17) at baseline.

[0521] 55. The myostatin-selective inhibitor for use according to embodiment 53 or embodiment 54, wherein the retention of motor function is as measured by time-to-rise (TTR) (e.g. TTR of between 4-8 seconds) at baseline.

[0522] 56. The myostatin-selective inhibitor for use according to any one of embodiments 53-55, wherein the subject is under the age of 10 years old.

[0523] 57. A myostatin inhibitor for use in the treatment of DMD in a patient, wherein the treatment comprises administration of a myostatin inhibitor to the patient diagnosed with DMD in an amount effective to treat DMD, wherein the patient has an NSAA score of 17 or greater at baseline (at screening).

[0524] 58. A myostatin inhibitor for use in the treatment of DMD in a patient, wherein the treatment comprises administration of a myostatin inhibitor to the patient diagnosed with DMD in an amount effective to treat DMD, wherein the patient has a TTR (“time to rise”) of 4-8 seconds at baseline (at screening).

[0525] 59. The myostatin inhibitor for use according to embodiment 57 or embodiment 58, wherein the patient is between the age of 4-7 years at the initiation of a myostatin inhibitor therapy.

[0526] 60. The myostatin inhibitor for use according to any one of embodiments 57-59, wherein the myostatin inhibitor is a myostatin-selective inhibitor, wherein optionally the myostatin-selective inhibitor is selected from: an antibody that selectively binds myostatin but does not bind Activin A or GDF11 ; an antibody thatAttorney Docket No. 15094.0067-00304

[0527] selectively binds pro / latent myostatin thereby inhibiting activation; or a nucleic acid-based agent that blocks expression of endogenous myostatin.

[0528] 61. The myostatin inhibitor for use according to embodiment 60, wherein the myostatin-selective inhibitor is selected from apitegromab, GYM329, trevogrumab, or any variant thereof.

[0529] 62. The myostatin inhibitor for use according to any one of embodiments 57-60, wherein the subject is treated with a dystrophin upregulator therapy, wherein the dystrophin upregulator therapy comprises a pharmaceutical agent aimed to increase dystrophin expression, wherein optionally the dystrophin upregulator comprises an exon-skipping agent or a gene therapy.

[0530] 63. The myostatin inhibitor for use according to any one of embodiments 57-62, wherein the amount effective to treat DMD is an amount sufficient to delay disease progression.

[0531] 64. A myostatin inhibitor for use in the treatment of facioscapulohumeral muscular dystrophy (FSHD) in a patient in need thereof, wherein the treatment comprises administration of a myostatin inhibitor to the patient in an amount effective to treat FSHD, wherein the patient has a Ricci Clinical Severity Scale score of 1.5 to 3.0 at baseline.

[0532] 65. A myostatin inhibitor for use in the treatment of facioscapulohumeral muscular dystrophy (FSHD) in a patient in need thereof, wherein the treatment comprises administration of a myostatin inhibitor to the patient in an amount effective to treat FSHD, wherein the patient has a 10-meter walk / run test (10MWRT) time of less than or equal to 5 seconds at baseline.

[0533] 66. A myostatin inhibitor for use in the treatment of facioscapulohumeral muscular dystrophy (FSHD) in a patient in need thereof, wherein the treatment comprises administration of a myostatin inhibitor to the patient in an amount effective to treat FSHD, wherein the patient has a Ricci Clinical Severity Scale score of 1.5 to 3.0 at baseline and has a 10-meter walk / run test (1 OMWRT) time of less than or equal to 5 seconds at baseline.

[0534] 67. A myostatin inhibitor for use in the treatment of FSHD in a patient in need thereof, wherein the treatment comprises administration of a myostatin inhibitor to the patient in an amount effective to treat FSHD, wherein the patient has a timed-up-and-go (TUG) time of 20 seconds or less (e.g., 16 seconds or less, 14 seconds or less, 12 seconds or less) at baseline.

[0535] 68. The myostatin inhibitor for use according to any one of embodiments 64-67, wherein the patient is between the age of 18-60 years at screening or at initiation of the administration of the myostatin inhibitor.

[0536] 69. The myostatin inhibitor for use according to any one of embodiments 64-68, wherein the patient has been genetically diagnosed with FSHD1 or FSHD2.

[0537] 70. The myostatin inhibitor for use according to any one of embodiments 64-69, wherein the myostatin inhibitor is a myostatin-selective inhibitor, wherein optionally the myostatin-selective inhibitor is selectedAttorney Docket No. 15094.0067-00304

[0538] from: an antibody that selectively binds myostatin but does not bind Activin A or GDF11 ; an antibody that selectively binds pro / latent myostatin thereby inhibiting myostatin activation; or a nucleic acid-based agent that blocks expression of endogenous myostatin.

[0539] 71. The myostatin inhibitor for use according to embodiment 70, wherein the myostatin-selective inhibitor is selected from apitegromab, GYM329, trevogrumab, SRK-439, or any variant thereof.

[0540] 72. The myostatin inhibitor for use according to embodiment 71, wherein the myostatin-selective inhibitor is apitegromab.

[0541] 73. The myostatin inhibitor for use according to embodiment 72, wherein apitegromab is administered to the patient at a dose of 2-20 mg / kg (e.g., 10 mg / kg).

[0542] 74. The myostatin inhibitor for use according to embodiment 73, wherein apitegromab is administered to the patient once every four weeks or once a month.

[0543] 75. The myostatin inhibitor for use according to any one of embodiments 64-74, wherein the myostatin inhibitor is administered to the patient intravenously.

[0544] 76. The myostatin inhibitor for use according to any one of embodiments 64-75, wherein the patient is further treated with a DUX4 inhibitor or is on a DUX4 inhibitor therapy.

[0545] 77. The myostatin inhibitor for use according to embodiment 76, wherein the DUX4 inhibitor is an siRNA that blocks DUX4 expression, wherein optionally, the DUX4 inhibitor is delpacibart braxlosiran (del-brax) or a TfR1-binding Fab conjugated to an siRNA against DUX4 (e.g., DYNE-302).

[0546] 78. The myostatin inhibitor for use according to embodiment 76, wherein the myostatin inhibitor and the DUX4 inhibitor are engineered into a single molecule construct.

[0547] 79. The myostatin inhibitor for use according to any one of embodiments 64-78, wherein the treatment increases total lean muscle volume (LMV) in the patient as compared to baseline, optionally wherein the total LMV is measured by a whole-body Magnetic Resonance Imaging (MRI) scan.

[0548] 80. The myostatin inhibitor for use according to any one of embodiments 64-79, wherein the treatment improves motor function in the patient as compared to baseline, optionally wherein motor function is measured by a quantitative myo metry test, a 10-meter walk / run test (10WMRT), a Timed Up and Go test, a 5x sit-to-stand test (5x STS), and / or an exercise questionnaire.

[0549] 81. The myostatin inhibitor for use according to any one of embodiments 64-80, wherein the treatment increases exercise capacity and / or endurance in the patient as compared to baseline, optionally wherein the increase in exercise capacity and / or endurance is measured by a graded treadmill test, e.g. as measured by time and / or distance to exhaustion.Attorney Docket No. 15094.0067-00304

[0550] 82. The myostatin inhibitor for use according to embodiment 81 , wherein the increase in exercise capacity and / or endurance is an increase in exercise capacity.

[0551] 83. The myostatin inhibitor for use according to embodiment 81 or embodiment 82, wherein the increase in exercise capacity comprises an increase in running capacity.

[0552] 84. The myostatin inhibitor for use according to any one of embodiments 81-83, wherein the increase in exercise capacity and / or endurance is an increase in endurance.

[0553] 85. The myostatin inhibitor for use according to any one of embodiments 64-84, wherein the treatment increases muscle function in the patient as compared to baseline, wherein optionally the increase in muscle function comprises an increase in a max force level of a muscle in the patient.

[0554] 86. The myostatin inhibitor for use according to embodiment 85, wherein the increase in muscle function is an increase in a max force level of a tibialis anterior (TA) muscle.

[0555] 87. The myostatin inhibitor for use according to any one of embodiments 64-86, wherein the treatment increases muscle mass in the patient as compared to baseline.

[0556] 88. The myostatin inhibitor for use according to embodiment 87, wherein the treatment increases the muscle mass of a gastrocnemius muscle in the patient.

[0557] 89. The myostatin inhibitor for use according to any one of embodiments 64-88, wherein the treatment increases grip strength in the patient as compared to baseline.

[0558] 90. The myostatin inhibitor for use according to any one of embodiments 64-89, wherein the treatment decreases a creatine / creatinine (Cr / Crn) ratio in the patient as compared to baseline.

[0559] 91. The myostatin inhibitor for use according to any one of embodiments 64-90, wherein the treatment reduces inflammation in the patient as compared to baseline.

[0560] 92. The myostatin inhibitor for use according to embodiment 91 , wherein the reduction in inflammation comprises a reduction of pro-inflammatory macrophage infiltration in a muscle in the patient.

[0561] 93. The myostatin inhibitor for use according to any one of embodiments 64-92, wherein the amount effective to treat FSHD is an amount sufficient to delay disease progression.

[0562] EXAMPLES

[0563] Example 1: Selection of preclinical muscular dystrophy model

[0564]

[0328] To date, commonly used preclinical models of muscular dystrophies include the BlO.mdx mouse model. BlO.mdx mice show relatively mild pathology with respect to skeletal muscle defect, as compared to clinical presentation. Although diaphragm pathology is considerable, in orderto induce weakness in other muscle groups, BlO.mdx mice need to be exercised.Attorney Docket No. 15094.0067-00304

[0565]

[0329] Previously, myostatin pathway inhibition as a monotherapy has been tested and has shown benefit in the BlO.mdx model. However, this has not translated into the clinic (e.g., Domagrozumab, T-Alfa (anti-myostatin Adnectin)).

[0566]

[0330] On the other hand, the D2.mdx model is the same dystrophin genetics as the BlO.mdx model but on a different background. In comparison, muscle pathology in D2.mdx mice is more severe with considerable fibrosis and strength loss early in life in muscles beyond the diaphragm, although the pathology in the D2 model is not progressive and tends to resolve somewhat at older age. Muscle calcification is also a feature of this model. Overall, a fair assumption is that the D2.mdx model provides a higher bar for evaluating myostatin inhibitors as a therapy.

[0567]

[0331] We therefore selected the more severe D2.mdx model and asked whether the myostatin-selective inhibition approach could enhance muscle strength either alone or in combination with an exon skipping agent that increases Dystrophin expression.

[0568] Example 2: Dystrophin expression in D2.mdx mice

[0569]

[0332] To confirm effects of the known Dystrophin exon skipping agent, “Vivo-Morpholino 23”, on Dystrophin expression, immunofluorescent study was performed in D2.mdx and wild type control mice. Vivo-Morpholino 23 is a stabilized splice modifying agent, in which the Morpholino moiety is covalently linked to a delivery moiety comprised of an octa-guanidine dendrimer and uses the active component of arginine-rich delivery peptides which confer enhanced stability.

[0570]

[0333] PMO treatment group received PMO for 5 weeks. At the study end, muscles (TA and gastroc) were dissected and were processed for IHC imaging.

[0571]

[0334] Results are shown in FIG.1. In D2.mdx mice treated with PMO, partial restoration of Dystrophin protein was observed, and this was more prominent in Gastroc (bottom panel, second from right). Wild type control (no PMO) showed strong immunoreactivity as expected (bottom left), while D2.mdx that did not receive PMO showed no detectable Dystrophin expression (bottom, second left). Corresponding upper panels show laminin and nucleus (DAPI) staining as reference. This study confirmed that Vivo-Morpholino 23 is capable of upregulating Dystrophin expression in D2.mdx mice.

[0572] Example 3: Effects of combination treatment of a myostatin-selective inhibitor and a dystrophin upregulator in D2.mdx mice

[0573]

[0335] We previously demonstrated in SMNA7 mouse model of SMA that enhancing the target muscle with a selective inhibitor of myostatin, while concurrently addressing motor neuron defects with an SMN upregulator, can build muscle and promote strength, and this approach has now been validated in a clinical setting. Here, we asked whether muscle defects in a DMD model may be addressed by a combination of two different agents that target the muscle, namely, muSRK-015P and a dystrophin upregulator in the tibialis anterior and gastrocnemius muscles. Our data demonstrate that the combination treatment leads to muscle mass and strength enhancement in D2.mdx mice, supporting the potential of selective myostatin inhibition with SRK-015 to build muscle mass and strength in moderately dystrophic muscle or in dystrophic muscle with partial dystrophin restoration, ed whether muscle defects in a DMD model may be addressed by a combination of two different agents that target the muscle, namely, muSRK-015P and a dystrophin upregulator in the tibialis anterior and gastrocnemius muscles. Our dataAttorney Docket No. 15094.0067-00304

[0574] demonstrate that the combination treatment leads to muscle mass and strength enhancement in D2.mdx mice, supporting the potential of selective myostatin inhibition with SRK-015 to build muscle mass and strength in moderately dystrophic muscle or in dystrophic muscle with partial dystrophin restoration.

[0575]

[0336] To examine in vivo effects of a myostatin-selective inhibition in the presence or absence of a Dystrophin upregulator in the D2.mdx mouse model of DMD, muSRK-015P, which is a monoclonal antibody that selectively inhibits myostatin activation by binding to the latent forms of myostatin (pro / latent myostatin), was employed. muSRK-015P is a murine counterpart of apitegromab. An isoform-matched IgG was used as negative control. A Morpholino (“PMO”) which was previously shown to increase Dystrophin expression in mice was used in conjunction with muSRK-015P to assess combination effects. The Morpholino has been shown to promote exon skipping such that the disease-causing exon is skipped during the splicing process, resulting in the production of a truncated but partially functional Dystrophin protein. The study design is summarized in Table 15 below.

[0576] Table 15: Study Design.

[0577]

[0578]

[0337] In this study, a more severe model of DMD (D2.mdx) was utilized to model muSRK-015P activity in combination with dystrophin upregulator (M23D-PMO). We have previously shown that muSRK-015P drives muscle mass in multiple models. The D2.mdx mice were dosed for 5 weeks with M32D-PMO or vehicle. Then, in addition to M23D-PMO or vehicle, muSRK-015P or IgG control was administered to the mice at 20 mg / kg weekly for 4 weeks.

[0579]

[0338] As shown in FIG.2A, improvement in muscle quality, as measured by % myocytes with centrally located nuclei in Gastroc, was seen in mice treated with combination of muSRK-015P and PMO.

[0580]

[0339] Next, we examined whether the myostatin-selective inhibitor, either alone or in combination with a Dystrophin upregulator, has an effect on muscle mass in D2.mdx mice. Additive (TA) or synergistic (Gastroc) effects of the combination treatment were seen on muscle mass (FIG. 2B-2C). The data include both left and right dissected muscle.

[0581]

[0340] We then evaluated effects on muscle strength, as measured by force generation as a function of stimulation frequency. Data are summarized in FIGs.3A-3B. In Tibialis Anterior, combination treatment significantly increased force generation, as compared to single-agent treatment groups (either muSRK-015P alone or PMO alone), indicating that muscle strength was enhanced by the combination therapy (FIG.

[0582] 3A). Effects of force improvement on gastroc were minimal (FIG. 3B). Similarly, no effect of muSRK-015P was seen in the diaphragm under the study condition tested (data not shown).

[0583] Specific force in TA muscleAttorney Docket No. 15094.0067-00304

[0584]

[0341] In addition, specific force (muscle force normalized to muscle weight) for tibialis anterior was determined (FIG. 3C) for mice treated with myostatin-selective inhibitor and / or PMO compared to wild-type and (vehicle) control. Animals treated with muSRK-015P in combination with PMO significantly increased TA specific force (“Force / Muscle Weight,” FIG.3C). Treatment with PMO alone resulted in a modest increase in strength of the TA muscle, while muSRK-015P alone had little effect.

[0585]

[0342] Thus, the data demonstrate synergistic effects of myostatin-selective inhibition and dystrophin upregulation in a preclinical DMD mouse model. Moreover, these results indicate that selective inhibition of myostatin in D2.mdx mice treated with a dystrophin upregulator can improve the quality of at least certain muscles, as evidenced by data showing increased specific force (FIG. 3C).

[0586] Total Latent Myostatin

[0587]

[0343] Total serum myostatin (a target engagement marker for muSRK-015P) was measured in treated mice and controls. As shown in FIG. 3D, serum total latent myostatin level was elevated in the samples from mice treated with muSRK-015P but not in those treated with control antibody, indicating target engagement by muSRK-015P. PMO had no effect on total latent myostatin levels. The presence of PMO did not significantly affect the level of muSRK-015P target engagement.

[0588] Gastroc cross-sectional area

[0589]

[0344] Cross-sectional area (CSA) is related to muscle size and volume. It is used as a measure of a muscle’s size and can be used as an indicator for maximal force generated by that muscle. Cross-sectional areas of gastrocnemius muscles of D2.mdx mice in this Example were histologically measured. Frozen tissue samples were embedded in cryomatrix and serially sectioned perpendicular to the fiber axis. Three fixed sections from the mid-belly of the muscle were incubated overnight at 4°C with anti-laminin rat monoclonal antibody (Invitrogen) to visualize cell membranes and an anti-dystrophin (MANDYS8, Peprotech) antibody to detect dystrophin, followed by appropriate secondary antibodies. Following washing steps, sections were digitized using fluorescent microscopy (Kinetix Camera on Nikon Ti2 Inverted Microscope), cell boundary traced using predictive software (Nikon NIS Elements AR GA), dystrophin positive cells counted, and cross-sectional area and myocytes with centrally located nuclei determined via unbiased automated measurements.

[0590]

[0345] Results were analyzed and expressed as the correlation between cumulative frequency of muscle fiber sized and cross-sectional area of muscle fibers. As shown in FIG. 3E, treatment with PMO and muSRK-015P resulted in modest improvements in myofiber cross-sectional area in the gastroc of D2.mdx mice. The observed right-shift indicates fewer small fibers in animals treated with the combination of PMO and muSRK-015P. These findings indicate that myostatin-selective inhibition can increase overall muscle fiber size, and this effect can be augmented by concurrent upregulation of dystrophin.

[0591] Dystrophin protein quantification in gastroc muscles

[0592]

[0346] Levels of dystrophin protein expression in gastroc muscles were determined by western blot. Muscles were homogenized in T-PER™ Tissue Protein Extraction Reagent (Thermo Scientific) using Lysing Matrix D zircon beads in a bead beater (Cryolys Evolution, Bertin Technology), and protein concentration determined. 20 pg of total protein was run on a 4-15% Tris-Glycine gel via SDS-PAGE andAttorney Docket No. 15094.0067-00304

[0593] transferred onto a nitrocellulose membrane. Membranes were stained for total protein per sample using Revert 700 Total Protein Stain and imaged on the LICOR Odyssey CL-x system. Immediately after imaging, the membrane was incubated with Revert destaining solution then rinsed in ultrapure water before blocking (SuperBlock PBS, Thermo Fisher Scientific) for 1 h at room temperature then incubated overnight at 4°C with anti-dystrophin antibody (MANDYS8 mouse monoclonal antibody, Peprotech) at 2pg / ml. Blots were then incubated with appropriate corresponding secondary antibody (1 :5000) for 1 h at room temperature, washed 3x for 5 min with 1x TBS + 0.1% tween 20 and imaged using the LICOR Odyssey CL-x system. Bands were quantified using Imaged and normalized to the total protein signal from 200kDa to 20kDa.

[0594]

[0347] As shown in FIG. 3F, as expected, PMO treatment alone enhanced dystrophin protein levels as assessed by western blot analysis. Surprisingly, the combination of PMO and muSRK-015P further boosted dystrophin protein levels. These findings show unexpected effects of myostatin inhibition on dystrophin protein expression.

[0595] Example 4: Effects of myostatin inhibition in a murine model of FSHD

[0596]

[0348] Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common forms of muscle dystrophy, affecting 1 in about 8,000 people worldwide. Symptoms can emerge anywhere from childhood to adulthood but typically manifest in the 2nd or 3rd decade of life. Disease severity is highly variable as roughly 20% of mutation carriers are asymptomatic. FSHD is characterized by progressive muscle weakness, often starting in the face, shoulders, and upper arms, but also affecting the abdomen, hips, and legs. A distinctive feature of FSHD is the uneven progression of muscle weakness — the condition typically progresses asymmetrically, often affecting only one side of the body at first.

[0597]

[0349] With respect to the genetic basis underlining the pathogenesis, FSHD is an autosomal dominant, gain-of-function disease caused by epigenetic dysregulation of the 4q35 D4Z4 macrosatellite repeats that lead to abnormal myogenic expression of the Double homeobox 4 (DUX4) gene. DUX4 encodes a paired homeobox domain transcriptional activator and regulator of cleavage-stage genes involved in germ cell and early development and is not typically expressed in healthy somatic cells.

[0598]

[0350] In FSHD, DUX4 misexpression in differentiated skeletal muscle ultimately initiates numerous potentially detrimental events including the induction of apoptosis, and activation of the inflammatory immune response.

[0599]

[0351] Here, a murine model of FSHD was used, in which a CRE-based inducible expression system was used to generate mice with controlled DUX4 expression. This transgenic mouse line with conditional floxed DUX4-fl expression is referred to as FLExDUX4. In the absence of ere, mice express a very low level of DUX4-fl mRNA, resulting in mild phenotypes. By contrast, when ere is expressed from the ACTA1 skeletal muscle-specific promoter, the double transgenic animals exhibit a developmental myopathy. When crossed with tamoxifen-inducible ere lines, DUX4-mediated pathology can be induced in adult animals. One of the advantages of this model is that the appearance and progression of pathology can be controlled to provide readily screenable phenotypes useful for assessing therapeutic approaches targeting DUX4-fl mRNA and protein.

[0600]

[0352] To examine the effect of myostatin inhibition in the murine FSHD model, female FLExDUX4.CRE and wild type mice (N=10 per group) were employed. Age of the mice at first dose of antibody (muSRK-Attorney Docket No. 15094.0067-00304

[0601] 015P or isotype-matched control IgG) was 8-10 weeks. A murine version of apitegromab, referred herein to as mSRK-015P or muSRK-015P, or isotype-matched control IgG (referred to as mlgG1 or HuNeg), was dosed at 20 mg / kg per week for 4 weeks. Study endpoints included whole body weight, muscle weight (tibialis anterior [TA] and gastrocnemius [Gastroc]), and in vivo muscle function.

[0602]

[0353] The study design is summarized in Table 16 below.

[0603] Table 16: Study Design.

[0604]

[0605]

[0354] Results are summarized in FIGs. 4A-7D.

[0606]

[0355] As shown in FIG. 4A, there was no significant difference in changes in body weight between study groups 1 and 2. Group 3 animals that received mSRK-015P (myostatin-selective inhibitor) showed increased body weight over the course of the study. Terminal body weight (at study day 28) is shown in FIG. 4B.

[0607]

[0356] At study end (day 28), muscle tissue (TA and Gastroc) was dissected and weighed. As shown in FIG.5A, Gastroc weight was significantly increased by mSRK-015P treatment, as compared to mice treated with IgG control. TA weight showed a trend toward an increase in muscle weight but was not statistically significant (FIG. 5B).

[0608]

[0357] On day 28, muscle force was measured from isolated TA and Gastroc muscles as a function of electrical stimulation at given frequencies.

[0609]

[0358] Results are summarized in FIGs. 6A-6D. Observed maximum force generation of TA was significantly increased by mSRK-015P treatment, as compared to IgG control, although the level of feree generation did not reach that of WT group (FIG. 6A). This was consistent when the measured maximum force was normalized to whole body weight (“Max / BW,” FIG. 6B) or normalized to muscle length (“Max / Length,” FIG. 6D). Surprisingly, when TA maximum force was normalized to muscle weight (e.g., “specific force”), however, the mSRK-015P group was indistinguishable from the WT group (“Max / MW,” FIG. 6C).

[0610]

[0359] Gastroc force was found to be slightly increased by mSRK-015P treatment, as shown in FIG.7.

[0611] Absolute / measured max force is shown in FIG. 7A. Gastroc max force was also normalized to body weight (“Max / BW,” FIG. 7B), to muscle weight (“Max / MW,” FIG. 7C); and to muscle length (“Max / Length,” FIG.

[0612] 7D).

[0613]

[0360] Furthermore, TUNEL+ apoptotic cells were reduced, indicating a reduction in cell death. These results suggest that muSRK-015P treatment may have positive effects on the extent of cell death in skeletal muscle in the FLExDUX4.CRE mice, in addition to increases in muscle size and strength.Attorney Docket No. 15094.0067-00304

[0614]

[0361] These results indicate that selective inhibition of myostatin in FSHD mice can improve the quality (e.g. improve the specific force generated by a muscle (muscle force normalized to muscle weight) as in FIG. 6C) of at least certain muscles.

[0615] Pharmacokinetic and pharmacodynamic analysis of muSRK-015P in FSHD mice.

[0616]

[0362] To evaluate PK, serum concentrations of muSRK-015P were measured in mice treated with 20 mg / kg of the antibody or the control IgG. FIG. 8, Left panel, shows mean concentrations of muSRK-015P of greater than 100 ng / mL at day 7 and day 28 (for latent myostatin, “Terminal”), showing consistent serum exposure over the course of the study duration.

[0617]

[0363] To assess target engagement, total serum latent myostatin (a target engagement marker for muSRK-015P) was measured in mice treated with 20 mg / kg muSRK-015P or control IgG. As shown in FIG.

[0618] 8, Right panel, serum total latent myostatin level was elevated in the samples from mice treated with muSRK-015P but not in those treated with control antibody (Isotype), indicating target engagement by muSRK-015P.

[0619] Example 5: Effects of myostatin inhibition in a murine model of FSHD (mice aged 8 months old): Improvements in muscle mass, strength and endurance

[0620]

[0364] The same FLExDUX4 mouse model of FSHD from Example 4 was used in this Example. A main difference between the studies is the use of older (aged 8 months) mice in this study, compared to 2-month old mice in Example 4.

[0621]

[0365] To examine the effect of myostatin inhibition in the murine FSHD model, female FLExDUX4.CRE and wild type mice (N=8 per group for FLExDUX4.CRE mice, N=7 for wild-type) were employed. Age of the mice at first dose of antibody (muSRK-015P or isotype-matched control IgG) was 8 months. A murine version of apitegromab, referred to as mSRK-015P or muSRK-015P, or isotype-matched control IgG (referred to as mlgG1 or HuNeg), was dosed at 20 mg / kg per week for 4 weeks. Study endpoints included whole body weight, muscle weight (tibialis anterior [TA] and gastrocnemius [Gastroc]), and in vivo muscle function.

[0622]

[0366] The study design is summarized in Table 17 below.

[0623] Table 17: Study Design.

[0624]

[0625]

[0367] Results are summarized in FIGs. 9-12 and below.

[0626] Muscle weight and muscle force

[0627]

[0368] At study end (day 28), muscle tissue (TA and Gastroc) was dissected and weighed. As shown in FIG. 9, Left panel, Gastroc weight was slightly increased by mSRK-015P treatment, as compared to miceAttorney Docket No. 15094.0067-00304

[0628] treated with IgG control, but was not statistically significant. TA weight showed a trend toward an increase in muscle weight compared to control but was not statistically significant (FIG. 9, Right panel).

[0629]

[0369] On day 28, maximum force was measured from isolated TA and Gastroc muscles as a function of electrical stimulation at given frequencies.

[0630]

[0370] Results are summarized in FIG. 10 and FIG. 11. Gastroc absolute / measured max force was found to be similar in mSRK-015P and control treated mice, as shown in FIG. 10. Left panel. Gastroc max force normalized to muscle weight (“Specific Force”) also was similar in mSRK-015P and control treated mice FIG. 10, Right panel). Observed maximum force generation of TA was significantly increased by mSRK-015P treatment, as compared to IgG control, although the level of force generation did not reach that ofWT group (FIG. 11, Left panel). Specific force (maximum force normalized to muscle weight) was also slightly increased, although this was a non-significant effect (FIG. 11, Right panel).

[0631]

[0371] These results indicate that selective inhibition of myostatin in FSHD mice can improve the quality (e.g. improve the specific force generated by a muscle (muscle force normalized to muscle weight) as in FIGs. 10 and 11) of at least certain muscles.

[0632] muSRK-015P Increases Treadmill Running Time and Distance to exhaustion in 8-month FLEx.DUX4 Mice

[0372] At study termination, FLEx.DUX4.CRE mice, or CRE WT littermates (control), were subjected to a treadmill test with increasing speeds over time. Whereas CRE WT control mice were able to run for an average of 31.2 minute and cover a distance of 458 m, FLEx.DUX4.CRE mice treated with isotype control were only able to run for an average of 23.2 minutes and cover a distance of 280.7 m, demonstrating that the genetic defects of the mice contributed to lower time and distance to exhaustion. By contrast, FLEx.DUX4.CRE mice who had received 20 mg / kg muSRK-015P once weekly for 4 weeks achieved improvements in both running time and distance, to averages of 28.4 minutes and 383.6 m respectively (FIG. 12, Table 18).

[0633] Table 18: Treadmill Running Time and Distance to Exhaustion.

[0634]

[0635]

[0373] These results suggest that selective myostatin inhibition with muSRK-015P was able to increase muscle function in 8-month old FLEx.DUX4 CRE mice, resulting in enhanced performance, as evidenced by greater running capacity during a graded treadmill test.Attorney Docket No. 15094.0067-00304

[0636] Pharmacokinetic and pharmacodynamic analysis ofmuSRK-015P in FSHD mice

[0637]

[0374] To evaluate PK, serum concentrations of muSRK-015P were measured in mice treated with 20 mg / kg of the antibody. FIG. 13, Left panel, shows mean concentrations of muSRK-015P of 100 ug / mL or greater at day 7 and end of study (“Term”), showing consistent serum exposure over the course of the study duration. These results are consistent with results from 8-10-week-old FLExDUX4 mice above.

[0638]

[0375] To assess target engagement, serum total latent myostatin (a target engagement marker for muSRK-015P) was measured in mice treated with 20 mg / kg muSRK-015P or control IgG. As shown in FIG. 13, Right panel, serum total latent myostatin level (ng / mL) was elevated in the samples from mice treated with muSRK-015P but not in those treated with control antibody (Isotype), indicating target engagement by muSRK-015P. Circulating latent myostatin levels are mildly reduced in the FLExDUX4 mice compared to wild-type, consistent with a dystrophic phenotype.

[0639] Example 6: Histological studies in a murine model of FSHD (mice aged 8-10 weeks old or 8 months old): Effects of myostatin inhibition on macrophages and satellite cells

[0640]

[0376] Histological analyses were performed on samples from the mice treated in Examples 4 and 5 (FLEx.DUX4 mouse model of FSHD, at 8-10 weeks of age [Example 4] and at 8 months of age [Example 5]).

[0641] Histology

[0642]

[0377] Samples frozen for histology were embedded in cryomatrix on a soft cork surface to enable easy sectioning. Briefly, frozen and embedded tissues were mounted in a cryotome and serially sectioned perpendicular to the fiber axis. The slices are then kept at -80°C until further use.

[0643] Sirius Red

[0644]

[0378] For fibrosis determination, sections from the mid-belly of the muscle (gastrocnemius) were stained with Sirius Red according to manufacturer’s instructions. Stained sections were digitized and %Area of Fibrosis quantified according to color via unbiased automated measurements.

[0645] Cross-sectional Area, Centrally Located Nuclei, Satellite Cells

[0646]

[0379] For cross-sectional area determination, three fixed sections from the midbelly of the right gastrocnemius muscle were stained with anti-laminin antibody conjugated to a fluorophore to visualize cell membranes. Nuclei were visualized with DAPI s...

Claims

1. Attorney Docket No. 15094.0067-00304CLAIMS1. A myostatin inhibitor for use in the treatment of facioscapulohumeral muscular dystrophy (FSHD) in a patient in need thereof,wherein the treatment comprises administration of a myostatin inhibitor to the patient in an amount effective to treat FSHD, andwherein the patient has a 10-meter walk / run test (1 OMWRT) time of less than or equal to 5 seconds at baseline; wherein optionally the patient has a Ricci Clinical Severity Scale score of 0.5 to 4 (e.g. 1.5 to 3.0) at baseline.

2. A myostatin inhibitor for use in the treatment of facioscapulohumeral muscular dystrophy (FSHD) in a patient in need thereof,wherein the treatment comprises administration of a myostatin inhibitor to the patient in an amount effective to treat FSHD, andwherein the patient has at least two of the following at baseline:a) a Ricci Clinical Severity Scale score of 0.5 to 4 (e.g. 1.5 to 3.0);b) a 10-meter walk / run test (1 OMWRT) time of less than or equal to 5 seconds; and c) a timed-up-and-go (TUG) time of 20 seconds or less (e.g., 16 seconds or less, 14 seconds or less, 12 seconds or less).

3. The myostatin inhibitor for use according to claim 1 or claim 2, wherein the patient is 16-65 years of age (e.g. 16-60 years of age, 18-60 years of age) at screening or at initiation of the administration of the myostatin inhibitor.

4. The myostatin inhibitor for use according to any one of claims 1-3, wherein the patient has a baseline Ricci Clinical Severity Scale score of less than 2.5, e.g., about 1.5 to 2.4, and / or has about 4-10 D4Z4 repeat units in at least one allele of the DUX4 gene.

5. The myostatin inhibitor for use according to any one of claims 1-4, wherein the patient has mild-to-moderate or later-onset FSHD.

6. The myostatin inhibitor for use according to any one of claims 1-3, wherein the patient is diagnosed with, exhibits, or progressed into a moderate to severe phenotype of FSHD, and / or the patient has early-onset (childhood-onset or infantile) FSHD.

7. The myostatin inhibitor for use according to any one of claims 1-6, wherein the patient has been genetically diagnosed with FSHD1 or FSHD2.

8. The myostatin inhibitor for use according to any one of claims 1-7, wherein the myostatin inhibitor is a myostatin-selective inhibitor, wherein optionally the myostatin-selective inhibitor is selected from:i) an antibody that selectively binds to myostatin but does not bind to Activin A or GDF11 ; ii) an antibody that selectively binds to mature myostatin;iii) an antibody that selectively binds to pro / latent myostatin, thereby inhibiting myostatin activation; orAttorney Docket No. 15094.0067-00304iv) a nucleic acid-based agent that blocks expression of endogenous myostatin.

9. The myostatin inhibitor for use according to claim 8, wherein the myostatin-selective inhibitor is selected from apitegromab, GYM329, trevogrumab, SRK-439, or a variant thereof.

10. The myostatin inhibitor for use according to claim 8 or claim 9, wherein the myostatin-selective inhibitor is apitegromab.

11. The myostatin inhibitor for use according to claim 10, wherein the apitegromab is administered to the patient at a dose of 2-20 mg / kg (e.g., 10 mg / kg).

12. The myostatin inhibitor for use according to claim 11 , wherein the apitegromab is administered to the patient once every four weeks or once a month.

13. The myostatin inhibitor for use according to any one of claims 1-12, wherein the myostatin inhibitor is administered to the patient intravenously.

14. The myostatin inhibitor for use according to any one of claims 1-13, wherein the myostatin inhibitor is administered to the patient as a monotherapy.

15. The myostatin inhibitor for use according to any one of claims 1 -13, wherein the patient is further administered a DUX4 inhibitor or wherein the patient is on a DUX4 inhibitor therapy, optionally wherein the DUX4 inhibitor is an siRNA that blocks DUX4 expression (e.g., delpacibart braxlosiran ora TfR1-binding Fab conjugated to an siRNA against DUX4 (e.g., DYNE-302)).

16. The myostatin inhibitor for use according to any one of claims 1-15, wherein the treatment increases total lean muscle volume (LMV) in the patient as compared to baseline, optionally wherein the total LMV is measured by a whole-body Magnetic Resonance Imaging (MRI) scan.

17. The myostatin inhibitor for use according to any one of claims 1-16, wherein the treatment improves motor function in the patient as compared to baseline, optionally wherein motor function is measured by a quantitative myometry test, a 10-meter walk / run test (10WMRT), a Timed Up and Go test, a 5xsit-to-stand test (5x STS), and / or an exercise questionnaire.

18. The myostatin inhibitor for use according to any one of claims 1-17, wherein the treatment increases exercise capacity and / or endurance in the patient as compared to baseline, optionally wherein the increase in exercise capacity and / or endurance is measured by a graded treadmill test, e.g. as measured by time and / or distance to exhaustion.

19. The myostatin inhibitor for use according to any one of claims 1-18, wherein the treatment increases muscle function in the patient as compared to baseline, wherein optionally the increase in muscle function comprises an increase in a max force level of a muscle (e.g., tibialis anterior (TA) muscle) in the patient.Attorney Docket No. 15094.0067-0030420. The myostatin inhibitor for use according to any one of claims 1-19, wherein the treatment increases muscle mass in the patient as compared to baseline, optionally wherein the treatment increases the muscle mass of a gastrocnemius muscle in the patient.

21. The myostatin inhibitor for use according to any one of claims 1-20, wherein the amount effective to treat FSHD is an amount sufficient to achieve any one or more of the following:i) increase muscle mass in the patient as compared to baseline,ii) increase muscle function in the patient as compared to baseline,iii) increase exercise capacity and / or endurance in the patient as compared to baseline, iv) increase total lean muscle volume (LMV) in the patient as compared to baseline, and v) delay disease progression.

22. The myostatin inhibitor for use according to any one of claims 1-21, wherein the patient has mild-to-moderate FSHD.

23. The myostatin inhibitor for use according to any one of claims 1-22, wherein the patient has later-onset FSHD.

24. A myostatin inhibitor for use in the treatment of Becker muscular dystrophy (BMD) in a patient in need thereof,wherein the treatment comprises administration of a myostatin-selective inhibitor in an amount effective to treat BMD, andwherein the myostatin-selective inhibitor is administered to the patient as a monotherapy.

25. A myostatin inhibitor for use in the treatment of Duchenne muscular dystrophy (DMD) in a patient in need thereof,wherein the treatment comprises administration of a myostatin-selective inhibitor in an amount effective to treat DMD,wherein the patient is treated with a dystrophin upregulator, andwherein the patient has a dystrophin expression level at screening or initiation of administration of the myostatin-selective inhibitor sufficient to retain motor function, optionally wherein the retention of motor function is as measured by a North Star Ambulatory Assessment (NSAA) score of 17 or greater prior to the administration of the myostatin-selective inhibitor and / or a TTR (“time to rise”) of 4-8 seconds prior to the administration of the myostatin-selective inhibitor, and wherein optionally the patient is 7 years of age or younger (e.g., between 4-7 years old) at the time of screening for or initiation of a myostatin inhibitor therapy.

26. A myostatin inhibitor for increasing exercise capacity and / or endurance in a subject suffering from a muscular dystrophy, comprising administration of a myostatin-selective inhibitor to the subject in an amount effective to increase the exercise capacity and / or endurance of the subject.Attorney Docket No. 15094.0067-0030427. The myostatin inhibitor for use according to claim 25, wherein the dystrophin upregulator comprises a pharmaceutical agent aimed to increase dystrophin expression, wherein optionally the dystrophin upregulator comprises an exon-skipping agent or a gene therapy.

28. The myostatin inhibitor for use according to any one of claims 24-27, wherein the myostatin inhibitor is a myostatin-selective inhibitor, wherein further optionally the myostatin-selective inhibitor is selected from apitegromab, GYM329, trevogrumab, SRK-439, ora variant thereof.

29. The myostatin inhibitor for use according to any one of claims 25-27, wherein the treatment increases muscle mass in the patient as compared to baseline, optionally wherein the treatment increases the muscle mass of a gastrocnemius or tibialis anterior (TA) muscle in the patient.

30. The myostatin inhibitor for use according to any one of claims 25-28, wherein the treatment increases muscle function in the patient as compared to baseline, optionally wherein the increase in muscle function comprises an increase in a max force level of a muscle (e.g., tibialis anterior (TA) muscle) in the patient.

31. The myostatin inhibitor for use according to any one of claims 24-30, wherein the amount effective to treat DMD is an amount sufficient to delay disease progression and / or an amount effective to enhance dystrophin expression as compared to dystrophin upregulator as monotherapy.

32. The myostatin inhibitor for use according to any one of the preceding claims, wherein the patient undergoes moderate strength training and / or aerobic exercise as part of the treatment regimen.

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