Selective and potent inhibitory antibodies for myostatin activation

Abstract

The present disclosure relates to antibodies and antigen-binding fragments that specifically bind to pro / latent myostatin and uses thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 476,908, filed December 22, 2022, U.S. Provisional Patent Application No. 63 / 477,552, filed December 28, 2022, U.S. Provisional Patent Application No. 63 / 515,267, filed July 24, 2023, and U.S. Provisional Patent Application No. 63 / 588,081, filed October 5, 2023, each of which is incorporated by reference herein in its entirety for all purposes.

[0002] This application relates generally to novel myostatin-inhibiting antibodies and their use in the treatment of disorders, including metabolic and neuromuscular disorders. The disclosure further relates to novel adjunctive and combination therapies for improving metabolic health. [Background technology]

[0003] Myostatin (also known as growth differentiation factor-8 or GDF8) is a member of the TGFβ superfamily of cytokines and, in humans, is encoded by the MSTN gene. Like other members of the TGFβ superfamily, myostatin is a homodimer initially expressed as an inactive precursor polypeptide (termed promyostatin). In the overall structure of promyostatin, the mature growth factor is held anchored in the prodomain, a cage-like structure composed of two α-helices connected by a loop termed the "latent lasso" (see, e.g., PCT / US2014 / 036933). The amino acid sequence of the human myostatin polypeptide corresponds to UniProt Accession No. O14793; the mouse counterpart corresponds to UniProt Accession No. O08689. Myostatin activation involves two separate protease cleavage steps. The first cleavage event in myostatin activation involves furin cleavage of promyostatin between the prodomain and the growth factor domain, resulting in "latent myostatin," while mature myostatin remains noncovalently associated with the prodomain and is protected from binding to its receptor by the prodomain. A second cleavage event by the BMP-1 / tolloid family of proteases (e.g., mammalian tolloid-like 2 (mTLL-2)) triggers activation, resulting in the release of mature, active myostatin growth factor from the latent complex. Following activation, mature myostatin signals by binding to a complex of type I and type II cell surface receptors (Alk4 / 5 and ActRIIB), the downstream signaling of which induces muscle atrophy and atrophy.

[0004] Due to its central role as a negative regulator of muscle mass and its involvement in metabolic regulation, myostatin has been implicated in muscle and metabolic disorders. However, clinical programs evaluating various myostatin inhibitors in many muscle indications have failed and been discontinued, calling into question their therapeutic potential. Myostatin inhibitors that have failed or been discontinued in clinical trials to date include neutralizing monoclonal antibodies against mature myostatin, such as stamulusab / MYO-029 (evaluated in Becker muscular dystrophy (BMD), facioscapulohumeral muscular dystrophy (FSHD), and limb-girdle muscular dystrophy (LGMD), domaglozumab / PF-06252616 (evaluated in Duchenne muscular dystrophy (DMD)), landlozumab / LY2495655 (evaluated in cachexia associated with pancreatic cancer and osteoarthritis after total hip replacement), trevoglumab / REGN1033 (evaluated in inclusion body myositis (sIBM)); soluble ActRIIB ligand traps, such as ramaglobin (RMA) These include Tercept / ACE-031 (evaluated in sIMB); follistatin-Fc constructs, such as ACE-083 (evaluated in FSHD and Charcot-Marie-Tooth disease); anti-myostatin Adnectins, such as BMS-986089 / RG6202 / RO-7239361 (evaluated in DMD); anti-ActRIIB antibodies, such as bimagrumab / BYM338 (evaluated in sIBM, etc.); follistatin gene therapy, such as AAAVI.CMV.F344 and rAAVI.CMV.huFollistatin334 (evaluated in BMD, sIBM, and DMD); and anti-myostatin peptibodies, such as AMG-745 (evaluated in age-related muscle weakness).

[0005] The anti-ActRIIB antibody, bimagrumab, has been shown to reduce total body fat mass and increase lean mass in obese patients with type 2 diabetes (Heymsfield et al. 2021). However, antagonizing the ActRII receptor with bimagrumab inhibits not only myostatin but also other structurally similar ligands, including GDF11 and activin, the latter of which plays a role in regulating follicle-stimulating hormone secretion. Therefore, it is unclear whether the observed effects were due to myostatin inhibition, other ligands, or a combination thereof. Notably, Muramatsu et al. (Sci Rep. 2021 Jan 25;11(1):2160) reported that GDF11 inhibition adversely affected muscle strength in preclinical models, raising the possibility that blockade of a common receptor may actually be harmful. While myostatin inhibition and follistatin overexpression have been shown to increase muscle mass, overexpression of follistatin in mice has been reported to result in altered bone structure and dysregulated bone metabolism. See Suh et al. (Proc Natl Acad Sci US A. 2020 Mar 3;117(9):4910-4920) and Chang et al. (JBMR Plus. 5(4):e10477). This may be due to follistatin's broad inhibition of myostatin, activin, and GDF11. Furthermore, based on knockout studies, there are concerns about toxicity associated with inhibiting GDF11 and activin A. For example, inhibiting GDF11 signaling can adversely affect bone (Suh et al. Proc Natl Acad Sci (2020) 117:4910). Patients with nonsense, frameshift, or missense variants in GDF11 have presented with craniofacial, vertebral, neurological, cardiac, auditory, and connective tissue abnormalities (Ravenscroft et al. Genet Med (2021) 23:1889).Furthermore, bimagrumab has been shown to significantly reduce follicle-stimulating hormone (FSH) in women, and clinical trials of bimagrumab have required women of childbearing age to use multiple forms of contraception (Garito et al. Diabetes Obes Metab. 2018; 20(1):94-102). Therefore, selectivity when targeting myostatin is beneficial to drive efficacy in increasing or maintaining muscle mass while avoiding potential toxicity resulting from inhibiting the signaling of closely related factors.

[0006] Currently, apitegromab remains the only selective myostatin inhibitor that has demonstrated efficacy and safety in a Phase 2 human clinical trial enrolling SMA patients (TOPAZ; NCT03921528). Apitegromab is being studied as an intravenous (iv) formulation in an ongoing Phase 3 trial (SAPPHIRE; NCT05156320), which is suitable for diseases such as SMA. However, the subcutaneous administration route may be a more attractive option for adults and / or outpatients or patients suffering from certain other diseases. Therefore, there remains an unmet need for potent and selective myostatin inhibitors to treat these diseases. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure provides, inter alia, novel antibodies and antigen-binding fragments thereof that are highly selective and highly potent inhibitors of myostatin activation, and therapeutic uses thereof. In some embodiments, the antibodies disclosed herein are suitable for subcutaneous administration, e.g., due at least in part to their high potency. Further disclosed herein are novel adjunctive and combination therapies that include myostatin-selective inhibitors (e.g., novel medical uses of myostatin-selective inhibitors) for the treatment of metabolic disorders such as obesity and type 2 diabetes, e.g., in combination with additional therapeutic agents and / or diet and exercise. Also disclosed are uses in the treatment of cardiometabolic diseases (e.g., cardiovascular disease, metabolic disorders such as obesity and type 2 diabetes, inflammatory diseases, chronic inflammation, chronic kidney disease, and fatty liver disease), as well as muscle disorders (e.g., spinal muscular atrophy, muscular dystrophy, and spinal cord injury), glycogen storage diseases, bone disorders (e.g., bone loss), and brain disorders (e.g., Alzheimer's disease, Parkinson's disease, and stroke).

[0008] Previously, the present applicant has disclosed antibodies that selectively bind to latent myostatin, thereby preventing the activation process of myostatin. See, for example, PCT / US2015 / 059468 and PCT / US2016 / 052014, the contents of which are incorporated herein in their entireties. The crystal structure of one such antibody bound to an antigen revealed that both arms of the antibody interact with the homodimeric prodomain to form a cyclic, stable complex with a 1:1 binding stoichiometry. See Dagbay et al. J Biol Chem. 2020 Apr 17;295(16):5404-5418, the contents of which are incorporated herein in their entirety. This is consistent with the observation that mAb (i.e., bivalent) affinity is significantly greater than Fab (i.e., monovalent) affinity, indicating that bivalent binding can provide substantial avidity leading to inhibitory activity. The epitope on the prodomain was found to be distal to the BMP-1 / thoroid proteolytic cleavage site in the prodomain required for myostatin activation, indicating that allosteric antibody binding inhibits protease-dependent activation of latent myostatin. Indeed, despite relatively weak monovalent affinity, the antibody shows potent efficacy in vivo in multiple preclinical models.

[0009] The identification of the inhibitory epitopes described above provided target regions for the development of additional antibodies, including those that compete (e.g., cross-block) with the above-described antibodies for binding. Accordingly, the present applicant sought to discover additional novel inhibitory antibodies, i.e., cross-competing antibodies, including those that bind to the same or substantially overlapping regions of the myostatin prodomain, particularly those that compete with Ab2 for binding to promyostatin (disclosed in PCT / US2016 / 052014 and PCT / US2015 / 059468). Over 30 distinct antibodies were identified, which were subsequently confirmed to exhibit greater inhibitory potency than Ab2 against protease-induced myostatin activation. Modifications to portions of these antibody sequences were also evaluated for various properties, as described herein. Among these, subclasses of antibodies with unexpected properties have been identified. Surprisingly, in addition to possessing higher affinity, these novel antibodies exhibit distinct properties, e.g., with respect to one or more of binding stoichiometry, pH sensitivity, and serum myostatin clearance behavior. In certain embodiments of the present disclosure, the novel antibodies or antigen-binding fragments thereof bind to the same or overlapping epitope as the prior art reference antibodies described above. In some embodiments, the novel antibodies bind to the same region of pro / latent myostatin as Ab2, but unexpectedly, bind in a "one-arm" manner while retaining high affinity and inhibitory potency (e.g., IC50 less than 1 nM as measured by functional ELISA as detailed herein). These surprising features raise the possibility that the novel antibodies / antigen-binding fragments disclosed herein may be utilized to engineer multispecific constructs, such as bispecific antibodies.

[0010] Thus, in some embodiments, the present disclosure encompasses antibodies or antigen-binding fragments thereof that bind to human pro / latent myostatin but do not bind to mature myostatin or GDF11, where binding can inhibit myostatin activation, and where the antibody or antigen-binding fragment binds to the same epitope as Ab2 provided in PCT / US2015 / 059468 and / or competes for antigen binding with such Ab2, and / or the antibody or antigen-binding fragment binds to human pro / latent myostatin at / or near amino acid positions 147-170 and / or amino acid positions 205-210, as numbered according to the pro-GDF8 sequence (SEQ ID NO: 52) provided herein. In some embodiments, any of the above-described antibodies or fragments may be characterized as follows: i) the sum of the combined heavy and light chain variable domains (i.e., cumulative VH+VL) shares less than 70% sequence identity with that of Ab2; ii) the heavy chain sequence shares less than 90%, 80%, or 70% sequence identity with that of Ab2; iii) the VL sequence of the antibody shares less than 50% sequence identity with the VL sequence of Ab2; iv) the L-CDR1 shares 25% or less (e.g., 20% or less) sequence identity with the L-CDR1 sequence of Ab2; v) the L-CDR2 shares less than 30% sequence identity with the L-CDR2 of Ab2; and / or vi) the L-CDR3 shares 20% or less (e.g., 10% or less) sequence identity with the L-CDR3 of Ab2.

[0011] In some embodiments, the antibodies or antigen-binding fragments disclosed herein bind to human pro / latent myostatin with a KD of less than 1 nM (e.g., a KD of less than 0.7 nM, less than 0.5 nM, or less than 0.2 nM) as measured by a suitable in vitro binding assay such as surface plasmon resonance (SPR) (e.g., Biacore™), biolayer interferometry (BLI) (e.g., Octet®), and / or solution equilibrium titration (e.g., MSD-SET). In some embodiments, the KD is determined by an SPR-based assay (e.g., Biacore™).

[0012] In some embodiments, the antibody or antigen-binding fragment has an IC50 (mTLL2 IC 50 ) can inhibit mammalian tolloid-like 2 (mTLL-2)-induced activation of myostatin.

[0013] In some embodiments, the antibodies or antigen-binding fragments disclosed herein bind to human pro / latent myostatin in a pH-dependent manner, optionally with a pH-dependence of greater than 10-fold as determined by comparing the dissociation rates at pH 5.5 / 7.4, where the dissociation rates are measured by a BLI-based assay (e.g., Octet®).

[0014] In some embodiments, the present disclosure provides antibodies or antigen-binding fragments that can monovalently bind to an antigen (e.g., a latent myostatin complex) with a monovalent KD of 50 nM or less, as measured, for example, by a BLI-based in vitro binding assay or an SPR-based in vitro binding assay. In some embodiments, a monoclonal antibody of the present disclosure binds to human pro / latent myostatin at a 1:2 antibody-to-antigen stoichiometry. A monoclonal antibody of the present disclosure binds to human pro / latent myostatin both at a 1:2 antibody-to-antigen stoichiometry and via daisy chain formation. In some embodiments, a monoclonal antibody of the present disclosure binds to human pro / latent myostatin via daisy chain formation. In some embodiments, a Fab fragment of a monoclonal antibody of the present disclosure binds to human pro / latent myostatin at a 2:1 Fab-to-antigen stoichiometry. In some embodiments, a Fab fragment of a monoclonal antibody of the present disclosure binds to human pro / latent myostatin at a 1:1 Fab-to-antigen stoichiometry. In some embodiments, the antibody binds to human pro / latent myostatin at a 1:2 mAb:Ag binding stoichiometry as measured by analytical SEC-MALS, where the mAb and Ag are present (e.g., in a mAb:Ag mixture) in a 1:1, 2:1, or 3:1 ratio, at a total protein concentration ranging from about 3.5 mg / mL (e.g., about 15 μM mAb and Ag, respectively) to about 8 mg / mL (e.g., about 45 μM mAb and about 15 μM Ag), and the immune complex is allowed to form at room temperature at neutral pH for a suitable period of time, such as 1 to 48 hours, preferably about 24 hours. In some embodiments, the mAb:Ag mixture further comprises an oligomeric complex comprising a 2:1 mAb:Ag complex and / or a 2:2 mAb:Ag complex. In some embodiments, the mAb:Ag mixture does not contain detectable levels of polydaisy chains as measured by analytical SEC-MALS. In some embodiments, when the antibody and antigen are mixed at 15 μM each and allowed to form an immune complex at neutral pH, the antibody can bind to the antigen at an antibody-to-antigen stoichiometry of 1:2, as measured by analytical size exclusion chromatography (SEC) (e.g., SEC-MALS).

[0015] In some embodiments, the antibodies or antigen-binding fragments disclosed herein (e.g., Ab109, Ab133, Ab141) do not cause the accumulation of circulating myostatin (e.g., total or latent myostatin in serum). While certain prior art myostatin-selective activation inhibitors, such as apitegromab, cause the accumulation of latent myostatin (e.g., latent myostatin-antibody immune complexes) in serum (i.e., circulating myostatin), the antibodies disclosed herein (e.g., Ab109) can, in some embodiments, reduce total serum myostatin levels in a subject compared to background levels. In some embodiments, the antibodies or antigen-binding fragments of the present disclosure cause a rapid decrease in circulating (i.e., serum) free latent myostatin. In some embodiments, when mice are administered a single dose of 2 to 20 mg / kg of antibody, free latent myostatin levels drop from background (e.g., about 50 ng / mL) to below detectable levels (e.g., within 1 day of administration) and remain at undetectable or nearly undetectable levels (e.g., for at least 42 days).

[0016] In some embodiments, the antibody binds to human pro / latent myostatin with a 1:2 mAb:AG binding stoichiometry as measured by analytical SEC-MALS. In some embodiments, serum myostatin levels can be determined in mice (e.g., as described in Example 2). In some embodiments, serum myostatin levels can be determined in humans. In some embodiments, the antibody or antigen-binding fragment binds with a 1:2 antibody / fragment to antigen stoichiometry. In some embodiments, an antibody disclosed herein (e.g., Ab109) can reduce serum concentrations of total or latent myostatin. Without wishing to be bound by theory, it is believed that faster serum clearance may correlate with larger immune complexes (e.g., polydaisy chains) formed in vivo, and that larger immune complex formation (e.g., oligomers) may facilitate clearance, for example, by increased FcRn interaction. Advantageously, such enhanced clearance can be achieved or improved without engineering the antibody by introducing mutations in the Fc region (e.g., Muramatsu et al. Sci Rep. 2021;11:2160), thus minimizing the risk of unwanted immunogenicity.

[0017] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, where CDRH1 comprises the sequence SYGMS (SEQ ID NO: 201), CDRH2 comprises the sequence SFTGSGGX1YYYPDSVKG (SEQ ID NO: 202), where X1 is T or A, CDRH3 comprises the sequence DLLIRFLEWSHYYGMDV (SEQ ID NO: 203), CDRL1 comprises the sequence RSSQSLLHSSGHNFLH (SEQ ID NO: 204), CDRL2 comprises the sequence EVSNRVS (SEQ ID NO: 205), and CDRL3 comprises the sequence X1QQTQYPX2T (SEQ ID NO: 206), where X1 is M or Q and X2 is P or G, and the CDR sequences are numbered according to the Kabat numbering system.

[0018] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, where CDRH1 comprises the sequence SYGMS (SEQ ID NO: 201), CDRH2 comprises the sequence SITGSGGETYYPDSVKG (SEQ ID NO: 207), CDRH3 comprises the sequence DLLVRFLEWSHYYGMDV (SEQ ID NO: 208), CDRL1 comprises the sequence RSSQSLLHSSGHNFLH (SEQ ID NO: 204), CDRL2 comprises the sequence EVSNRVS (SEQ ID NO: 205), and CDRL3 comprises the sequence X1QATQFPRP (SEQ ID NO: 210), where X1 is M or Q, and the CDR sequences are numbered according to Kabat.

[0019] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, where CDRH1 comprises the sequence SYGMS (SEQ ID NO: 201), CDRH2 comprises the sequence SINPSGGTTYYAQKFKG (SEQ ID NO: 211), CDRH3 comprises the sequence DLLVRFLEWSHYYGMDV (SEQ ID NO: 208), and CDRL1 comprises the sequence RX1SQS X2LHSX3X4HNFLH (SEQ ID NO: 212), where X1 is S or A; X2 is I or L; X3 is S or L; and X4 is G or A, CDRL2 comprises the sequence EX1SNX2X3S (SEQ ID NO: 213), where X1 is A or V; X2 is R or L; and X3 is V or A, and CDRL3 comprises the sequence QQX1TQYPPT (SEQ ID NO: 214), where X1 is Q or Y, and the CDR sequences are numbered according to Kabat.

[0020] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, where CDRH1 comprises the sequence SYGMS (SEQ ID NO:201), CDRH2 comprises the sequence SX1TGSGGX2TYYPDSVKG (SEQ ID NO:275), X1 is F or I, and X2 is E or A, and CDRH3 comprises the sequence DLXLEFLEWSHYYGMDV (SEQ ID NO:272), and CD RL1 comprises the sequence RSSQSLLHSSGHNFLH (SEQ ID NO: 204), CDRL2 comprises the sequence ETSNRX1X2 (SEQ ID NO: 276), where X1 is V or A and X2 is P or S, CDRL3 comprises the sequence X1QQX2TQX3PX4X5 (SEQ ID NO: 277), where X1 is M or Q, X2 is Q or A, X3 is Y or F, X4 is R, P, or G, and X5 is T or P, and the CDR sequences are numbered according to the Kabat numbering system.

[0021] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, where CDRH1 comprises the sequence GFTFX1SY (SEQ ID NO: 278), and X is S or T; CDRH2 comprises the sequence TGSGG (SEQ ID NO: 279); and CDRH3 comprises the sequence LLX1RFLEWSHYYGMD (SEQ ID NO: 280), and X is I or V. wherein CDRL1 comprises the sequence SQSLLHSSGHNF (SEQ ID NO:281), CDRL2 comprises the sequence EX1S, where X1 is T or V, CDRL3 comprises the sequence X1X2X3X4X5X6, where X1 is Q, R, or A, X2 is T or P, X3 is Q or F, X4 is Y, F, or G, X5 is P or G, and X6 is G, P, or R, and the CDR sequences are numbered according to the Chothia numbering system. In some embodiments, CDRL3 comprises the sequence QTQYPX1 (SEQ ID NO:293), where X1 is P or G.

[0022] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, where CDRH1 comprises the sequence GFTFTSSYG (SEQ ID NO: 284), CDRH2 comprises the sequence X1TGSGGX2T (SEQ ID NO: 285), where X1 is F or I and X2 is E, T, or A, and CDRH3 comprises the sequence ARDLLVRFLEWSHYYGMDV (SEQ ID NO: 286). ), wherein CDRL1 comprises the sequence QSLLHSGHNF (SEQ ID NO:287), CDRL2 comprises the sequence EX1S (wherein X is T or V), or the sequence EVSNRVS (SEQ ID NO:205), and CDRL3 comprises X1QX2TQX3PX4X5 (SEQ ID NO:288), where X1 is Q or M, X2 is Q or A, X3 is Y or F, X4 is Y, P, or G, and X5 is P or T, and the CDR sequences are numbered according to the IMGT numbering system.

[0023] In some embodiments, the antibody or antigen-binding fragment, when numbered according to the Kabat numbering system, comprises an HCDR1 of SEQ ID NO: 201; an HCDR2 of SEQ ID NO: 202 (wherein X1 is T or A); an HCDR3 of SEQ ID NO: 203; an LCDR1 of SEQ ID NO: 204; an LCDR2 of SEQ ID NO: 205; and an LCDR3 of SEQ ID NO: 206 (wherein X1 is M or Q and X2 is P or G).

[0024] In some embodiments, the antibody or antigen-binding fragment, when numbered according to the Chothia numbering system, comprises an HCDR1 of SEQ ID NO: 293; an HCDR2 of SEQ ID NO: 279; an HCDR3 of SEQ ID NO: 296; an LCDR1 of SEQ ID NO: 281; an LCDR2 of EVS; and an LCDR3 of SEQ ID NO: 297 (wherein X1 is P or G).

[0025] In some embodiments, the antibody or antigen-binding fragment, when numbered according to the IMGT numbering system, comprises an HCDR1 of SEQ ID NO: 293; an HCDR2 of SEQ ID NO: 294 (wherein X1 is T or A); an HCDR3 of SEQ ID NO: 257; an LCDR1 of SEQ ID NO: 258; an LCDR2 of EVS; and an LCDR3 of SEQ ID NO: 292 (wherein X1 is M or Q and X2 is P or G).

[0026] In some embodiments, an anti-pro / latent myostatin antibody or antigen-binding portion thereof suitable for implementing various embodiments of the present disclosure comprises the following six CDRs: CDRH1 comprising GFTFSSYG (SEQ ID NO: 3); CDRH2 comprising FTGSGGX1 (SEQ ID NO: 291) (wherein X1 is selected from T and A); CDRH3 comprising ARDLIRFLEWSHYYGMDV (SEQ ID NO: 257); CDRL1 comprising QSLLHSSGHNF (SEQ ID NO: 258); CDRL2 comprising EVSNRVS (SEQ ID NO: 289); and CDRL3 comprising X1QQTQYPX2T (SEQ ID NO: 292) (wherein X1 is selected from M and Q, and X2 is selected from P and G). In preferred embodiments, CDRH2 comprises FTGSGGT (SEQ ID NO: 256) or FTGSGGA (SEQ ID NO: 262), and / or CDRL3 comprises QQQTQYPGT (SEQ ID NO: 261), MQQTQYPPT (SEQ ID NO: 260), or MQQTQYPGT (SEQ ID NO: 290).

[0027] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, where, as defined by the Kabat numbering system, CDRH1 comprises SEQ ID NO: 201, CDRH2 comprises SEQ ID NO: 214, CDRH3 comprises SEQ ID NO: 215, CDRL1 comprises SEQ ID NO: 216, CDRL2 comprises SEQ ID NO: 217, and CDRL3 comprises any one of SEQ ID NOs: 218 or 224. In some embodiments, preferred antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or a combination thereof, where, as defined by the Kabat numbering system, CDRH1 comprises SEQ ID NO: 201, CDRH2 comprises any one of SEQ ID NOs: 219 or 226, CDRH3 comprises SEQ ID NO: 220, CDRL1 comprises SEQ ID NO: 216, CDRL2 comprises SEQ ID NO: 222, and CDRL3 comprises any one of SEQ ID NOs: 223, 225, or 227.

[0028] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, where, as defined by the Kabat numbering system, CDRH1 comprises SEQ ID NO: 201, CDRL2 comprises SEQ ID NO: 214, CDRH3 comprises SEQ ID NO: 215, CDRL1 comprises SEQ ID NO: 216, CDRH2 comprises SEQ ID NO: 217, and CDRL3 comprises SEQ ID NO: 218. In some embodiments, the antibody or antigen-binding fragment comprises CDRH1 comprising the sequence of SEQ ID NO: 201, CDRH2 comprising the sequence of SEQ ID NO: 214, CDRH3 comprising the sequence of SEQ ID NO: 215, CDRL1 comprising the sequence of SEQ ID NO: 216, CDRL2 comprising the sequence of SEQ ID NO: 217, and CDRL3 comprising the sequence of SEQ ID NO: 224, as defined by the Kabat numbering system. In some embodiments, the antibody or antigen-binding fragment comprises a CDRH1 comprising the sequence of SEQ ID NO: 201, a CDRH2 comprising any one of the sequences of SEQ ID NO: 219 or 226, a CDRH3 comprising the sequence of SEQ ID NO: 220, a CDRL1 comprising the sequence of SEQ ID NO: 216, a CDRL2 comprising the sequence of SEQ ID NO: 222, and a CDRL3 comprising any one of SEQ ID NO: 223, 225, or 227. In some embodiments, the antibody or antigen-binding fragment comprises a CDRH1 comprising the sequence of SEQ ID NO: 201, a CDRH2 comprising the sequence of SEQ ID NO: 219, a CDRH3 comprising the sequence of SEQ ID NO: 220, a CDRL1 comprising the sequence of SEQ ID NO: 216, a CDRL2 comprising the sequence of SEQ ID NO: 222, and a CDRL3 comprising the sequence of SEQ ID NO: 223, as defined by the Kabat numbering system. In some embodiments, the antibody or antigen-binding fragment comprises a CDRH1 comprising the sequence of SEQ ID NO: 201, a CDRH2 comprising the sequence of SEQ ID NO: 219, a CDRH3 comprising the sequence of SEQ ID NO: 220, a CDRL1 comprising the sequence of SEQ ID NO: 216, a CDRL2 comprising the sequence of SEQ ID NO: 222, and a CDRL3 comprising the sequence of SEQ ID NO: 225, as defined by the Kabat numbering system.In some embodiments, the antibody or antigen-binding fragment comprises a CDRH1 comprising the sequence of SEQ ID NO: 201, a CDRH2 comprising the sequence of SEQ ID NO: 226, a CDRH3 comprising the sequence of SEQ ID NO: 220, a CDRL1 comprising the sequence of SEQ ID NO: 216, a CDRL2 comprising the sequence of SEQ ID NO: 222, and a CDRL3 comprising the sequence of SEQ ID NO: 227, as defined by the Kabat numbering system.

[0029] The novel antibodies and antigen-binding fragments thereof of the present disclosure are suitable for therapeutic use in human patients in the treatment of one or more myostatin-related diseases and disorders, including, but not limited to, muscle disorders (e.g., neuromuscular disorders such as atrophy and SMA) and cardiometabolic disorders (e.g., obesity, diabetes, prediabetes, fatty liver, bone disorders, and heart failure). Due in part to their high potency and favorable developability, such antibodies are particularly suitable for subcutaneous formulation. In some embodiments, pharmaceutical compositions comprising such antibodies (or engineered constructs comprising antigen-binding fragments of such antibodies) formulated for subcutaneous administration are used to treat metabolic disorders, optionally including obesity, metabolic syndrome, diabetes, and / or prediabetes. In some embodiments, the antibody is Ab101, Ab102, Ab103, Ab104, Ab105, Ab106, Ab107, Ab108, Ab109, Ab110, Ab111, Ab112, Ab113, Ab114, Ab115, Ab116, Ab117, Ab118, Ab119, Ab120 , Ab121, Ab122, Ab123, Ab124, Ab125, Ab126, Ab127, Ab128, Ab129, Ab130, Ab131, Ab132, Ab133, Ab134, Ab135, Ab136, Ab137, Ab138, Ab139, Ab140 and Ab141. In a preferred embodiment, the antibody is Ab109, Ab133 or Ab141.

[0030] Currently available obesity treatments, such as GLP-1 receptor agonists, primarily focus on weight loss. In contrast, the present disclosure takes into account the quality of weight management beyond simple weight loss (e.g., healthier weight loss) to achieve improved metabolic health. Thus, myostatin inhibitors are incorporated into weight management regimens with the goal of achieving preferential fat mass reduction over lean mass; maintaining reduced fat mass; preventing muscle loss; increasing lean mass; increasing endurance; reducing fatigue; preventing bone loss; improving blood glucose levels; and / or improving liver health. Thus, myostatin inhibitors, such as the novel antibodies and antigen-binding fragments disclosed herein, may contribute to safe and sustainable weight management, particularly when used in conjunction with another therapy aimed at addressing metabolic dysregulation.

[0031]

[0010] Accordingly, the present disclosure provides a myostatin-selective inhibitor for use in treating a metabolic disorder in a patient, the treatment comprising administering to the patient a myostatin-selective inhibitor, alone or together with an additional agent, such as a GLP-1 pathway activator (e.g., a GLP-1 receptor agonist), in an amount effective to treat the metabolic disorder, wherein the myostatin-selective inhibitor is any one of the antibodies or antigen-binding fragments thereof described herein. In some embodiments, the metabolic disorder is obesity, prediabetes, diabetes (e.g., T2D), metabolic syndrome, and / or fatty liver disease. In some embodiments, the antibody is Ab101, Ab102, Ab103, Ab104, Ab105, Ab106, Ab107, Ab108, Ab109, Ab110, Ab111, Ab112, Ab113, Ab114, Ab115, Ab116, Ab117, Ab118, Ab119, Ab120 , Ab121, Ab122, Ab123, Ab124, Ab125, Ab126, Ab127, Ab128, Ab129, Ab130, Ab131, Ab132, Ab133, Ab134, Ab135, Ab136, Ab137, Ab138, Ab139, Ab140 and Ab141. In preferred embodiments, the antibody is Ab109, Ab133 or Ab141, and optionally the GLP-1 receptor agonist is semaglutide, tirzepatide, AMG-133 (a GLP-1 receptor agonist / GIP-1 receptor antagonist being developed by Amgen), or danugliplon (an oral GLP-1 receptor agonist being developed by Pfizer). In some embodiments, the dosage and / or frequency of a GLP-1 receptor agonist effective in treating a metabolic disorder may be reduced when used in combination with a myostatin selective inhibitor disclosed herein.

[0032] In some embodiments, the present disclosure provides methods of treating a metabolic disorder in a subject, the method comprising administering to the subject a myostatin-selective inhibitor (e.g., any one of the antibodies or antigen-binding fragments described herein), and optionally, the subject is or has been administered at least one dose of a GLP-1 receptor agonist and / or metformin. In some embodiments, the subject is administered metformin and is not administered a GLP-1 receptor agonist. In some embodiments, the metabolic disorder is diabetes, obesity, or obesity with diabetes. In some embodiments, the GLP-1 receptor agonist comprises semaglutide. In some embodiments, the antibody is Ab101, Ab102, Ab103, Ab104, Ab105, Ab106, Ab107, Ab108, Ab109, Ab110, Ab111, Ab112, Ab113, Ab114, Ab115, Ab116, Ab117, Ab118, Ab119, Ab120 , Ab121, Ab122, Ab123, Ab124, Ab125, Ab126, Ab127, Ab128, Ab129, Ab130, Ab131, Ab132, Ab133, Ab134, Ab135, Ab136, Ab137, Ab138, Ab139, Ab140, and Ab141. In some embodiments, the myostatin-selective inhibitor comprises Ab109, Ab133, or Ab141. In some embodiments, the myostatin-selective inhibitor is Ab109. In some embodiments, the dosage and / or frequency of the GLP-1 receptor agonist is reduced when used in combination with a myostatin-selective inhibitor disclosed herein.

[0033] In some embodiments, the present disclosure provides methods of treating obesity or improving body composition, comprising administering a myostatin-selective inhibitor (e.g., any one of the antibodies or antigen-binding fragments described herein) to a subject, and optionally, the subject is or has been administered at least one dose of a GLP-1 receptor agonist and / or metformin. In some embodiments, the subject is receiving a GLP-1 receptor agonist. In some embodiments, the subject has discontinued the GLP-1 receptor agonist. In some embodiments, the GLP-1 receptor agonist comprises semaglutide. In some embodiments, the antibody is Ab101, Ab102, Ab103, Ab104, Ab105, Ab106, Ab107, Ab108, Ab109, Ab110, Ab111, Ab112, Ab113, Ab114, Ab115, Ab116, Ab117, Ab118, Ab119, Ab120 , Ab121, Ab122, Ab123, Ab124, Ab125, Ab126, Ab127, Ab128, Ab129, Ab130, Ab131, Ab132, Ab133, Ab134, Ab135, Ab136, Ab137, Ab138, Ab139, Ab140, and Ab141. In some embodiments, the myostatin selective inhibitor comprises Ab109, Ab133, or Ab141. In some embodiments, the myostatin selective inhibitor is Ab109. In some embodiments, the myostatin selective inhibitor is Ab109. In some embodiments, the dosage and / or frequency of the GLP-1 receptor agonist is reduced when used in combination with a myostatin selective inhibitor disclosed herein.

[0034] In some embodiments, the present disclosure provides methods of treating obesity or improving body composition, comprising administering a myostatin-selective inhibitor to a subject who has discontinued treatment with a GLP-1 receptor agonist. In some embodiments, the myostatin-selective inhibitor is selected from the group consisting of Ab101, Ab102, Ab103, Ab104, Ab105, Ab106, Ab107, Ab108, Ab109, Ab110, Ab111, Ab112, Ab113, Ab114, Ab115, Ab116, Ab117, Ab118, Ab119, Ab120, and Abl The antibody or antigen-binding fragment thereof is selected from b121, Ab122, Ab123, Ab124, Ab125, Ab126, Ab127, Ab128, Ab129, Ab130, Ab131, Ab132, Ab133, Ab134, Ab135, Ab136, Ab137, Ab138, Ab139, Ab140, and Ab141. In a preferred embodiment, the antibody is Ab109, Ab133, or Ab141.

[0035] In some embodiments, the present disclosure provides a method for reducing body fat mass regain in a subject after discontinuing treatment with a GLP-1 receptor agonist, comprising administering to the subject an amount of a myostatin inhibitor effective to reduce body fat mass gain compared to a subject who has discontinued GLP-1 receptor agonist therapy but is not treated with a myostatin inhibitor. In some embodiments, the myostatin inhibitor treatment reduces the extent of body fat mass regain after discontinuing GLP-1 receptor agonist therapy. In some embodiments, the myostatin inhibitor treatment reduces the rate of body fat mass regain after discontinuing GLP-1 receptor agonist therapy. In a preferred embodiment, the myostatin inhibitor is a myostatin-selective inhibitor, such as any one of the antibodies or antigen-binding fragments disclosed herein (such as Ab109, Ab133, or Ab141), trevoglumab (REGN1033), and GYM329 (RO7204239) (an anti-latent myostatin Fc-engineered antibody discovered by Chugai and developed by Roche). In a preferred embodiment, the myostatin-selective inhibitor is an antibody or antigen-binding fragment selective for pro / latent myostatin, such as any one of Ab101-141. In a most preferred embodiment, the myostatin-selective inhibitor is Ab109, Ab133, or Ab141.

[0036] In some embodiments, the present disclosure provides methods for reducing liver fat in a subject, e.g., an obese subject and / or a subject with fatty liver disease, comprising administering to the subject an amount of a myostatin-selective inhibitor effective to reduce liver fat. In preferred embodiments, the myostatin-selective inhibitor is used in combination with a GLP-1 receptor agonist for a period sufficient to synergistically reduce relative liver weight (e.g., for a period greater than 5 weeks). In some embodiments, the myostatin-selective inhibitor is selected from the group consisting of Ab101, Ab102, Ab103, Ab104, Ab105, Ab106, Ab107, Ab108, Ab109, Ab110, Ab111, Ab112, Ab113, Ab114, Ab115, Ab116, Ab117, Ab118, Ab119, Ab120, Ab121, Ab122, Ab123, Ab124, Ab125, Ab126, Ab127, Ab128, Ab129, Ab130, Ab131, Ab132, Ab133, Ab134, Ab135, Ab136, Ab137, Ab138, Ab139, Ab140, Ab141, Ab142, Ab143, Ab144, Ab145, Ab146, Ab147, Ab148, Ab149, Ab150, Ab151, Ab152, Ab153, Ab154, Ab155, Ab156, Ab157, Ab158, Ab159, Ab160, Ab161, Ab162, Ab163, Ab164, Ab165, Ab166, Ab167, Ab168, Ab169, Ab170, Ab171, Ab172, Ab173, Ab174, Ab175, The antibody or antigen-binding fragment thereof is selected from b121, Ab122, Ab123, Ab124, Ab125, Ab126, Ab127, Ab128, Ab129, Ab130, Ab131, Ab132, Ab133, Ab134, Ab135, Ab136, Ab137, Ab138, Ab139, Ab140, and Ab141. In a preferred embodiment, the antibody is Ab109, Ab133, or Ab141.

[0037] In some embodiments, the present disclosure provides a myostatin-selective inhibitor for use in treating a metabolic disorder in a patient, wherein the treatment comprises administering to the patient a myostatin-selective inhibitor alone or together with an additional agent suitable for treating the metabolic disorder. In some embodiments, the metabolic disorder is obesity, prediabetes, diabetes (e.g., T2D), metabolic syndrome, and / or fatty liver disease. In some embodiments, the myostatin-selective inhibitor is selected from the group consisting of Ab101, Ab102, Ab103, Ab104, Ab105, Ab106, Ab107, Ab108, Ab109, Ab110, Ab111, Ab112, Ab113, Ab114, Ab115, Ab116, Ab117, Ab118, Ab119, Ab120, Ab121, Ab122, Ab123, Ab124, Ab125, Ab126, Ab127, Ab128, Ab129, Ab130, Ab131, Ab132, Ab133, Ab134, Ab135, Ab136, Ab137, Ab138, Ab139, Ab140, Ab141, Ab142, Ab143, Ab144, Ab145, Ab146, Ab147, Ab148, Ab149, Ab150, Ab151, Ab152, Ab153, Ab154, Ab155, Ab156, Ab157, Ab158, Ab159, Ab160, Ab161, Ab162, Ab163, Ab164, Ab165, Ab166, Ab167, Ab168, Ab169, Ab170, Ab171, Ab172, Ab173, Ab174, Ab175, Ab176, Ab177, Ab178, Ab17 and an antibody or antigen-binding fragment thereof selected from b121, Ab122, Ab123, Ab124, Ab125, Ab126, Ab127, Ab128, Ab129, Ab130, Ab131, Ab132, Ab133, Ab134, Ab135, Ab136, Ab137, Ab138, Ab139, Ab140, and Ab141. In preferred embodiments, the antibody is Ab109, Ab133, or Ab141. In some embodiments, the amount and / or frequency of administration of an additional agent suitable for treating a metabolic disorder may be reduced when used in combination with a myostatin-selective inhibitor disclosed herein.

[0038] In some embodiments, the present disclosure provides methods of improving bone strength and / or preventing bone loss in a subject (e.g., an obese subject), comprising administering to the subject an amount of a myostatin-selective inhibitor effective to improve bone strength and / or prevent bone loss compared to a subject (e.g., an obese subject) not administered the myostatin-selective inhibitor. In some embodiments, bone strength is measured by bone mineral density and / or frequency or severity of fractures. In some embodiments, the subject is undergoing or has undergone GLP-1 receptor agonist therapy. In some embodiments, the subject is undergoing a weight loss regimen. In some embodiments, the subject is undergoing or has undergone a treatment that results in GDF11 inhibition (e.g., a selective or non-selective inhibitor of GDF11). In some embodiments, GDF11 inhibitor treatment is replaced with a myostatin-selective inhibitor treatment (such as the novel antibodies disclosed herein). In some embodiments, the myostatin selective inhibitor is selected from the group consisting of Ab101, Ab102, Ab103, Ab104, Ab105, Ab106, Ab107, Ab108, Ab109, Ab110, Ab111, Ab112, Ab113, Ab114, Ab115, Ab116, Ab117, Ab118, Ab119, Ab120, A The antibody or antigen-binding fragment thereof is selected from b121, Ab122, Ab123, Ab124, Ab125, Ab126, Ab127, Ab128, Ab129, Ab130, Ab131, Ab132, Ab133, Ab134, Ab135, Ab136, Ab137, Ab138, Ab139, Ab140, and Ab141. In a preferred embodiment, the antibody is Ab109, Ab133, or Ab141.

[0039] In some embodiments, the present disclosure provides methods of improving blood glucose or hemoglobin A1C (A1C) levels in a prediabetic or diabetic subject who is or has been receiving a GLP-1 receptor agonist, comprising administering to the subject an amount of a myostatin selective inhibitor effective to reduce blood glucose (e.g., fasting blood glucose) or A1C levels compared to baseline (i.e., before administration of the myostatin selective inhibitor). In some embodiments, the myostatin selective inhibitor is selected from the group consisting of Ab101, Ab102, Ab103, Ab104, Ab105, Ab106, Ab107, Ab108, Ab109, Ab110, Ab111, Ab112, Ab113, Ab114, Ab115, Ab116, Ab117, Ab118, Ab119, Ab120, Ab121, Ab122, Ab123, Ab124, Ab125, Ab126, Ab127, Ab128, Ab129, Ab130, Ab131, Ab132, Ab133, Ab134, Ab135, Ab136, Ab137, Ab138, Ab139, Ab140, Ab141, Ab142, Ab143, Ab144, Ab145, Ab146, Ab147, Ab148, Ab149, Ab150, Ab151, Ab152, Ab153, Ab154, Ab155, Ab156, Ab157, Ab158, Ab159, Ab160, Ab161, Ab162, Ab163, Ab164, Ab165, Ab166, Ab167, Ab168, Ab169, Ab170, Ab171, Ab172, Ab173, Ab174, Ab175, Ab176, Ab1 The antibody or antigen-binding fragment thereof is selected from b121, Ab122, Ab123, Ab124, Ab125, Ab126, Ab127, Ab128, Ab129, Ab130, Ab131, Ab132, Ab133, Ab134, Ab135, Ab136, Ab137, Ab138, Ab139, Ab140, and Ab141. In a preferred embodiment, the antibody is Ab109, Ab133, or Ab141.

[0040] In any one of the embodiments disclosed herein, the GLP-1 receptor agonist may include semaglutide, tirzepatide, AMG-133 (a GLP-1 receptor agonist / GIP-1 receptor antagonist being developed by Amgen), or danugliplon (an oral GLP-1 receptor agonist being developed by Pfizer).

[0041] In any one of the embodiments disclosed herein, the myostatin-selective inhibitor for use according to the present disclosure may be an antibody or antigen-binding fragment described herein, such as Ab109, Ab133, or Ab141 or an antigen-binding fragment thereof, trevoglumab (REGN1033), or GYM329 (RO7204239), which is an anti-latent myostatin, Fc-engineered antibody discovered by Chugai and being developed in SMA by Roche. In some embodiments, myostatin selective inhibitors for use according to the present disclosure include Ab101, Ab102, Ab103, Ab104, Ab105, Ab106, Ab107, Ab108, Ab109, Ab110, Ab111, Ab112, Ab113, Ab114, Ab115, Ab116, Ab117, Ab118, Ab119, Abl The myostatin-selective inhibitor is an antibody or antigen-binding fragment thereof selected from b120, Ab121, Ab122, Ab123, Ab124, Ab125, Ab126, Ab127, Ab128, Ab129, Ab130, Ab131, Ab132, Ab133, Ab134, Ab135, Ab136, Ab137, Ab138, Ab139, Ab140, and Ab141. In preferred embodiments, the antibody is Ab109, Ab133, or Ab141. In some embodiments, the myostatin-selective inhibitor for use according to the present disclosure is Ab109.

[0042] The present disclosure further encompasses combination or adjunctive (add-on) therapies comprising a myostatin inhibitor and a biguanide (e.g., metformin) that do not include a GLP-1 receptor agonist. Such combination or adjunctive (add-on) therapies may be used to treat a metabolic disorder in a patient, where the metabolic disorder is, optionally, obesity, diabetes, prediabetes, and / or metabolic syndrome. In some embodiments, the patient has a low response to GLP-1 receptor agonist therapy, a low tolerance to GLP-1 receptor therapy, and / or is at risk of developing depression, suicidal ideation, or cancer. In some embodiments, the myostatin inhibitor is a non-selective inhibitor, such as an agent that inhibits both myostatin and GDF11 but not activin A, or an agent that inhibits both myostatin and activin A but not GDF11. In some embodiments, the non-selective inhibitor is an ActRII receptor antagonist (such as bimagrumab), an anti-myostatin Adnectin® (such as taldef globep alfa), or a ligand trap comprising a ligand-binding fragment / portion of ActRII or follistatin. In preferred embodiments, the myostatin inhibitor is a myostatin-selective inhibitor that does not inhibit GDF11 or activin A. In some embodiments, the myostatin-selective inhibitor used in combination with a biguanide (e.g., metformin) is selected from the novel antibodies or antigen fragments disclosed herein (such as Ab109, Ab133, and Ab141), trevogrumab (REGN1033), and GYM329 (RO7204239), an anti-latent myostatin Fc-engineered antibody discovered by Chugai and being developed by Roche in SMA.In some embodiments, myostatin selective inhibitors for use according to the present disclosure include Ab101, Ab102, Ab103, Ab104, Ab105, Ab106, Ab107, Ab108, Ab109, Ab110, Ab111, Ab112, Ab113, Ab114, Ab115, Ab116, Ab117, Ab118, Ab119, Abl The antibody or antigen-binding fragment thereof is selected from b120, Ab121, Ab122, Ab123, Ab124, Ab125, Ab126, Ab127, Ab128, Ab129, Ab130, Ab131, Ab132, Ab133, Ab134, Ab135, Ab136, Ab137, Ab138, Ab139, Ab140, and Ab141. [Brief explanation of the drawings]

[0043] [Figure 1A-1B] 1 shows inhibition of myostatin activation by antibodies of the present disclosure. [Figure 2A] Binding of Ab101-109, Ab135, Ab2, and a control to purified promyostatin from human, mouse, or cynomolgus monkey is shown. [Figure 2B-D] Binding of antibodies Ab122-128 to human promyostatin (Figure 2B), mouse promyostatin (Figure 2C), and cynomolgus monkey promyostatin (Figure 2D) is shown. [Figure 3] 1 shows the binding of antibodies disclosed herein to human or mouse GDF-11. [Figure 4A-B] 1 shows the pH-dependent dissociation of antibodies disclosed herein at pH 7.4 compared to pH 5.5. The numbers to the right of the curves represent the calculated fold difference in off-rate at the various pH values ​​tested. [Figure 5A-F] Binding stoichiometry of antibody:promyostatin or Fab:myostatin is shown. Diagram is a simplified depiction and is not drawn to scale. [Figure 6A-C]Figure 6 shows the in vivo effects of Ab2, Ab102, Ab105, Ab121, and Ab123 in mice with dexamethasone-induced muscle atrophy. Figure 6A shows the percent change in body weight from baseline. Figure 6B shows the percent change in lean mass from baseline. Figure 6C shows the percent change in gastrocnemius muscle mass from baseline. The dashed line in each figure indicates the mean value for Ab2 treatment at 10 mg / kg. ****p<0.0001; ***p<0.005; **p<0.01; *p<0.05. [Figure 7A-D] Figure 7 shows the in vivo effects of Ab2, Ab130, and Ab109 in mice with dexamethasone-induced muscle atrophy. Figure 7A shows the change in body weight. Figure 7B shows the change in lean mass. Figure 7C shows the change in gastrocnemius muscle mass. Figure 7D shows the change in quadriceps muscle mass. ****p<0.0001; ***p<0.005; **p<0.01; *p<0.05. [Figure 8] Figure 1 shows the levels of total serum myostatin in mice treated with Ab2, Ab109, or Ab130. Statistical analysis was performed using one-way ANOVA (Dunnett's multiple comparison test). [Figure 9A-D] Figure 9 shows the in vivo effects of Ab2, Ab112, Ab109, and Ab127 in mice with dexamethasone-induced muscle atrophy. Figure 9A shows the change in body weight. Figure 9B shows the change in lean mass as measured by qNMR. Figure 9C shows the change in gastrocnemius muscle mass. Figure 9D shows the change in quadriceps muscle mass. The dashed line in each figure corresponds to the mean value for Ab2 treatment at 10 mg / kg. ****p<0.0001; ***p<0.005; **p<0.01; *p<0.05. [Figure 10A-F]Figure 10 shows the in vivo effects of Ab2, Ab109, and Ab133 in mice with dexamethasone-induced muscle atrophy. Figure 10A shows the percent change in body weight from baseline. Figure 10B shows the percent change in lean mass from baseline. Figure 10C shows the percent change in gastrocnemius weight from control. Figure 10D shows the percent change in quadriceps weight from control. Figure 10E shows gastrocnemius weight. Figure 10F shows quadriceps weight. The dashed line in each figure corresponds to the mean value for Ab2 treatment at 3 mg / kg. ****p<0.0001; ***p<0.005; **p<0.01; *p<0.05. [Figures 11A-H] Figures 11A-C show the in vivo effects in DIO mice treated with Ab2, Ab109, or Ab130 along with liraglutide. Figures 11A-C show body weight over the treatment period. Figure 11D shows the percent change in body weight from baseline. Figure 11E shows the percent change in lean mass from baseline; ****p<0.0001; ***p<0.005; **p<0.01; *p<0.05. Figure 11F shows the percent change in gastrocnemius muscle mass compared to liraglutide alone (left panel) or IgG control (right panel). Figure 11G shows the percent change in fat mass from baseline from day 15 to day 1 (left panel) or from day 29 to day 1 (right panel). Figure 11H shows the serum exposure of Ab2, Ab109, and Ab130. [Figure 12A-B] Figure 12 shows the in vivo effects of combined treatment with Ab109 and metformin in mice fed a high-fat diet. Figure 12A shows the effect of Ab109 and / or metformin on fat mass in mice fed a 60% high-fat diet and switched to a 45% high-fat diet. Figure 12B shows the effect of Ab109 and / or metformin on lean mass in mice fed a 60% high-fat diet and switched to a 45% high-fat diet. [Figure 13A-B]Figure 13 shows the in vivo effects of semaglutide treatment in combination with IgG control, Ab109, or Ab141 in mice fed a high-fat diet. Figure 13A shows the effect on subcutaneous adipose tissue weight. Figure 13B shows the effect on epididymal adipose tissue weight. [Figure 14A-K] Figure 14 shows the in vivo effects of semaglutide treatment in combination with IgG control or Ab109 in DIO mice fed a high-fat diet. Figure 14A shows the effect on body weight. Figures 14B-14C show the effect on lean mass. Figure 14D shows the effect on fat mass. Figure 14E shows the effect on gastrocnemius muscle weight. Figure 14F shows the effect on inguinal fat pad weight. Figure 14G shows the effect on epididymal fat pad weight. Figure 14H shows the effect of Ab109 and semaglutide on lean mass (left panel) and fat mass (right panel) as measured by qNMR; statistical analysis was performed using one-way ANOVA (Dunnett's multiple comparison test; ****p<0.0001; ***p<0.005; **p<0.01; *p<0.05). Figure 14I shows fasting serum glucose throughout the study and on days 18 and 64 after treatment with Ab109 alone or in combination with semaglutide. Figure 14 J shows the change in relative liver weight (expressed as % liver weight of body weight) after treatment with semaglutide in combination with 2 mg / kg or 20 mg / kg Ab109. ****p<0.0001; ***p<0.005; **p<0.01; *p<0.05. Figure 14K shows the relative fat mass and circulating leptin in mice from this study; ****p<0.0001; **p<0.01; *p<0.05. Statistical analysis was performed using one-way ANOVA (Tukey's multiple comparison test). [Figure 15A-B] Total serum myostatin levels are shown in mice treated with dexamethasone (FIG. 15A) or liraglutide (FIG. 15B) in combination with Ab2, Ab109, or Ab130. [Figure 16A] Figure 1 shows the dose-dependent serum exposure of Ab109 and Ab141 when administered alone or in combination with semaglutide at 0.1-3 mg / kg. [Figure 16B]Figure 1 shows total myostatin after treatment with Ab109 or Ab141 when administered alone or in combination with semaglutide at 0.1-3 mg / kg. [Figure 16C] Shows free latent myostatin (latent myostatin not bound by antibody) levels after 22 days of treatment with Ab109 or Ab141 when administered alone or in combination with semaglutide at 0.1-3 mg / kg. [Figure 17] 1 shows the levels of free latent myostatin in mice treated with Ab109, Ab133, and Ab141 compared to a control mIgG antibody. Data for individual animals are shown in each plot. [Figure 18A-B] Figure 18A shows the change in body weight (Figure 18A) and percentage change in body weight (Figure 18B) in mice treated with Ab109 and semaglutide. The upper panel of each figure shows the effect for a semaglutide dose of 0.04 mg / kg, and the lower panel of each figure shows the effect for a semaglutide dose of 0.01 mg / kg. [Figure 19A-B] Figure 19 shows the change in body fat mass in mice treated with Ab109 and semaglutide. Figure 19A shows the effect for 0.04 mg / kg semaglutide. Figure 19B shows the effect for 0.01 mg / kg semaglutide. The upper panel of each figure shows the absolute change in body fat mass, and the lower panel of each figure shows the percentage change in body fat mass. Figure 19C compares the percentage change in body fat mass between the semaglutide 0.04 mg / kg group and the semaglutide 0.01 mg / kg group. [Figure 20A-B] Figure 20 shows the lean mass change and lean mass percentage change in mice treated with Ab109 and semaglutide. Figure 20A shows the effect for 0.04 mg / kg semaglutide. Figure 20B shows the effect for 0.01 mg / kg semaglutide. Figure 20C compares the lean mass percentage change between the semaglutide 0.04 mg / kg group and the semaglutide 0.01 mg / kg group. [Figure 21A-B]Figure 21 shows the effect of Ab109 and semaglutide treatment on the weight of specific muscle tissues. Figure 21A shows the relative weight (left) and percent weight change (right) of the quadriceps muscle. Figure 21B shows the relative weight (left) and percent weight change (right) of the gastrocnemius muscle. [Figure 22A-B] Figure 22 shows the effect of Ab109 and semaglutide treatment on the weight of specific adipose tissues. Figure 22A shows the relative weight (left) and percent weight change (right) of the perigonadal fat pad. Figure 22B shows the relative weight (left) and percent weight change (right) of the inguinal fat pad. [Figure 23A-C] Negative stain electron microscopy 2D class averages are shown for: promyostatin in a 1:1 complex from the Ab2:1:1 input sample (Figure 23A), promyostatin in a 1:1 complex from the Ab109:1:2 input sample (Figure 23B), and promyostatin in a 1:1 complex from the Ab133:2:1 input sample (Figure 23C). [Figure 24] 1 shows the mean serum concentrations of Ab109 in female cynomolgus monkeys (Cynomolgus macaques) up to 28 days post-dose. DETAILED DESCRIPTION OF THE INVENTION

[0044] The present disclosure encompasses novel antibodies and antigen-binding fragments thereof that selectively bind to pro / latent myostatin with high affinity and can inhibit protease-dependent activation of myostatin with high potency (e.g., an IC50 of less than 1 nM, e.g., an IC50 of less than 0.5 nM, preferably as measured by functional ELISA). Such antibodies and antigen-binding fragments thereof specifically bind to pro / latent myostatin but do not bind to free mature myostatin or GDF11. In some embodiments, the antibodies and fragments bind to a region (e.g., an epitope) within the prodomain of the pro / latent myostatin complex that confers robust inhibitory potency, e.g., a region similar or identical to the region bound by Ab2. However, unlike the inhibitors described above, in certain embodiments, the antibodies / fragments disclosed herein can bind to the antigen (i.e., pro / latent myostatin) with high monovalent affinity without compromising inhibitory potency. These features provide the opportunity to engineer constructs such as bispecific antibodies that contain a first antibody arm that selectively binds to pro / latent myostatin and inhibits activation, and a second antibody arm that binds to a second target of interest. In some embodiments, the high binding affinity of the antibodies and fragments disclosed herein facilitates effective subcutaneous formulations and / or therapeutic uses.

[0045] In some embodiments, the present disclosure also encompasses methods of treating or preventing diseases associated with myostatin dysregulation using a myostatin inhibitor disclosed herein, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin and blocks myostatin activation, in an amount effective to treat or prevent such disease. In some embodiments, the present disclosure provides a method of treating or preventing a metabolic disorder, e.g., obesity and / or type 2 diabetes, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment disclosed herein that specifically binds to pro / latent myostatin and blocks myostatin activation. In some embodiments, the present disclosure encompasses the use of such antibodies or antigen-binding fragments as monotherapy or in combination with at least one other therapy to treat or prevent metabolic disorders, e.g., obesity and / or type 2 diabetes. In some embodiments, the antibodies and fragments disclosed herein may also be used to treat other indications, e.g., muscle disorders such as various types of dystrophies, spinal cord injuries, or spinal muscular atrophies.

[0046] The present disclosure provides antibodies and antigen-binding fragments that can selectively inhibit myostatin by binding to pro / latent myostatin with high potency and specificity. Such highly potent antibodies and antigen-binding fragments thereof that specifically bind to pro / latent myostatin may not only be effective in treating diseases associated with myostatin signaling, but may also provide an improved therapeutic profile (including improved safety and tolerability) and / or facilitate ease of administration (e.g., at concentrations suitable for subcutaneous administration). The lack of specificity observed with myostatin antagonists described elsewhere may pose a higher risk to certain patient populations due to off-target effects. For example, 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 between the mature myostatin protein and other members of the TGFβ superfamily (e.g., activin A or GDF11). Thus, such off-target effects could potentially limit the patient population that can safely receive treatment 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)). For example, activin A is involved in both wound healing and reproductive biology; therefore, inhibition of activin A would limit use in patients who have recently undergone surgery or injury, or in women of childbearing age. Such increased risk of adverse effects or toxicity may be particularly relevant if i) the patient population requires long-term treatment (such as a chronic disease); and / or ii) the 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 specifically and potently target pro / latent myostatin, thereby potentially offering a higher safety profile.

[0047] Additionally, the antibodies or antigen-binding fragments thereof disclosed herein may provide further surprising improvements over antibodies known in the art. In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein provide one or more (e.g., all) of the following effects: increased affinity to facilitate lower administration concentrations, increased myostatin binding stoichiometry, increased serum clearance of myostatin, reduced levels of circulating latent myostatin, and greater pH sensitivity of antigen binding (i.e., binding to pro / latent myostatin with greater affinity at physiological pH compared to acidic pH), which can prevent muscle atrophy and preserve muscle during weight loss. In some embodiments, the antibodies and antigen-binding fragments thereof disclosed herein provide improved subcutaneous bioavailability, e.g., at least 80%, 81%, 82%, 83%, 84%, or 85% or greater, as measured, for example, by serum exposure levels in animals (e.g., monkeys) administered equivalent doses of antibody via intravenous and subcutaneous routes.

[0048] definition As used herein, the articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0049] Except in the examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." The term "about," when used in connection with a percentage, can mean ±1%. Additionally, the term "about" can mean within ±1% of a value.

[0050] Adjunctive Therapy: The terms "adjunctive therapy" and "add-on" therapy are used interchangeably herein and are intended to refer to a treatment regimen in which a second agent (used as adjunctive therapy) is administered to a subject who is receiving, has received, or will be treated with a first agent (e.g., background therapy). The terms "in conjunction with" and "complementary to" are used interchangeably herein and are intended to refer to therapies used together, whether used simultaneously or overlapping in time.

[0051] Administer / Administration: The terms "administer," "administering," or "administration" include any method or act of delivery of a pharmacological agent (e.g., a drug) to an intended subject (e.g., a patient). The pharmacological agent can be any suitable therapeutic agent, such as a biologic agent such as an antibody or antigen-binding fragment thereof (e.g., a pharmaceutical composition comprising such an antibody or antigen-binding fragment), a peptide agent (e.g., a hormone or modified analog thereof), or a low molecular weight drug (e.g., a structurally defined small molecule or chemical entity). Administration can be systemic or local. In some embodiments, administration can include one or more agents, which can be administered concurrently, simultaneously, or sequentially.

[0052] Affinity: Affinity (or "binding affinity") is the strength of binding of a molecule (such as an antibody) to its ligand (such as an antigen). It is typically measured and reported by the equilibrium dissociation constant (KD). In the context of antibody-antigen interactions, KD is the ratio of the antibody's dissociation rate ("off rate" or Koff) to the antibody's binding rate ("on rate" or Kon). Koff is how rapidly an antibody dissociates from its bound antigen, and Kon is how rapidly an antibody binds to its antigen. For example, an antibody with an affinity of ≦5 nM has a KD value of 5 nM or less (i.e., an affinity of 5 nM or greater) as determined by a suitable in vitro binding assay. A suitable in vitro binding assay can be used to measure the KD value of an antibody for its antigen. Suitable assays include, but are not limited to, biolayer interferometry (BLI)-based assays (such as Octet®), surface plasmon resonance (SPR)-based assays (such as Biacore™), and MesoScale Discovery (MSD) immunoassays (such as MSD solution equilibrium titration or MSD-SET). In some embodiments, the KD is determined by a BLI-based assay (such as Octet®). In preferred embodiments, the KD is determined by an SPR-based assay (such as Biacore™).

[0053] Antibody: As used herein, the term "antibody" refers to a full-length immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, inter-connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The antibodies provided in this disclosure include human and humanized antibodies.

[0054] Antigen-binding fragment: The terms "antigen-binding fragment," "antigen-binding fragment," "antigen-binding portion," "antibody fragment," or "antibody portion" are used interchangeably herein and refer to one or more fragments of an antibody (e.g., pro / latent myostatin) that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a Fab')2 fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity-determining regions (CDRs); and (vii) adnectins. Furthermore, the two domains of an Fv fragment, VL and VH, are encoded by separate genes but can be linked using recombinant techniques by a synthetic linker, which allows them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent molecule known as a single-chain Fv (scFv) (see, e.g., Bird et al. (1988), Science, 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also encompassed.Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain but use a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing pairing with complementary domains on another chain and generating two antigen-binding sites (see, e.g., Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ et al. (1994) Structure 2:1121-1123). Antigen-binding fragments can be incorporated into engineered constructs, such as multifunctional constructs comprising antigen-binding fragments. Non-limiting examples of such engineered constructs include multispecific antibodies, such as bispecific antibodies. In some embodiments, the bispecific antibody comprises a Fab fragment of any one of the novel antibodies disclosed herein that allows single-arm binding to pro / latent myostatin.

[0055] Biolayer Interferometry (BLI): BLI is a label-free technique for optically measuring biomolecular interactions, for example, between a ligand immobilized on a biosensor tip surface and an analyte in solution. BLI offers the ability to monitor binding specificity, binding and dissociation rates, and / or concentration. BLI platform instruments are commercially available, for example, from Pall / ForteBio and are commonly referred to as the Octet® System. Unless otherwise specified, BLI-based assays are performed according to the manufacturer's instructions (e.g., binding assays are performed at ambient / room temperature, e.g., approximately 20-25°C).

[0056] Body composition: The term "body composition" refers to the relative components that make up the body, including fat mass, muscle (lean) mass, bone, and water. Particularly in the context of weight management, body composition refers to the ratio of muscle mass to fat mass in the body. Unless otherwise specified, body composition refers to the total body composition. Body composition can be measured by a variety of suitable methods known in the art, including, but not limited to, body density, dual-energy x-ray absorptiometry (DEXA), air displacement plethysmography (ADP), bioelectrical impedance analysis (BIA), body volume index (BVI), skinfold thickness (using measuring calipers), ultrasound, quantitative magnetic resonance (QMR), and abdominal circumference measurement (e.g., measured by waist circumference).

[0057] Body Mass Index (BMI): The term "Body Mass Index" or "BMI" is a number derived from a person's weight and height, calculated as weight in kilograms divided by height in meters squared (kg / m 2 BMI is defined as the body mass index (BMI) expressed in units of 1 / 2. BMI provides a general weight-height relationship that can be used to classify people as underweight, normal weight, overweight, obese, or extremely obese based on tissue mass (muscle, fat, and bone) relative to height.

[0058] Combination therapy: As used herein, "combination therapy" refers to a therapeutic regimen that includes the administration of two or more active agents (e.g., two or more pharmacological agents) intended to treat a given indication and / or disease associated therewith. The two or more agents may be formulated as separate compositions (e.g., formulations) or as a single composition (formulation). "Combination therapy" encompasses complementary therapies that are used in conjunction with each other.

[0059] Competition: As used herein, the terms "compete" or "block" with respect to antigen binding by an antibody or antigen-binding fragment refer to when a first antibody or antigen-binding fragment binds to an epitope of a protein (e.g., latent myostatin) in a manner sufficiently similar to that of a second antibody or antigen-binding fragment such that the result of binding of the first antibody or antigen-binding fragment to its epitope is detectably reduced in the presence of the second antibody or antigen-binding fragment compared to binding of the first antibody or antigen-binding fragment in the absence of the second antibody or antigen-binding fragment. An alternative example is when binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody, although this may not be the case. That is, a first antibody can inhibit binding of a second antibody to its epitope without the second antibody inhibiting binding of the first antibody to its respective epitope. Competition between antibodies can be determined using any method known in the art, including biolayer interferometry (BLI)-based techniques (e.g., Octet®) or enzyme-linked immunosorbent assays (ELISAs). In some embodiments, epitope binning experiments can be used to assess competitive binding between antibodies or antigen-binding fragments.

[0060] Cross-competition: The terms "cross-compete" or "cross-blocking," as used herein with respect to antigen binding by antibodies or antigen-binding fragments, refer to when a first antibody or antigen-binding fragment binds to an epitope of a protein (e.g., latent myostatin) in a manner sufficiently similar to the binding of a second antibody or antigen-binding fragment such that each antibody detectably inhibits binding of the other antibody to its epitope or ligand, whether to the same, a greater extent, or a lesser extent. For example, a first antibody cross-competes with a second antibody if it measurably inhibits antigen binding by the second antibody, and vice versa. This differs from a first antibody that competes with but does not cross-compete with a second antibody, where the first antibody inhibits antigen binding by the second antibody, but the second antibody does not necessarily inhibit antigen binding by the first antibody. Cross-competition between antibodies can be determined using any method known in the art, including biolayer interferometry (BLI)-based techniques (e.g., Octet®) or enzyme-linked immunosorbent assays (ELISAs). In some embodiments, epitope binning experiments can be used to assess competitive binding between antibodies or antigen-binding fragments.

[0061] Both competing and cross-competing antibodies are within the scope of the present disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), one of skill in the art will understand that such competing and / or cross-competing antibodies are encompassed and may be useful for the methods and / or compositions provided herein. In some embodiments, competition or cross-blocking (cross-competition) is determined using a biolayer interferometry (BLI)-based assay. In some embodiments, a first antibody or antigen-binding fragment is immobilized on a biosensor, and binding between the first antibody or antigen-binding fragment and the antigen is determined using a premixed complex containing a second antibody or antigen-binding fragment bound to the antigen. In some embodiments, a first antibody or antigen-binding fragment is immobilized on a biosensor, and then the stepwise binding of the antigen and the second antibody or antigen-binding fragment is measured.

[0062] Decrease / Reduction: The terms "decrease" or "reducing," as used herein in reference to disease symptoms, refer to a statistically significant decrease in such level. The decrease can be, for example, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. 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, an individual with a disorder can achieve a reduced effect level that is comparable to or within the normal range for that effect in an individual without such disorder.

[0063] Diet / Dietary Therapy: In the context of the present disclosure, a particular dietary therapy may be incorporated as part of weight management, e.g., obesity treatment, including pharmacological intervention. Dietary therapy may involve changes in calories / calorie restriction (i.e., reduced calorie intake or reduced calorie absorption), as well as selection of the type of food consumed (e.g., high-protein, low-fat, and / or low-carbohydrate regimens), and / or adjusted timing / schedule of food intake (e.g., intermittent fasting). Thus, a patient is undergoing a "diet or reduced-calorie regimen" when the patient incorporates or is instructed by a physician or equivalent to incorporate a diet into their overall treatment regimen, e.g., as part of weight management.

[0064] Effective amount: As used herein, the terms "effective amount," "effective dose," and "therapeutically effective amount" are used interchangeably and refer to any amount or dose of a compound or composition sufficient to produce a desired biological or medical effect in a tissue or subject. For example, in certain embodiments of the present disclosure, the intended purpose may be to inhibit myostatin activation in vivo or to achieve a clinically meaningful outcome associated with myostatin inhibition. For any particular pharmaceutical agent, the therapeutically effective amount (and / or appropriate unit dose within an effective dosing regimen) may vary, for example, depending on the route of administration and in combination with other pharmaceutical agents. In some embodiments, the specific therapeutically effective amount (and / or unit dose) for any particular patient may depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the particular pharmaceutical agent used; the particular composition used; the patient's age, weight, general health, sex, and diet; the timing, route of administration, and / or excretion or metabolic rate of the particular pharmaceutical agent used; the duration of treatment; and similar factors well known in the medical arts. In some embodiments, an effective amount may refer to an amount that, when administered according to a particular regimen, produces a positive physiological or clinical outcome with a reasonably tolerable level of adverse effects (e.g., toxicity), such that if adverse effects are present, they are well tolerated for the experiment to continue or are well tolerated for the patient to continue the treatment regimen and the benefits of treatment outweigh the toxicity. One of skill in the art will understand that in some embodiments of the present disclosure, an administered amount may be considered an effective amount if it contains an amount appropriate for administration that correlates with a positive result.

[0065] Epitope: As used herein, the term "epitope" refers to the region of an antigen bound by an antibody or fragment thereof. It includes any polypeptide determinant capable of specific binding to an antibody or fragment. In certain embodiments, epitopic determinants include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphonyl, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. In certain embodiments, an antibody or fragment is said to specifically bind to an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. An epitope can be a linear epitope or a conformational epitope. An epitope can be determined, for example, by crystallography of an antigen complexed with an antibody. Antibodies are said to "bind to the same or similar epitope" if they cross-compete with each other.

[0066] Epitope binning: The term "epitope binning" (sometimes referred to as antibody binning or epitope mapping) refers to the process of sorting a set (e.g., a "library") of monoclonal antibodies generated against a target protein or protein complex (i.e., an antigen) based on competition for binding to the target. Antibodies in the library are tested pairwise to assess whether they block / cross-block each other's binding to the antigen. Closely related binning profiles indicate that the antibodies have identical or closely related (e.g., overlapping) epitopes and are "binned" together. Because the biological activity (e.g., intervention; efficacy) resulting from the binding of an antibody to its target is likely to be inherited by other antibodies in the same bin, binning can provide a useful structure-function profile of antibodies sharing similar binding regions within the same antigen. Thus, among antibodies within the same epitope bin, those with higher affinity (lower KD) typically have higher efficacy.

[0067] Exercise / Exercise Regimen: As used herein, the term "exercise" includes any physical activity. The term "exercise regimen" refers to a treatment regimen that incorporates physical activity as a component.

[0068] Fc variant: As used herein, an "Fc variant" of a reference antibody refers to an antibody that contains one or more mutations in the Fc region compared to the reference antibody. In some embodiments, the Fc variant antibody retains the same CDR sequences as the reference antibody. Fc variants may be generated to have altered (e.g., increased) affinity for an Fc receptor (FcR), such as the neonatal Fc receptor (FcRn). In some embodiments, an antibody that binds to FcRn with increased affinity may result in a longer serum half-life of the Fc variant compared to a reference antibody without the Fc mutations.

[0069] GLP-1 Analog: As used herein, the term "GLP-1 Analog" or "Incretin Mimetic" refers to a peptide or modified peptide that has structural similarity to native GLP-1 and can bind to and activate the GLP-1 receptor. The GLP-1 analog can be an extenzin-based therapy or a DPP-IV-resistant analog. Non-limiting examples of GLP-1 analogs include albiglutide, veinaglutide, cotadutide, danugliplon, dulaglutide, exenatide, exenatide ER, liraglutide, lixisenatide, PEG-loxenatide, mazudutide, MEDI0382, neuglutide, orforgliplon, pembidutide, PF-07081532, retatortide, semaglutide, taspoglutide, tirzepatide, and XW003. GLP-1 analogs include substances that delay renal excretion, such as fatty acids, albumin, α-aminoisobutyric acid, and the like; they may be acylated. GLP-1 analogs include peptides or modified peptides containing the amino acid sequence EGTFTSD (SEQ ID NO: 116). GLP-1 analogs may also include peptides or modified peptides containing the amino acid sequence HXXGXFTXD (SEQ ID NO: 117), where X is any amino acid residue.

[0070] GLP-1 receptor agonist: As used herein, the term "GLP-1 receptor agonist" or "GLP-1R agonist" or "GLP-1 RA" refers to an agent that can bind to and activate the GLP-1 receptor. GLP-1 is a natural agonist of the GLP-1 receptor. GLP-1 receptor agonists include GLP-1 analogs. The GLP-1 receptor agonist may be a small molecule GLP-1 receptor agonist. In some embodiments, the GLP-1 receptor agonist is a long-acting small molecule GLP-1 receptor agonist. In some embodiments, the GLP-1 receptor agonist is a GLP-1 analog.

[0071] GLP-1 Pathway Activator: The terms "GLP-1 pathway activator" and "activator of the GLP-1 signaling pathway" are used interchangeably herein and include any agent that increases or enhances the activity of the GLP-1 signaling pathway, regardless of mechanism of action. Increased or enhanced activity of the GLP-1 signaling pathway can be the result of, for example, higher activity, a longer duration of activity, increased availability of one or more components of the signaling pathway, etc. In some embodiments, GLP-1 pathway activators include agents that modulate upstream regulators of GLP-1 (e.g., dipeptidyl peptidase (DPP-IV) inhibitors). As used herein, the term GLP-1 pathway activator encompasses GLP-1 receptor agonists and GLP-1 analogs. In some embodiments, GLP-1 pathway activators include agents that modulate the amount or activity of GLP-1 (e.g., agents that increase the production or secretion of GLP-1; GLP-1 stabilizers). In some embodiments, the GLP-1 pathway activator includes an agent that increases activation of the GLP-1 receptor (GLP-1R). In some embodiments, the agent that increases activation of the GLP-1R includes a GLP-1 agonist, including a GLP-1 analog. In some embodiments, the GLP-1 pathway activator includes an agent that activates downstream signaling of the GLP-1R (e.g., an activator of PI3K, PKC, cAMP, etc.). In some embodiments, the GLP-1 pathway activator includes an agent that modulates receptor GLP-1R expression and / or trafficking (e.g., an inhibitor of GLP-1R internalization; see, e.g., Jones et al., Nat. Comm. (2018) 9:1602). The GLP-1 pathway activator includes an activator of the GLP-1R. In a preferred embodiment, the GLP-1 pathway activator is a GLP-1 receptor agonist. GLP-1 pathway activators include, but are not limited to, antibodies and antigen-binding fragments thereof, engineered protein constructs (such as Fc conjugates and multifunctional molecules including GLP-1 analogs), peptides, GLP-1 gene therapy, and small molecules.

[0072] Human antibody: As used herein, the term "human antibody" refers to antibodies and fragments thereof having variable and constant regions derived from human germline immunoglobulin sequences.

[0073] Humanized antibody: As used herein, the term "humanized antibody" refers to an antibody derived from a non-human species whose protein sequence has been modified to increase its similarity to human antibodies. "Humanized antibody" may also refer to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0074] Inhibit or Inhibition of: As used herein, the terms "inhibit" or "inhibition of" mean to reduce by a measurable amount and can include, but does not require, complete prevention or inhibition.

[0075] Insulin sensitivity; insulin resistance: The term "insulin sensitivity" refers to the metabolic action of insulin to promote glucose disposal in a subject's body. If a subject requires a lower amount of insulin to lower blood glucose levels compared to the average for the human population, the subject is said to have increased insulin sensitivity. In contrast, if a subject requires a higher amount of insulin to lower blood glucose levels, the subject is said to have decreased insulin sensitivity. If the amount of exogenous or endogenous insulin required to increase glucose uptake and utilization in the subject is significantly greater than that in a healthy subject, the subject is said to have "insulin resistance." For example, if the amount of exogenous or endogenous insulin required to increase glucose uptake and utilization in the subject is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more compared to that in a healthy subject, the subject is said to have "insulin resistance."

[0076] Circulating latent myostatin: As used herein, the phrases "circulating latent myostatin" or "circulating latent myostatin" refer to latent myostatin in the blood, plasma, or serum.

[0077] Lean / Lean Mass: As used herein, "lean" mass or tissue refers to muscle mass or muscle tissue, as opposed to fat mass or adipose tissue (e.g., fat).

[0078] Mature myostatin: The term "mature myostatin," also known as GDF8, refers to the dimeric growth factor released from the latent myostatin complex. Mature myostatin is a soluble, biologically active ligand capable of binding to and activating its receptor. Unless otherwise specified, the term "mature myostatin" refers to a fully processed, biologically active form of myostatin or a fragment of full-length mature myostatin that retains biological activity. The wild-type sequence of the mature myostatin polypeptide sequence (i.e., single chain) is provided below (SEQ ID NO: 134). In some cases, mature myostatin may contain one or more mutations that may indicate altered structure / function or stability. DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS (SEQ ID NO: 134).

[0079] Metabolic Disorder: The term "metabolic disorder" is used interchangeably with the terms "metabolic disease" or "metabolic condition" and encompasses any condition involving dysregulation of the body's metabolic functions due to metabolic alterations (anabolism and / or catabolism) that result in a disruption of the normal physiological state of homeostasis. Metabolic disorders can be inherited or acquired. Non-limiting examples of metabolic disorders include obesity or overweight, type 2 diabetes, obesity-associated type 2 diabetes, and metabolic syndrome.

[0080] Metabolic Rate: The term "metabolic rate" refers to the amount of energy expended over a specific period of time. It is typically measured in calories, kilocalories, or joules. Metabolic rate can be expressed as oxygen consumed or carbon dioxide produced per unit time.

[0081] Metabolism: The term "metabolism" refers to the processes involved in the biosynthesis and breakdown of the building blocks of the body, such as fat (e.g., adipose tissue), muscle, and bone. Thus, "fat metabolism" refers to the processes of biosynthesis and breakdown of fat.

[0082] Myostatin: In the context of this disclosure, unless otherwise clearly defined, the term "myostatin" may refer to any form of the myostatin protein, such as promyostatin, latent myostatin, and mature myostatin, each of which exists as a dimer in vivo.

[0083] Myostatin inhibitor: As used herein, the term "myostatin inhibitor" refers to any agent that inhibits one or more forms of myostatin (e.g., promyostatin, latent myostatin, and / or mature myostatin). The term myostatin inhibitor encompasses any molecular modality, such as large molecules (biologics such as antibodies and engineered protein constructs) and small molecules (structurally defined low molecular weight chemicals, etc.). The term myostatin inhibitor encompasses both selective inhibitors of myostatin and non-selective inhibitors of myostatin. A myostatin inhibitor can be an anti-myostatin antibody or antigen-binding fragment thereof that binds to pro- and / or latent myostatin and / or mature myostatin. In some embodiments, a myostatin inhibitor can be an anti-pro / latent myostatin antibody or antigen-binding fragment thereof that preferentially (e.g., selectively) binds to pro- and / or latent myostatin over mature myostatin. In various embodiments, the myostatin inhibitor can be an antibody (such as a neutralizing antibody), an activation inhibitor (e.g., an antibody that inhibits activation of pro- and / or latent myostatin), an adnectin, a peptibody, a receptor trap, or a ligand trap. In some embodiments, the myostatin inhibitor is a small molecule inhibitor. In other embodiments, the myostatin inhibitor refers to gene therapy.

[0084] Myostatin-selective inhibitor: The term "myostatin-selective inhibitor" is used interchangeably with "selective myostatin inhibitor" and refers to a myostatin inhibitor that inhibits myostatin but not other members of the TGFβ superfamily (e.g., GDF11 or activin A). In some embodiments, a myostatin-selective inhibitor inhibits at least one activity of myostatin signaling (e.g., inhibits myostatin activation and / or inhibits or prevents subsequent downstream signaling by myostatin) with at least 100-fold, 200-fold, 500-fold, 1,000-fold, or more potency (e.g., affinity) for myostatin compared to another member of the TGFβ superfamily (e.g., GDF11 or activin A) at biologically or clinically relevant concentrations as measured by any suitable in vitro assay, such as functional ELISA. In preferred embodiments, a myostatin-selective inhibitor exhibits no detectable binding to other TGFβ family members. In some embodiments, the myostatin-selective inhibitor is a neutralizing antibody that binds to mature myostatin and inhibits its activity. In some embodiments, the myostatin-selective inhibitor is an antibody that binds to pro / latent myostatin and inhibits the activation process of myostatin. In some embodiments, the myostatin-selective inhibitor is an antibody or antigen-binding fragment provided herein (e.g., any one of Ab101-Ab141). In some embodiments, the myostatin-selective inhibitor is an antibody or antigen-binding fragment thereof that comprises all six CDRs of any one of Ab101-Ab141, e.g., the set of SEQ ID NOs identified for a particular antibody in Tables 2d-f. In some embodiments, the myostatin-selective inhibitor is an antibody or antigen-binding fragment thereof that comprises the heavy and light chain variable domains of any one of Ab101-Ab141, e.g., the pair of SEQ ID NOs identified for a particular antibody in Table 3. In some embodiments, the myostatin selective inhibitor is an antibody comprising the heavy and light chains of any one of Ab101 to Ab141, for example, the pair of sequence numbers identified for that particular antibody in Table 4.In some embodiments, the antibody sequence is that of Ab109, Ab133, or Ab141, or an antigen-binding fragment thereof.

[0085] Overweight / Obesity: A person is described as overweight or obese if they weigh more than what is considered normal weight adjusted for height. Using BMI-based classification, for human adults (ages 20 and older), a BMI of 18.5-24.9 is considered normal weight; a BMI of 25-29.9 is considered overweight; a BMI of 30+ is considered obese (including extreme obesity); and a BMI of 40+ is considered extremely obese. For children and adolescents (ages 2-19), a BMI of 85-90 on the CDC growth charts is considered normal weight; a BMI of 25-29.9 is considered overweight; a BMI of 30+ is considered obese (including extreme obesity); and a BMI of 40+ is considered extremely obese. th A BMI at or above the 95th percentile on the CDC growth charts is considered overweight or obese. th A BMI at or above the 95th percentile on the CDC growth charts is considered obese (including extremely obese). th A BMI above 120 percentile is considered extremely obese.

[0086] Percent identity: As used herein, the term "percent identity" refers to the similarity between two amino acid sequences or two nucleic acid sequences. Percent identity can be determined using any available alignment tool that attempts to match as many residues as possible over the entire length of the two sequences. For example, percent identity can be determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, as modified in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the BLASTN and BLASTX programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. Protein alignment by BLAST can be performed using the BLASTX program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecule of interest. When gaps exist between two sequences, gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. In embodiments where cumulative or total sequence identity percentage is required, the identity percentage can be determined using the Needle algorithm of the European Molecular Biology Open Software Suite ("EMBOSS") program.

[0087] Potency: As used herein, the term "potency" refers to the activity of a drug, such as an antibody (or antigen-binding fragment thereof) with inhibitory activity, in relation to the concentration or amount of drug that produces a specified effect. For example, an antibody that can produce a particular effect at a given dosage is more potent than another antibody that requires twice the amount (dosage) to produce the same effect. Potency can be measured using any suitable functional assay, such as functional ELISA and cell-based assays, and the extent of myostatin activation, such as activation induced by a protease (e.g., mTLL2), can be measured in the presence or absence of a test substance (e.g., an inhibitory antibody).

[0088] Preventing / Preventing: As used herein, the terms "preventing" and "preventing" refer to preventing or delaying the onset of a condition or disease in a subject, or preventing or delaying the onset of at least one symptom of a condition or disease in a subject.

[0089] Pro / Latent Myostatin: As used herein, the term "pro / latent myostatin" refers to promyostatin, latent myostatin, or both (i.e., the pro-form or precursor of myostatin), but excludes the free form of mature myostatin that is not associated with the prodomain. Promyostatin and latent myostatin are dimers (e.g., homodimers) composed of two promyostatin polypeptides. During biosynthesis, the N-terminal signal peptide is cleaved. The promyostatin homodimer is a proteolytic substrate for intracellular furin, which cleaves between the prodomain and the growth factor domain. The furin-cleaved homodimer complex remains bound until activation ("latent myostatin"), which releases growth factors from the latent complex. The human sequence of each promyostatin polypeptide is provided as SEQ ID NO: 52.

[0090] The terms "pro-myostatin," "pro myostatin," or "promyostatin," also known as "pro-GDF8," refer to an inactive precursor of mature myostatin comprising a disulfide-linked homodimer, in which each molecule of the homodimer contains an amino-terminal prodomain covalently linked to a carboxyl-terminal mature myostatin domain. In one embodiment, "promyostatin" has not been cleaved by either proprotein convertases or proteases from the BMP / thoroid family. Exemplary promyostatin sequences, variants thereof, and methods for producing promyostatin are well known in the art and are described in more detail herein. In the context of a polypeptide sequence, the term "human pro-GDF8" or "human promyostatin" refers to the amino acid sequence set forth in SEQ ID NO: 52, which reflects a single polypeptide chain.

[0091] As used herein, the term "latent myostatin" or "latent-myostatin" refers to an inactive precursor of mature myostatin comprising a disulfide-linked homodimer, each molecule of which contains an amino-terminal prodomain noncovalently linked to a carboxyl-terminal mature myostatin domain. In one embodiment, "latent myostatin" is generated from promyostatin that has been cleaved by proprotein convertases but not by proteases from the BMP / thoroid family. In another embodiment, "latent myostatin" can be generated in vitro by combining and properly folding the prodomain and carboxy-terminal mature myostatin domain. See, e.g., Sengle et al., J. Biol. Chem., 286(7):5087-5099, 2011. Exemplary latent myostatin sequences, variants thereof, and methods for producing latent myostatin are well known in the art and are described in more detail herein.

[0092] Pro-GDF8 (human): [ka]

[0093] pro-GDF8 (rat): [ka]

[0094] pro-GDF8 (mouse): [ka]

[0095] Pro-GDF8 (cynomolgus monkey): [ka]

[0096] Exemplary pro-GDF8 sequences for human, rat, mouse, and cynomolgus monkey are provided above. In these pro-GDF8 sequences, the proprotein convertase cleavage site is shown in bold and the toroidal protease site is underlined. In some embodiments, the proprotein convertase cleavage site comprises amino acid residues 240-243 of SEQ ID NOs: 52-55. In some embodiments, the female protease site comprises amino acid residues 74-75 of SEQ ID NOs: 52-55. The exemplary pro-GDF8 sequences provided herein are not intended to be limiting, and it should be understood that additional pro-GDF8 sequences from other species, including any isoforms thereof, are within the scope of the present disclosure.

[0097] The prodomain of a myostatin polypeptide is composed of several structural domains as described above (see, e.g., PCT / US2014 / 036933). These include, for example, the straight jacket region, zipper region, arm region, finger region 1, finger region 2, cryptic loop, alpha-1 helix region, and bowtie region. In some embodiments, preferred antibodies or fragments thereof that specifically bind to promyostatin bind to an epitope within the arm region of the myostatin prodomain. In some embodiments, the epitope is located within a "" region within the arm region of the prodomain. [ka] " (SEQ ID NO: 118) polypeptide stretch. In some embodiments, the amino acid residues within the arm regions of the prodomain that contact the antibody when bound to antigen are residues that are not conserved between myostatin and GDF11. In some embodiments, such residues are K, E, and / or N of the polypeptide stretch (shown in bold above). In some embodiments, the epitope comprises at least one amino acid residue from the " [ka] " (SEQ ID NO: 57) polypeptide stretch. In some embodiments, such residues are F, Q, L, Y, R, S, and / or K (shown in bold above) of the polypeptide stretch. See Dagbay et al. (J. Biol. Chem. (2020) 295(16):5404-5418), the contents of which are incorporated herein in their entirety.

[0098] Serum clearance: As used herein, the term "serum clearance" or "clearance" refers to the relative pharmacokinetic / pharmacodynamic behavior of an analyte (e.g., a target protein or protein complex) with respect to changes in serum concentration (e.g., circulating level) over time. When an analyte being measured accumulates in serum, it is said to have slow clearance. In contrast, when an analyte being measured is rapidly removed ("cleared") from serum, it is said to have fast clearance. In vivo serum clearance can occur by multiple mechanisms, including, for example, targeted degradation, Fc-mediated internalization, etc. For example, serum clearance of circulating myostatin can be measured using an assay to determine the binding of an antibody to free myostatin in serum (circulating myostatin not bound by the antibody). Such an assay can involve immobilizing a biotinylated capture antibody known to bind to pro- and latent myostatin, followed by adding a sample containing myostatin to test the antibody's ability to bind to free myostatin, and then adding a detection antibody bearing a detectable marker. The capture antibody known to bind to myostatin may be a biotinylated antibody capable of binding to latent myostatin, such as biotinylated Ab2 or a biotinylated antibody of the present disclosure (e.g., biotinylated Ab109, Ab133, or Ab141), and the detection antibody may be a ruthenium-labeled antibody known to bind to both latent and mature myostatin.

[0099] Slow-twitch muscle: As used herein, the terms "slow-twitch muscle," "slow-twitch type 1," or "type I" muscle refer to muscles rich in type I muscle fibers, which are frequently used, are more postural, and help enable long-term endurance, such as long-distance running. As used herein, the terms "fast-twitch muscle," "fast-twitch type 2," or "type II" muscle refer to muscles that provide higher energy output and strength and are used for powerful, explosive movements, such as sprinting, but such muscles fatigue more quickly and cannot be used repeatedly. Fast-twitch muscle fibers are divided into two categories: intermediate-speed fibers (type IIA) and fast-twitch fibers (type IIB or IIx). Intermediate-speed fibers are thicker, contract faster, and wear out more rapidly than slow-twitch fibers. Fast-twitch fibers, which are the most powerful and have the least endurance, are activated as the body approaches maximal exertion. While most muscles tend to be composed of a mixture of various fiber types, different muscles contain different ratios of fiber types. During development or in response to certain events (e.g., exercise, disease, injury, etc.), fiber types within a muscle or muscle group can undergo fiber type switching, which can result in phenotypic changes in muscle physiology.

[0100] Solution equilibrium titration (SET): SET is an assay in which the binding between two molecules (such as an antigen and an antibody that binds to the antigen) can be measured at equilibrium in solution. For example, Meso-Scale Discovery ("MSD")-based SET or MSD-SET is a method for determining the dissociation constants of particularly high-affinity protein-protein interactions at equilibrium, such as picomolar affinity antibodies that bind to their antigens (see, e.g., Ducata et al. (2015) J. Biomolecular Screening 20(10):1256-1267). SET-based assays can be particularly useful for determining KD values ​​for antibodies with subnanomolar (e.g., picomolar) affinities.

[0101] Specific / Specificity: The terms "specific" or "specificity," when used in reference to interactions between members of a specific binding pair (e.g., ligand and binding site, antibody and antigen, biotin and avidin), refer to the selective reactivity of the interaction. In reference to antibodies, the phrase "specifically binds" and similar phrases refer to the ability of an antibody (or antigen-reactive fragment thereof) to bind (i.e., "specifically bind") to an intended target antigen (or fragment thereof) as opposed to other entities. Specific binding is understood as a preference for binding to a particular antigen, epitope, receptor ligand, or binding partner, e.g., at least 100-fold, 200-fold, 500-fold, or 1000-fold preference, over a control nonspecific antigen, epitope, receptor ligand, or binding partner. As used herein, "specific binding" refers to a binding activity that is greater than or equal to the K on , K. off , and K D For example, a ligand may have a binding kinetics of 10 or more, as measured by a suitable in vitro binding assay, such as BLI (e.g., Octet®), surface plasmon resonance (SPR) (e.g., Biacore™), and ELISA. -3 sec -1 Below, 10 -4 sec -1 Below, 10 -5 sec -1 Below, or 10 -6 sec -1 The following K off and / or 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, or 10 -11 M or less, or 10 -12 K below M DA target site can be considered to specifically bind if it has a specific binding affinity to that target site. It is understood that various proteins can share common epitopes or other binding sites (e.g., kinase reaction sites). In certain embodiments, a binding site can bind to more than one ligand and still be considered specific based on binding preference compared to nonspecific antigens and / or by having particular binding kinetic parameters. Methods for selecting an appropriate nonspecific control are within the capabilities of one of ordinary skill in the art. Binding assays are typically performed under physiological conditions.

[0102] Stoichiometry: As used herein, the term "stoichiometry" or "binding stoichiometry" refers to the configuration in which an antibody (or antigen-binding fragment) interacts with its antigen under defined conditions (e.g., the total mass of the complex composed of a specific ratio of components). The binding stoichiometry between an antibody ("Ab") and an antigen ("Ag") can be determined using, for example, whole immunoglobulins such as monoclonal antibodies ("mAbs"), or fragments such as Fab (monovalent) and F(ab')2 (bivalent). In the context of this disclosure, the antigen is the pro / latent myostatin complex, which is a homodimer containing two binding sites per antigen (one on each monomer). For example, a mAb (e.g., Ab2) can bind to an Ag in a 1:1 Ab:Ag configuration, such that a first arm of the mAb interacts with a first binding site on the Ag and a second arm of the mAb interacts with a second binding site on the Ag (Dagbay et al. (J. Biol. Chem. (2020) 295(16):5404-5418)). In contrast, in a 2:1 Ab:Ag configuration, on average, two molecules of antibody can simultaneously interact with one molecule of antigen. Similarly, in a 1:2 Ab:Ag configuration, on average, one molecule of antibody can simultaneously interact with two molecules of antigen. In some embodiments, in the context of protein complex formation (e.g., protein-protein interactions), such as immune complexes (e.g., antibody-antigen complexes), the concept of stoichiometry takes into account both the ratio of components forming the complex and the total mass of the complex. For example, a mAb and an antigen (pro / latent myostatin complex) can form an immune complex consisting of one mAb molecule and one antigen molecule, one mAb molecule and two antigen molecules, two mAb molecules and two antigen molecules, or a mixture thereof when the mAb and Ag are mixed in solution at a total protein concentration of approximately 3.5-8.0 mg / mL. The stoichiometry can be measured by analytical SEC-MALS.For example, the mAb and Ag may be present in a 1:1, 2:1, or 3:1 ratio (e.g., as a mAb:Ag mixture), or vice versa, with a total protein concentration ranging from about 3.5 mg / mL (e.g., about 15 μM mAb and Ag, respectively) to about 8 mg / mL (e.g., about 45 μM mAb and about 15 μM Ag), and allowed to form an immune complex at room temperature at neutral pH for a suitable period of time, e.g., 1 to 48 hours, preferably about 24 hours.

[0103] Subject: As used herein, the term "subject" refers to a target to which one or more of the therapies described herein may be administered. In clinical contexts, the terms "subject" and "patient" may be used interchangeably. In some embodiments, the subject is a mammalian subject, such as a companion animal (e.g., dog, cat, etc.), livestock (e.g., cow, pig, horse, sheep, goat, poultry, etc.), and laboratory animal (e.g., rat, mouse, guinea pig, etc.). In a preferred embodiment, the subject is a human subject.

[0104] Surface Plasmon Resonance (SPR): Surface plasmon resonance is an optical phenomenon that allows for real-time detection of unlabeled interactors. SPR-based biosensors, such as those commercially available from Biacore™, can be used to measure biomolecular interactions, including protein-protein interactions such as antigen-antibody binding. This technique is widely known in the art and is useful for determining parameters such as binding affinity, rate constants, and thermodynamics.

[0105] Total fat mass: The term "total fat mass" refers to the cumulative fat content in a subject's body. Total fat mass includes fat composed of various types of adipocytes, such as white fat, brown fat, and beige fat, and includes fat stores in various body compartments, such as essential fat, subcutaneous fat, and visceral fat. Total fat mass can be measured or estimated by any method known in the art, including measuring subcutaneous fat thickness using calipers, measuring the circumference of specific body parts, dual-energy X-ray absorptiometry (DXA), underwater weighing, air-displacement plethysmography, bioelectrical impedance analysis, bioimpedance spectroscopy, electrical impedance myography, three-dimensional body scanners, multicompartment models, or magnetic resonance imaging. The terms "body fat mass gain" or "body fat mass loss" refer to a change in the amount of measured body fat mass compared to a baseline measurement. For example, a subject with a metabolic disorder may show a decrease in body fat mass or a decrease in visceral fat mass after treatment of the metabolic disorder (e.g., treatment with a myostatin inhibitor). The term "subcutaneous fat" refers to the fat found just below the skin. The term "visceral fat" refers to the fat content that is primarily composed of fat found deep within the abdominal organs, e.g., the abdomen of a subject's body and surrounding the subject's major organs, such as the liver, kidneys, pancreas, intestines, and heart.

[0106] Treatment or Prevention: The terms "treating," "treat," and "treatment" are used interchangeably herein. The term "treating" a condition or disease in a subject refers to the act of providing a therapeutic regimen intended to improve, ameliorate, or affect at least one symptom of a medical condition or disease, including curing, curing, palliating, alleviating, altering, correcting, slowing the progression, slowing or retarding its progression. Thus, the term treating does not necessarily require a complete cure of the disease or disorder. In one embodiment, treating a subject alleviates the symptoms of the disease or disorder by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0107] Weight loss: Weight loss refers to a loss of body mass, regardless of the specific tissue lost. For example, weight loss itself does not distinguish between a loss of fat mass and a loss of muscle mass. An overall loss of total body weight does not necessarily reflect an improvement in body composition.

[0108] Weight Management: As used herein, the term "weight management" encompasses measures taken to lose weight, maintain weight, and to reduce adipose tissue, increase lean mass, or otherwise improve or maintain body composition. Clinically meaningful weight management success may or may not involve overall weight loss. Thus, weight management can include diet (e.g., a calorie-restricted diet, e.g., reducing calorie intake or reducing calorie absorption), exercise therapy, and / or pharmacotherapy (e.g., treatment with a myostatin inhibitor) to reduce total body mass, reduce total fat mass, reduce visceral fat mass, increase metabolic rate, increase lean mass, and / or increase muscle-to-fat ratio, or otherwise improve body composition in a subject.

[0109] Weight-related condition: As used herein, the term "weight-related condition" or "weight-related problem" refers to one or more medical conditions associated with excess body fat mass (i.e., in addition to being overweight or obese), and the subject's excess body fat mass is a contributing factor.Non-limiting examples of weight-related conditions include type 2 diabetes, high blood pressure, high triglyceride or cholesterol levels, heart disease, stroke, kidney disease, fatty liver (e.g., non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) also known as metabolic dysfunction-associated steatohepatitis (MASH)), and sleep apnea.

[0110] General structural features of novel antibodies and their antigen-binding fragments The present disclosure provides a novel class of antibodies capable of inhibiting myostatin activation. Such antibodies bind to the pro / latent myostatin complex but not to free mature myostatin unbound to the prodomain. In some embodiments, these antibodies bind to an epitope comprising one or more residues of the amino acid stretch FVQILRLIKPMKDGTRYTGIRSLK (SEQ ID NO: 57) (amino acid residues 147-170 of human promyostatin) and / or KALDEN (SEQ ID NO: 118) (amino acid residues 205-210 of human promyostatin). This is an epitope previously identified, for example, in a previously identified myostatin inhibitor (Ab2) to confer inhibitory activity against protease-induced activation of myostatin. In some embodiments, these antibodies bind to a conformational epitope in the arm region of the prodomain that is distinct from the proteolytic site. See Dagbay et al. (J. Biol. Chem. (2020) 295(16):5404-5418).

[0111] While retaining the common binding region, in some embodiments, the novel antibodies and antigen-binding fragments disclosed herein share 70% or less sequence identity with Ab2 when the VH and VL sequences are combined. In particular, in some embodiments, 2, 3, 4, or 5 of the 6 CDRs of the novel antibodies or fragments share less than 50% sequence identity with the corresponding CDRs of Ab2.

[0112] In some embodiments, the antibodies and fragments disclosed herein share 70% or less cumulative VH+VL sequence identity with Ab2. In some embodiments, the VL sequence of the antibody shares less than 50% identity with the VL sequence of Ab2. In some embodiments, the L-CDR1 of the antibody shares 25% or less (preferably 20% or less) sequence identity with the sequence of Ab2. In some embodiments, the L-CDR2 of the antibody shares less than 30% sequence identity with the sequence of Ab2. In some embodiments, the L-CDR3 of the antibody shares 20% or less (preferably 10% or less) sequence identity with the sequence of Ab2. Preferred antibodies or fragments according to the present disclosure are fully human antibodies / fragments. In various embodiments, preferred antibodies and fragments disclosed herein exhibit at least the properties of Category 1 from Table 1 below. In some embodiments, preferred antibodies and fragments exhibit the properties of Category 1 and at least one additional category from Table 1 below. In some embodiments, preferred antibodies and fragments exhibit the properties of Category 1 and at least two additional categories from Table 1 below. In some embodiments, preferred antibodies and fragments exhibit properties of category 1 and properties of at least three additional categories from Table 1 below. In some embodiments, preferred antibodies and fragments exhibit properties of category 1 and properties of at least four additional categories from Table 1 below. In some embodiments, preferred antibodies and fragments exhibit properties of category 1 and properties of at least five additional categories from Table 1 below. In some embodiments, preferred antibodies and fragments exhibit properties of all of the categories from Table 1 below.

[0113] [Table 1]

[0114] [Table 2]

[0115] Characterization of novel antibodies and antigen-binding fragments A. Binding selectivity When determining binding selectivity, antibody-antigen interactions may be measured using any suitable in vitro binding assay technique, such as BLI (e.g., Octet®), SPR (e.g., Biacore™), or ELISA. Typically, recombinantly expressed and purified protein is used as the antigen to perform binding assays (see, e.g., PCT / US2014 / 036933). As demonstrated herein, the novel antibodies and antigen-binding fragments disclosed herein selectively target pro- and / or latent myostatin dimer complexes but do not bind to free mature myostatin when the growth factor is not bound to the prodomain. There is no detectable binding to pro-GDF11, pro-activin A, pro-activin B, mature GDF11, mature activin A, or mature activin B. In some embodiments, the antibodies or antigen-binding fragments selectively bind to pro- and latent myostatin but do not bind to mature GDF11 as measured by ELISA.

[0116] In a preferred embodiment, selective binding to pro / latent myostatin over mature myostatin, for example, provided by any one of Ab101-141, preemptively prevents activation, whereas antibodies that bind to mature myostatin may exert their effect only after an activation event and / or may exhibit more off-target binding.

[0117] In some embodiments, preferred antibodies have similar binding characteristics and exhibit species cross-reactivity with human, cynomolgus monkey, rat, and / or mouse pro / latent myostatin. Most preferably, such antibodies have similar binding characteristics and exhibit species cross-reactivity with human, cynomolgus monkey, and mouse pro / latent myostatin.

[0118] B. Binding region, epitope In certain embodiments, the disclosure encompasses antibodies or antigen-binding fragments thereof that bind to a region of the prodomain of the pro / latent myostatin complex at an epitope comprising one or more amino acid residues of the sequence FVQILRLIKPMKDGTRYTGIRSLK (SEQ ID NO: 57) (amino acid positions 147-170 of human promyostatin when numbered according to SEQ ID NO: 52) and / or KALDEN (SEQ ID NO: 118) (amino acid positions 205-210 of human promyostatin when numbered according to SEQ ID NO: 52).

[0119] In some embodiments, binding to one or more of the above residues is determined by assessing cross-blocking with an antibody, e.g., Ab2, known to bind to that epitope. A cross-blocking antibody pair indicates that the two antibodies have substantially overlapping binding regions. In some embodiments, epitope binning can be performed to determine whether two antibodies cross-block each other. In some embodiments, epitope binning can be determined using a premix binning assay in which a first antibody or antigen-binding fragment is immobilized on a biosensor and binding between the first antibody or antigen-binding fragment is determined using a premix complex containing a second antibody or antigen-binding fragment bound to the antigen. In some embodiments, epitope binning can use a sandwich binning assay in which a first antibody or antigen-binding fragment is immobilized on a biosensor and then stepwise binding between the antigen and the second antibody or antigen-binding fragment is measured. In some embodiments, cross-blocking antibodies of the present disclosure bind to one or more of the above amino acid residues.

[0120] C. Binding affinity In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding fragments thereof suitable for use in practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments thereof that bind to pro / latent myostatin with high affinity as determined by SPR (e.g., Biacore™), by measuring the equilibrium dissociation constant by solution equilibrium titration (SET), or by determining the KD using a BLI-based assay (e.g., Octet™). In some embodiments, the binding affinity is determined by a BLI-based assay (e.g., Octet™). In some embodiments, the binding affinity is determined by SET. In some embodiments, SET can be used to measure levels of circulating myostatin (see, e.g., Example 3). Preferably, the binding affinity is determined by SPR (e.g., Biacore™).

[0121] In some embodiments, the antibodies or antigen-binding fragments provided herein preferably have a binding affinity of 10 or more, as measured by an SPR-based in vitro binding assay such as Biacore™. -8 M, 10 -9 M, 10 -10 M, 10 -11 The equilibrium dissociation constant (K D In some embodiments, the antibody or antigen-binding fragment binds to pro / latent myostatin with a nanomolar or sub-nanomolar K DFor example, an anti-pro / latent myostatin antibody, or antigen-binding fragment thereof, can bind to pro / latent myostatin with an affinity of 5 pM to 500 nM, e.g., 50 pM to 100 nM, e.g., 500 pM to 50 nM, e.g., 50 pM to 5 nM, e.g., 0.5 nM to 2 nM. In some embodiments, the present disclosure encompasses antibodies or antigen-binding fragments that compete or cross-compete with any of the antibodies described herein for binding to pro / latent myostatin and have an affinity of 50 nM or less (e.g., 20 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less). In a preferred embodiment, the antibody binds to pro or latent myostatin with a KD of less than 1.0 nM as measured by an SPR-based in vitro binding assay, such as Biacore™.

[0122] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein preferably have a binding affinity of 10 or more, as measured by an SPR-based in vitro binding assay such as Biacore™. -11 M~10 -8 K in the M range D In some embodiments, the antibody or antigen-binding fragment thereof binds to human pro / latent myostatin with a K of less than 5 nM. D In some embodiments, the antibody or antigen-binding fragment thereof binds to pro / latent myostatin with a K of less than 1 nM. D In some embodiments, the antibody or antigen-binding fragment thereof binds to pro / latent myostatin with a KD of less than 0.5 nM. In some embodiments, the antibody or antigen-binding fragment thereof binds to pro / latent myostatin with a KD of less than 0.1 nM. In some embodiments, the antibody or antigen-binding fragment thereof binds to pro / latent myostatin with a KD that is at least 10-fold lower than the Ab2 disclosed in PCT / US2015 / 059468. DIn some embodiments, affinity or binding kinetics is determined by a BLI-based in vitro binding assay. When such binding profiles are measured using Octet® or Biacore™, the assays are performed according to the manufacturer's instructions unless otherwise specified. In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof provided herein that binds to pro / latent myostatin with a KD of less than 1 nM, e.g., selected from Ab101, Ab102, Ab103, Ab104, Ab105, Ab106, Ab107, Ab108, Ab109, Ab121, Ab123, Ab125, Ab127, Ab128, Ab133, Ab134, Ab135, Ab136, Ab137, Ab138, Ab139, Ab140, and Ab141. In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof provided herein that binds to pro / latent myostatin with a KD of less than 0.7 nM, e.g., less than 0.6 nM (e.g., selected from Ab102, Ab105, Ab109, Ab130, Ab131, Ab133, Ab138, Ab139, and Ab140). In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof provided herein that binds to pro / latent myostatin with a KD of less than 0.5 nM, e.g., selected from Ab102, Ab105, Ab109, Ab130, Ab131, Ab133, Ab138, Ab139, and Ab140. In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment provided herein that binds to pro / latent myostatin with a KD of less than 0.2 nM, e.g., Ab109, Ab133, Ab138, Ab139, or Ab140. In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment provided herein that binds to pro / latent myostatin with a KD of less than 0.1 nM, e.g., Ab133.

[0123] In some embodiments, novel antibodies or antigen-binding fragments thereof encompassed by the present disclosure bind to recombinant human pro / latent myostatin with a bivalent KD of less than 1 nM (i.e., <1 nM) as measured by an SPR-based in vitro binding assay such as Biacore™ according to the manufacturer's instructions (e.g., using the exemplary protocol set forth in Example 1). In some embodiments, novel anti-pro / latent myostatin antibodies or antigen-binding fragments thereof bind to recombinant human pro / latent myostatin with a KD of 1 nM or less, e.g., less than 0.1 nM.

[0124] In some embodiments, the KD may be determined by a biolayer interferometry (BLI)-based assay (such as Octet®), a surface plasmon resonance (SPR)-based assay (such as Biacore™), or a Mesoscale Discovery (MSD) immunoassay (such as MSD solution equilibrium titration or MSD-SET). In some embodiments, the KD is determined by a BLI-based assay, such as Octet®. In preferred embodiments, the KD may be determined by an SPR-based assay, such as Biacore™.

[0125] D. Inhibitory Efficacy In some embodiments, the antibodies or antigen-binding fragments thereof described herein can bind to pro / latent myostatin, thereby inhibiting proteolytic activation of pro / latent myostatin to mature myostatin. In some cases, the antibodies or antigen-binding fragments thereof described herein can inhibit proteolytic activation of pro / latent myostatin by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some cases, the antibodies described herein can inhibit proteolytic cleavage of promyostatin by proprotein convertases (e.g., furin) by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some cases, the antibodies, or antigen-binding fragments thereof, described herein can inhibit proteolytic cleavage of promyostatin or latent myostatin by a toroid protease (e.g., mTLL2) by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the antibodies (e.g., Ab109 and Ab130) can inhibit proteolytic cleavage of pro / latent myostatin by a toroid protease (e.g., mTLL2) with an IC50 of less than 0.4 nM.

[0126] In some embodiments, the antibodies, or antigen-binding fragments thereof, described herein can bind to pro / latent myostatin and inhibit myostatin activity. In some embodiments, the antibodies, or antigen-binding fragments thereof, described herein can inhibit myostatin signaling by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, inhibition of myostatin signaling can be measured routinely using a myostatin activation assay, for example, as described in PCT / US2015 / 059468, the entire contents of which are expressly incorporated herein by reference. However, it should be understood that additional methods can be used to measure myostatin signaling activity.

[0127] It should be understood that the extent of proteolytic cleavage of myostatin, for example, by proprotein convertases and / or thrombin proteases, can be measured and / or quantified using any suitable method. In some embodiments, the extent of proteolytic cleavage of myostatin is measured and / or quantified using an enzyme-linked immunosorbent assay (ELISA). For example, an ELISA may be used to measure the level of released growth factors (e.g., mature myostatin). As another example, an antibody or antigen-binding fragment thereof that specifically binds to promyostatin, latent myostatin, and / or mature myostatin may be used in an ELISA to measure the level of a particular form of myostatin (e.g., pro / latent / mature myostatin) or to quantitate the extent of proteolytic cleavage of myostatin. In some embodiments, the extent of proteolytic cleavage of myostatin is measured and / or quantified using immunoprecipitation followed by SDS-PAGE or mass spectrometry of tryptic peptides, fluorescence anisotropy-based techniques, FRET assays, hydrogen-deuterium exchange mass spectrometry, and / or NMR spectroscopy.

[0128] The novel antibodies and antigen-binding fragments thereof according to the present disclosure, such as any of Ab101-Ab141, are highly potent in inhibiting the activation step of myostatin from the latent complex. Such antibodies or fragments inhibit myostatin activation with an IC50 of less than 1 nM as measured by functional ELISA, and exemplary uses thereof are provided in the Examples section below. In some embodiments, any one of Ab102, Ab105, Ab109, Ab123, Ab112, Ab130, Ab131, Ab132, Ab133, Ab134, Ab135, Ab136, Ab137, Ab138, Ab139, Ab140, or Ab141, or an antigen-binding fragment thereof, can be used to inhibit myostatin activation with an IC50 of less than 1 nM as measured by functional ELISA. More generally, to measure the inhibitory potency of myostatin activation inhibitors, such as those disclosed herein, in some embodiments, an ELISA-based in vitro potency assay ("functional ELISA") can be used. In some embodiments, a test substance (such as a test antibody) can be preincubated with recombinant human latent myostatin to form an immune complex. A tolloid protease, preferably mTLL-2, can then be added to the immune mixture to induce the release of mature myostatin by proteolytic cleavage. If the test antibody is able to block mTLL-2-induced activation, mature myostatin will not be released from the latent myostatin complex. On the other hand, if the test antibody does not inhibit myostatin activation, mTLL-2 treatment will release myostatin from the latent myostatin complex. After the tolloid / mTLL-2 treatment step, the amount of free (released) mature myostatin in the presence of the test antibody can be measured by ELISA. The ELISA assay can include a plate coated with a myostatin capture reagent. In some embodiments, the myostatin capture reagent is an antibody or fusion construct that binds to mature myostatin, hi some embodiments, the fusion construct is an ActRII-Fc fusion protein that is a ligand trap.Free mature myostatin present in the assay mixture is captured on an ELISA plate, and the amount of bound mature myostatin can be measured by any suitable method, such as using a biotin-streptavidin-based detection reagent. Functional ELISA experiments can be performed at room temperature (e.g., 20-25°C).

[0129] In some embodiments, highly potent antibodies and antigen-binding fragments thereof include Ab102, Ab109, Ab130, Ab132, Ab133, and Ab141.

[0130] E. Bond stoichiometry In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding fragments thereof suitable for use in practicing various embodiments of the present disclosure can bind to pro / latent myostatin at an antibody:pro / latent myostatin stoichiometry of about 1: 2. The antibody:pro / latent myostatin stoichiometry can be determined using any method known in the art, including SEC-MALS (size exclusion chromatography with multi-angle light scattering).

[0131] In some embodiments, monoclonal antibodies of the disclosure bind to human pro / latent myostatin in a 1:2 antibody-to-antigen stoichiometry to . In some embodiments, monoclonal antibodies of the disclosure bind to human pro / latent myostatin in both a 1:2 antibody-to-antigen configuration to and in daisy chain formation. In some embodiments, monoclonal antibodies of the disclosure bind to human pro / latent myostatin in daisy chain formation. In some embodiments, Fab fragments of monoclonal antibodies of the disclosure bind to human pro / latent myostatin in a 2:1 Fab-to-antigen stoichiometry to . In some embodiments, the antibody binds to human pro / latent myostatin at a 1:2 mAb:Ag binding stoichiometry as measured by analytical SEC-MALS, where the mAb and Ag are pre-assigned at a 1:1, 2:1, or 3:1 stoichiometry (e.g., as a mAb:Ag mixture) at a total protein concentration ranging from about 3.5 mg / mL (e.g., about 15 μM mAb and Ag, respectively) to about 8 mg / mL (e.g., about 45 μM mAb and about 15 μM Ag), and allowed to form immune complexes at room temperature for 24 hours at neutral pH. In some embodiments, the mAb:Ag mixture further comprises an oligomeric complex comprising a 2:1 mAb:Ab complex and / or a 2:2 mAb:Ag complex. In some embodiments, the mAb:Ag mixture does not contain detectable levels of polydaisy chains as measured by analytical SEC-MALS.

[0132] In some embodiments, the antibodies or antigen-binding fragments disclosed herein can reduce total serum myostatin levels in a subject relative to background levels where the total serum myostatin comprises antibody-antigen immune complexes. Although myostatin is thought to function locally rather than through a circulating pool, it is believed that high levels of circulating immune complexes (therapeutic antibodies bound to latent myostatin) can reach tissues, where the bound latent myostatin may at some point dissociate from the inhibitory antibody, leading to unintended activation in the tissue. In such cases, antibodies capable of rapid serum clearance of myostatin may reduce the risk of unintended myostatin activation.

[0133] In some embodiments, the antibody binds to human pro / latent myostatin at a 1:2 mAb:Ag binding stoichiometry as measured by analytical SEC-MALS, where the mAb and Ag are present at a 1:1, 2:1, or 3:1 stoichiometry at a total protein concentration ranging from 3.5 mg / mL to 8 mg / mL, and the complex is allowed to form at room temperature, for example, for 24 hours. In some embodiments, the antibodies or antigen-binding fragments disclosed herein form larger immune complexes with pro / latent myostatin (e.g., 1:2 mAb:Ag or greater). In some embodiments, these larger complexes may be greater than 1 mAb to the antigen but contain smaller oligomers than daisy chains (e.g., oligomers of 500 kDa or less). Such larger immune-oligomeric complexes may be advantageous, for example, in promoting faster clearance and / or better target binding in tissues with higher local concentrations of myostatin. In some embodiments, larger immune complexes (e.g., polydaisy chains), including complexes greater than 500 kDa in size, may facilitate, for example, faster serum clearance. Without being bound by theory, it is believed that larger immune complex formations (e.g., oligomers or daisy chains) may facilitate clearance, for example, through increased FcRn interaction.

[0134] F.pH dependence In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding fragments thereof suitable for use in practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments thereof that exhibit pH-sensitive binding to pro / latent myostatin, such that the antibodies or antigen-binding fragments thereof bind to pro / latent myostatin with higher affinity at a pH in the range of 7.0 to 7.6 (e.g., physiological pH, e.g., pH 7.4) compared to binding at a pH in the range of 4.0 to 6.5 (e.g., acidic pH, e.g., pH 5.5). In some embodiments, the antibodies or antigen-binding fragments exhibit pH-sensitive affinity such that the off-rate (i.e., dissociation rate or Kd) is at least 10-fold greater at acidic pH than at neutral pH. In one embodiment, the pH-sensitive binding characteristics of an antibody or antigen-binding fragment thereof can be measured using a BLI-based assay such as Octet® (e.g., Octet Red 384®). In some embodiments, pH sensitivity is measured by measuring the off-rate (Kd) of the antibody at two or more pH levels. off In some embodiments, antibodies or fragments bound to pro / latent myostatin dissociate from the antigen at a faster rate at acidic pH (e.g., pH 5.5) than at neutral pH (e.g., pH 7.4).

[0135] In some embodiments, the pH dependence can be expressed as the ratio of the first dissociation rate at a first pH level to the second dissociation rate at a second pH level. In some embodiments, the first pH level is an acidic pH level, such as pH 5.5. In some embodiments, the second pH level is a neutral pH level, such as pH 7.4. In some embodiments, the pH sensitivity of an antibody can be expressed as the first dissociation rate divided by the second dissociation rate.

[0136] In some embodiments, such as those shown in Example 1, the dissociation rate is measured by known in vitro binding techniques, such as a BLI-based assay (eg, Octet®).

[0137] In some embodiments, an antibody or antigen-binding fragment thereof according to the present disclosure is a pH-dependent binder characterized by a ratio of dissociation rate at acidic compared to neutral pH levels of 9 or greater, e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or greater, as determined by a BLI-based binding assay, determined by dividing the dissociation rate at pH 5.5 by the dissociation rate at pH 7.4.

[0138] In some embodiments, antibodies with high pH sensitivity (e.g., faster dissociation in acidic conditions compared to neutral conditions) may promote a more robust in vivo recycling capability compared to antibodies with lower pH sensitivity, which may contribute to an increased serum half-life of the antibody in vivo.

[0139] Accordingly, the present disclosure includes antibodies that selectively bind to human pro / latent myostatin at an epitope comprising one or more amino acid residues of the sequence FVQILRLIKPMKDGTRYTGIRSLK (SEQ ID NO: 57) (amino acid residues 147-170 of human promyostatin) and / or KALDEN (SEQ ID NO: 118) (amino acid residues 205-210 of human promyostatin), thereby inhibiting myostatin activation with an IC50 of less than 1 nM as measured by a functional ELISA measuring the inhibition of myostatin by mTLL2, wherein the antibody has a K50 that is at least 10-fold higher at acidic pH than at neutral pH. off It dissociates from pro / latent myostatin at a rate, optionally at an acidic pH of 5.5 and a neutral pH of 7.4.

[0140] G. Development Potential With regard to antibody engineering, the antibodies or antigen-binding fragments disclosed herein can be fully human antibodies of the IgG1 or IgG4 subtype. In the latter case, in some embodiments, the antibody contains the Adair mutation (S228P), which provides a hinge-stabilizing scaffold for reduced binding to Fc gamma receptors to minimize effector function. In some embodiments, the variable regions used are on a preferred framework utilizing only germline amino acids to reduce the potential for undesired immunogenicity.

[0141] With respect to expression profiles, in some embodiments, candidate antibodies are selected based at least in part on their ability for high transient expression (e.g., 100-200 mg / L) using suitable mammalian cells, such as 293 Expi cells, at research scale in shake flasks. In addition, preferred antibodies may exhibit high monomer content after Protein A purification. In preferred embodiments, Protein A-purified antibody samples exhibit greater than 85% monomer content based on small-scale transient expression.

[0142] The developability profile can be assessed by well-known parameters. In some embodiments, the antibody exhibits no measurable multireactivity as measured by baculovirus particle ELISA or polyspecific reagent (PSR). In some embodiments, the antibody is tested for aggregation behavior by affinity capture self-interacting nanoparticle spectroscopy (AC-SINS). In some embodiments, the antibody exhibits no measurable self-association (Δλmax (nM)<5) as measured by affinity capture self-interacting nanoparticle spectroscopy (AC-SINS). Typically, gold nanoparticles are coated with a polyclonal antibody specific for a human monoclonal antibody, and the monoclonal antibody is captured by the conjugate. The multivalency of the monoclonal antibody conjugate amplifies attractive self-interactions, i.e., aggregation between the adsorbed antibodies. This results in a decrease in the interparticle separation distance, which can be detected by a color change of the colloidal gold solution and quantified by a shift in the wavelength of maximum absorbance (plasmon wavelength). A plasmon wavelength of 530 nm can be used as the reported value for unagglomerated gold nanoparticles. Antibodies that are prone to self-aggregation shift their plasmon wavelength to the red end of the spectrum. In some embodiments, a shift in plasmon wavelength of more than 5 nm (i.e., >5 nm) can be used as a cutoff indicative of a self-interacting antibody. In some embodiments, antibodies that do not exhibit measurable self-association (e.g., aggregation) as measured by AC-SINS are selected from Ab101, Ab102, Ab103, Ab134, Ab104, Ab105, Ab106, Ab107, Ab108, Ab109, Ab133, Ab135, and Ab141.

[0143] In some embodiments, the polyspecificity of novel antibodies can be assessed using ELISA detection of nonspecific binding to baculovirus particles (BV-ELISA). In some embodiments, an arbitrary cutoff of 1000 RFU can be set based on the mean + 5 x standard deviation of control IgG and no antibody. Antibodies that do not exhibit measurable polyreactivity using baculovirus particle ELISA include, but are not limited to, Ab101, Ab102, Ab103, Ab134, Ab104, Ab105, Ab106, Ab107, Ab108, Ab109, Ab133, Ab135, and Ab141.

[0144] In some embodiments, the antibodies exhibit relatively low hydrophobic interactions as determined by retention time in a hydrophobic chromatography column, indicating a low likelihood of self-interactions, and in some embodiments, minimal aggregation is observed in a 4-week accelerated degradation / stability study.

[0145] Regarding in vivo disposition, in some embodiments, pharmacokinetic studies in non-human primates (e.g., cynomolgus monkeys) are predictive of half-life in humans. Preferably, such studies predict a half-life of about 28 days in humans. In some embodiments, a suitable assay can be used to determine the pharmacokinetics of a myostatin inhibitory antibody. This assay can be performed by immobilizing promyostatin on a surface, such as a microplate, and detecting binding of the antibody to promyostatin using a detection agent. Suitable detection agents include goat anti-human antibodies conjugated to horseradish peroxidase (HRP), or, for increased specificity, particularly for human clinical applications, a suitable detection agent, such as a mouse anti-human IgG4 Fc fragment conjugated to ruthenium red, can be used.

[0146] In some embodiments, novel antibodies and antigen-binding fragments thereof according to the present disclosure are modified to reduce their susceptibility to deamination and oxidation. In some embodiments, such modifications include one or more modifications in or around a region of the protein containing amino acid residue NG. In some embodiments, novel antibodies and antigen-binding fragments thereof according to the present disclosure are modified to reduce their susceptibility to isomerization. In some embodiments, such modifications include one or more modifications in or around a region of the protein containing amino acid residue DG.

[0147] H. Reducing or preventing serum accumulation It has previously been observed that certain myostatin-selective activation inhibitors, such as apitegromab, can cause elevated levels of circulating latent myostatin (e.g., latent myostatin-antibody immune complexes) in subjects treated with the antibody. See, e.g., PCT / US2016 / 052014, the contents of which are incorporated herein in their entirety. Accumulation of serum latent myostatin above baseline was, in fact, used as a pharmacodynamic biomarker for apitegromab. However, certain antibodies disclosed herein (e.g., Ab102, Ab130, Ab109, Ab132, Ab133, Ab141) unexpectedly exhibited enhanced serum clearance or reduced accumulation of circulating latent myostatin in serum samples (e.g., Ab102, Ab130, Ab133, Ab141) from subjects administered the antibodies, as evidenced by little or no accumulation of total myostatin (e.g., latent myostatin) in serum samples collected from subjects administered the antibodies (e.g., Ab109, Ab132) compared to the increased accumulation seen with apitegromab. Without wishing to be bound by theory, it is believed that the enhanced serum clearance of latent myostatin associated with certain antibodies disclosed herein (e.g., Ab102, Ab130, Ab109, Ab132, Ab133, Ab141) may be due, in part, to the greater pH differential exhibited by certain antibodies disclosed herein (e.g., Ab102, Ab130, Ab109, Ab132, Ab133, Ab141). It is also believed that differences in binding stoichiometry may contribute to the enhanced serum clearance of latent myostatin. In some embodiments, the higher stoichiometry of certain antibodies disclosed herein may contribute to their enhanced serum clearance or reduced serum accumulation. For example, in some embodiments, the single 1:2 antibody-to-antigen stoichiometry peak observed when measuring the stoichiometry of Ab109 and Ab132 is believed to contribute to enhanced serum clearance of latent myostatin compared to apitegromab, which binds at a 1:1 antibody-to-antigen stoichiometry, and may prevent accumulation of circulating latent myostatin above baseline levels.In some embodiments, the 1:2 and 2:1 antibody-to-antigen stoichiometric ratios of Ab102, Ab130, Ab133, and Ab141 may contribute to enhanced serum clearance of latent myostatin compared to apitegromab and may prevent the accumulation of circulating latent myostatin levels in serum samples from subjects using the antibodies compared to apitegromab.

[0148] Despite the observation of higher antibody:promyostatin stoichiometries (e.g., 1:2 and / or 2:1) with certain antibodies of the present disclosure (e.g., Ab102, Ab130, Ab109, Ab132, Ab133, Ab141), negative stain electron microscopy performed at lower protein concentrations (e.g., 0.01-0.015 mg / ml) demonstrates that certain antibodies (e.g., Ab2, Ab102, Ab130, Ab109, Ab132, Ab133, Ab141) can also form a 1:1 stoichiometry. In some embodiments, these antibodies (e.g., Ab102, Ab130, Ab109, Ab132, Ab133, Ab141) can form both higher and 1:1 stoichiometries (the antibodies can form both). Without wishing to be bound by theory, it is believed that in the circulation, subjects administered Ab109 may have circulating levels of myostatin:antibody complexes that may promote the formation of a 1:1 stoichiometry, whereas in other instances where target myostatin concentrations are higher, e.g., in target tissues such as muscle and other depots, and on cell surfaces where the antibody is cleared and local concentrations of immune complexes are formed, 1:2 and 2:1 stoichiometries may be favored, promoting target clearance and / or preventing target accumulation.]

[0149] In some embodiments, the antibodies disclosed herein can reduce serum concentrations of total or latent myostatin. In some embodiments, the antibody can include Ab102, Ab130, or an antigen-binding fragment thereof (e.g., to provide higher clearance and lower accumulation than Ab2). In some embodiments, the antibody can include Ab133, Ab141, or an antigen-binding fragment thereof (e.g., to provide higher clearance and lower accumulation than Ab2). In some embodiments, the antibody can include Ab109, Ab132, or an antigen-binding fragment thereof (e.g., to provide even higher clearance and lower accumulation than Ab102, Ab130, Ab133, or Ab141). Without wishing to be bound by theory, it is believed that faster serum clearance may correlate with larger immune complexes (e.g., polydaisy chains) formed in vivo, and that larger immune complex formation (e.g., oligomers) may facilitate clearance, for example, by increased FcRn interaction. In some embodiments, such enhanced clearance of total or latent myostatin associated with the antibodies disclosed herein is achieved without manipulating (e.g., introducing mutations into) the Fc region (see, e.g., Muramatsu et al. Sci Rep. 2021;11:2160), thereby minimizing the risk of undesirable immunogenicity.

[0150] In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding fragments thereof suitable for use in practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments that reduce circulating total myostatin or latent myostatin. In some embodiments, administration of an antibody or antigen-binding fragment provided herein to a subject can reduce the subject's circulating total latent myostatin by at least 10% (e.g., at least 20%, 30%, 40%, 50%, 75%, 80%, 90% or more) compared to baseline.

[0151] In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding fragments thereof suitable for use in practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments that increase the clearance of myostatin from serum. In some embodiments, administration of an antibody or antigen-binding fragment provided herein to a subject can increase the clearance of myostatin from the subject's serum by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90% or more) compared to before administration. In some embodiments, administering an anti-pro / latent myostatin antibody or antigen-binding fragment thereof provided herein can provide greater clearance of myostatin from a subject's serum (e.g., at least 10%, 20%, 30%, 40%, 50%, 75%, 80%, 90%, or more clearance) compared to administering a similar dose of another anti-pro / latent myostatin antibody or antigen-binding fragment known in the art (e.g., compared to Ab2 provided in PCT / US2015 / 059468).

[0152] In some embodiments, the inventors have discovered that certain antibodies or antigen-binding fragments of the present disclosure that share common features with Ab2, such as selectivity for binding to pro / latent myostatin, pH-dependent binding, and binding regions (i.e., epitopes), can surprisingly result in distinct serum clearance profiles in vivo (e.g., faster clearance of immune complexes compared to Ab2), such antibodies including, for example, Ab109 and Ab130.

[0153] Assays of antibody target binding, i.e., pharmacodynamic assays, can be performed by measuring antibody binding to myostatin. In some embodiments, myostatin is detected as described by Lakshman et al. (Mol. Cell. Endocrinol. (2009) 302(1):26-32), the contents of which are incorporated herein in their entirety. In some embodiments, samples are treated with acid to convert all myostatin forms to mature growth factors; a biotinylated capture antibody specific for mature myostatin is added to a streptavidin-coated plate and detected with a labeled antibody specific for mature myostatin.

[0154] In another embodiment, a pharmacodynamic assay can be performed by measuring antibody binding to serum-free latent myostatin (i.e., circulating latent myostatin not bound by antibody). Such an assay can test the ability of an antibody to bind to free latent myostatin by immobilizing streptavidin, binding to a biotinylated antibody known to bind to latent myostatin, adding latent myostatin, and labeling the test antibody with a detectable marker. In one embodiment, the biotinylated antibody known to bind to latent myostatin is biotinylated Ab2, and the test antibody is a ruthenium-labeled antibody of the present disclosure. In such embodiments, the detection range of the assay can be in the nanogram range, e.g., 0.1 ng / ml to 750 ng / ml, 1.0 ng / ml to 500 ng / ml, or 3.0 ng / ml to 500 ng / ml, e.g., 3.9 to 500 ng / mL. When mice are administered a single dose (e.g., 2-20 mg / kg) of the novel myostatin inhibitors disclosed herein, a rapid decrease in serum-free latent myostatin is observed within one day, which remains at undetectable or nearly undetectable levels for at least 42 days, demonstrating sustained target engagement and inhibitory activity of the antibody.

[0155] I. In Vivo Efficacy In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding fragments thereof suitable for use in practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments that can produce one or more of the following effects in vivo: 1) preventing muscle atrophy; 2) maintaining or increasing muscle mass; and / or 3) maintaining overall body weight. In some embodiments, the one or more effects can be tested in vivo using a dexamethasone-induced injury model of atrophy (e.g., as described in Example 2 of the present disclosure). In some embodiments, administration of one of the novel anti-pro / latent myostatin antibodies or antigen-binding fragments thereof disclosed herein can produce one or more of the following effects: 1) inducing overall weight loss; 2) reducing weight gain; 3) maintaining or increasing lean muscle mass; 4) reducing body fat mass; and / or 5) altering the muscle-to-fat ratio.

[0156] In some embodiments, administration of one of the novel anti-pro / latent myostatin antibodies or antigen-binding fragments thereof disclosed herein in combination with a standard of care for diabetes and / or obesity (e.g., a GLP-1 pathway activator, such as semaglutide, tirzepatide, AMG-133 (a GLP-1 receptor agonist / GIP-1 receptor antagonist being developed by Amgen) or danugliplon (an oral GLP-1 receptor agonist being developed by Pfizer) may result in greater efficacy than administration of the standard of care alone. For example, administration of the combination therapy results in increased weight loss or a further decrease in weight gain, an increase in lean muscle mass or a decrease in lean muscle mass, and / or a decrease in body fat mass compared to administration of the standard of care alone.

[0157] Non-limiting examples of novel antibodies and antigen-binding fragments thereof In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding fragments thereof suitable for use in practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments that exhibit one or more surprising and distinct properties compared to prior art antibodies, such as Ab2 provided in PCT / US2015 / 059468. In some embodiments, the properties include one or more (e.g., all) of the following: 1) binding to pro / latent myostatin with a bivalent KD (e.g., F(ab')2 or mAb) of less than 1 nM as measured by an SPR-based in vitro binding assay, e.g., Biacore™; 2) pH-sensitive binding to pro / latent myostatin, e.g., such that the binding affinity to pro / latent myostatin at physiological pH (e.g., pH 7.0-7.5, e.g., pH 7.4) is at least 9-fold, e.g., 10-fold, higher than the binding rate at acidic pH (e.g., pH 4.0-6.5, e.g., pH 5.5); 3) a 1:2 mAb:pro / latent myostatin binding affinity. 3) capable of binding to pro / latent myostatin in a stoichiometric ratio; 4) ability to inhibit protease-induced activation of myostatin in vitro with an IC50 of less than 1 nM as measured by functional ELISA described herein; 5) not causing serum accumulation of total or latent myostatin levels or reducing circulating total or latent myostatin levels (e.g., enhancing serum clearance of myostatin); and / or 6) ability to monovalently bind to latent myostatin with a KD of less than 50 nM as measured by an SPR-based in vitro binding assay, e.g., Biacore™. In some embodiments, the antibody or antigen-binding fragment further comprises an IgG4 constant domain.

[0158] In some embodiments, anti-myostatin antibodies or antigen-binding fragments thereof suitable for use in practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments that exhibit one of the six defining characteristics described above. In some embodiments, the antibodies or antigen-binding fragments thereof exhibit two of the defining characteristics described above. In some embodiments, the antibodies or antigen-binding fragments thereof exhibit three of the defining characteristics described above. In some embodiments, the antibodies or antigen-binding fragments thereof exhibit four of the defining characteristics described above. In some embodiments, the antibodies or antigen-binding fragments thereof exhibit five of the defining characteristics described above. In some embodiments, the antibodies or antigen-binding fragments thereof exhibit all six of the defining characteristics described above. Combinations of any group of the six characteristics are contemplated herein.

[0159] The inventors have made the surprising discovery that a subset of antibodies that originate from or bind to the same epitope as Ab2 provided in PCT / US2015 / 059468 possess all six of the above characteristics. These antibodies, described in further detail below, are particularly useful in practicing various embodiments of the present disclosure. In some embodiments, the antibody or antigen-binding fragment thereof comprises Ab102, Ab109, Ab130, Ab132, Ab133, or Ab141 (e.g., as defined by their respective heavy and light chain sequences), or a set of six CDRs and / or a set of variable domains derived from any of these antibodies.

[0160] In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding fragments thereof suitable for use in practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments that specifically bind to pro / latent myostatin. In some embodiments, such antibodies and antigen-binding fragments bind to an epitope within the prodomain, the epitope including one or more (e.g., all) of amino acid residues F147, Q149, L151, Y183, S168, Q149, L151, Y163, S168, K170, K205, and L207, when numbered according to SEQ ID NO: 52 (Dagbay et al. J Biol Chem. 2020 Apr 17;295(16):5404-5418). In some embodiments, such antibodies and antigen-binding fragments bind to an epitope within the prodomain, the epitope including one or more (e.g., all) of amino acid residues F147, Q149, L151, Y186, S168, K170, K205, and / or L207, when numbered according to SEQ ID NO:52.

[0161] In some embodiments, the antibody or antigen-binding fragment has an off-rate that is at least 10-fold higher than the on-rate. In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment provided herein, e.g., Ab109, Ab130, Ab132, Ab133, or Ab141.

[0162] In some embodiments, the antibody or antigen-binding fragment has an antibody:pro / latent myostatin binding stoichiometry of 1: 2. In some embodiments, the antibody is an antibody provided herein, e.g., Ab105, Ab109, Ab130, Ab133, or Ab141.

[0163] In some embodiments, the antibody or antigen-binding fragment binds to pro / latent myostatin with a Kd of binding of 0.1 nM or less as measured by an SPR-based in vitro binding assay, such as Biacore™. In some embodiments, the antibody is an antibody provided herein, e.g., Ab101, Ab102, Ab104, Ab105, Ab107, Ab109, Ab133, or Ab141.

[0164] In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding fragments thereof suitable for use in practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising a constant domain of the IgG1 or IgG4 subtype. In some embodiments, antibodies comprising an IgG1 or IgG4 constant domain further comprise an Adair mutation (S228P). In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment provided herein, such as Ab102, Ab109, Ab130, Ab132, Ab133, or Ab141.

[0165] In any one of the embodiments disclosed herein, the antibody or antigen-binding fragment may comprise an HCDR1 of SEQ ID NO: 201; an HCDR2 of SEQ ID NO: 202 (wherein X1 is T or A); an HCDR3 of SEQ ID NO: 203; an LCDR1 of SEQ ID NO: 204; an LCDR2 of SEQ ID NO: 205; and an LCDR3 of SEQ ID NO: 206 (wherein X1 is M or Q and X2 is P or G), when numbered according to the Kabat numbering system.

[0166] In any one of the embodiments disclosed herein, the antibody or antigen-binding fragment, when numbered according to the Chothia numbering system, may comprise an HCDR1 of SEQ ID NO: 293; an HCDR2 of SEQ ID NO: 279; an HCDR3 of SEQ ID NO: 296; an LCDR1 of SEQ ID NO: 281; an LCDR2 of EVS; and an LCDR3 of SEQ ID NO: 297 (wherein X1 is P or G).

[0167] In any one of the embodiments disclosed herein, the antibody or antigen-binding fragment, when numbered according to the IMGT numbering system, may comprise an HCDR1 of SEQ ID NO: 293; an HCDR2 of SEQ ID NO: 294 (wherein X1 is T or A); an HCDR3 of SEQ ID NO: 257; an LCDR1 of SEQ ID NO: 258; an LCDR2 of EVS; and an LCDR3 of SEQ ID NO: 292 (wherein X1 is M or Q and X2 is P or G).

[0168] In some embodiments for carrying out various embodiments of the present disclosure, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs), CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, where CDRH1 comprises the sequence SYGMS (SEQ ID NO:201), CDRH2 comprises the sequence SFTGSGGX1YYPDSVKG (SEQ ID NO:202), where X1 is T or A, CDRL3 comprises the sequence DLLIRFLEWSHYYGMDV (SEQ ID NO:203), CDRL1 comprises the sequence RSSQSLLHSSGHNFLH (SEQ ID NO:204), CDRL2 comprises the sequence EVSNRVS (SEQ ID NO:205), and CDRH3 comprises the sequence X1QQTQYPX2T (SEQ ID NO:206), where X1 is M or Q and X2 is P or G, and the CDR sequences are numbered according to the Kabat numbering system.

[0169] In some embodiments for carrying out various embodiments of the present disclosure, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs), CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, where CDRH1 comprises the sequence SYGMS (SEQ ID NO:201), CDRH2 comprises the sequence SITGSGGETYYPDSVKG (SEQ ID NO:207), CDRH3 comprises the sequence DLLVRFLEWSHYYGMDV (SEQ ID NO:208), CDRL1 comprises the sequence RSSQSLLHSSGHNFLH (SEQ ID NO:204), CDRL2 comprises the sequence EVSNRVS (SEQ ID NO:205), and CDRL3 comprises the sequence X1QATQFPRP (SEQ ID NO:210), where X1 is M or Q, and the CDR sequences are numbered according to Kabat.

[0170] In some embodiments for carrying out various embodiments of the disclosure, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs), CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, where CDRH1 comprises the sequence SYGMS (SEQ ID NO: 201), CDRH2 comprises the sequence SINPSGGTTYYAQKFKG (SEQ ID NO: 211), and CDRH3 comprises the sequence DLLVRFLEWSHYYGMDV (SEQ ID NO: 208). X2 is R or L; X3 is V or A; and CDRL3 comprises the sequence QQX1TQYPPT (SEQ ID NO: 214), where X1 is Q or Y, and the CDR sequences are numbered according to Kabat.

[0171] In some embodiments for carrying out various embodiments of the present disclosure, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs), CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, where CDRH1 comprises the sequence SYGMS (SEQ ID NO:201) and CDRH2 comprises the sequence SX1TGSGGX2X3YX4X5X6X7X8X9 (SEQ ID NO:512), where X1 is I or F; X2 is E, T, or A; and X X3 is Y or T; X4 is P or Y; X5 is D or P; X6 is S or D; X7 is V or S; X8 is K or V; X9 is G or K, CDRH3 comprises the sequence DLLX1RFLEWSHYYGMDV (SEQ ID NO:513), X1 is V or I, CDRL1 comprises the sequence RSSQSLLHSSGHNFLH (SEQ ID NO:204), CDRL2 comprises the sequence EX1SNRX2X3 (SEQ ID NO:514), X1 is T or V; X2 is A or V; X 3 is P or S, and CDRL3 has the sequence X 1 QX 2 TQX 3 PX 4 X 5 (SEQ ID NO: 515), wherein X1 is Q or M; X2 is Q or A; X3 is Y or F; X4 is P or R or G; and X5 is P or T, and the CDR sequences are numbered according to Kabat.

[0172] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, where CDRH1 comprises the sequence SYGMS (SEQ ID NO:201), CDRH2 comprises the sequence SX1TGSGGX2TYYPDSVKG (SEQ ID NO:275), where X1 is F or I, and X2 is E or A, and CDRH3 comprises the sequence DLLX1RFLEWSHYYGMDV (SEQ ID NO:272), where X1 is I or V. wherein CDRL1 comprises the sequence RSSQSLLHSSGHNFLH (SEQ ID NO: 204), CDRL2 comprises the sequence ETSNRX1X2 (SEQ ID NO: 276), where X1 is V or A and X2 is P or S, and CDRL3 comprises the sequence X1QQX2TQX3PX4X5 (SEQ ID NO: 277), where X1 is M or Q, X2 is Q or A, X3 is Y or F, X4 is R, P, or G, and X5 is T or P, and the CDR sequences are numbered according to the Kabat numbering system.

[0173] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, where CDRH1 comprises the sequence GFTFX1SY (SEQ ID NO: 278), X is S or T, CDRH2 comprises the sequence TGSGG (SEQ ID NO: 279), and CDRH3 comprises the sequence LLX1RFLEWSHYYGMD (SEQ ID NO: 280), and X is I or V, CDRL1 comprises SQSLLHSSGHNF (SEQ ID NO: 281), CDRL2 comprises the sequence EX1S, where X1 is T or V, CDRL3 comprises the sequence X1X2X3X4X5X6, where X1 is Q, R, or A, X2 is T or P, X3 is Q or F, X4 is Y, F, or G, X5 is P or G, and X6 is G, P, or R, and the CDR sequences are numbered according to the Chothia numbering system.

[0174] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, where CDRH1 comprises the sequence GFTFTSSYG (SEQ ID NO: 284), CDRH2 comprises the sequence X1TGSGGX2T (SEQ ID NO: 285), where X1 is F or I and X2 is E, T, or A, and CDRH3 comprises the sequence ARDLLVRFLEWSHYYGMDV (SEQ ID NO: 286). wherein CDRL1 comprises the sequence QSLLHSGHNF (SEQ ID NO:287), CDRL2 comprises the sequence EX1S (wherein X is T or V), or the sequence EVSNRVS (SEQ ID NO:205), and CDRL3 comprises the sequence X1QX2TQX3PX4X5 (SEQ ID NO:288), where X1 is Q or M, X2 is Q or A, X3 is Y or F, X4 is Y, P, or G, and X5 is P or T, and the CDR sequences are numbered according to the IMGT numbering system.

[0175] In some embodiments, an anti-pro / latent myostatin antibody or antigen-binding portion thereof suitable for implementing various embodiments of the present disclosure comprises the following six CDRs: CDRH1 comprising GFTFSSYG (SEQ ID NO: 3); CDRH2 comprising FTGSGGX1 (SEQ ID NO: 291) (wherein X1 is selected from T and A); CDRH3 comprising ARDLIRFLEWSHYYGMDV (SEQ ID NO: 257); CDRL1 comprising QSLLHSSGHNF (SEQ ID NO: 258); CDRL2 comprising EVSNRVS (SEQ ID NO: 289); and CDRL3 comprising X1QQTQYPX2T (SEQ ID NO: 292) (wherein X1 is selected from M and Q, and X2 is selected from P and G). In preferred embodiments, CDRH2 comprises FTGSGGT (SEQ ID NO: 256) or FTGSGGA (SEQ ID NO: 262), and / or CDRL3 comprises QQQTQYPGT (SEQ ID NO: 261), MQQTQYPPT (SEQ ID NO: 260), or MQQTQYPGT (SEQ ID NO: 290). In some embodiments, CDRL3 comprises the sequence QTQYPX1 (SEQ ID NO: 293), and X1 is P or G.

[0176] In some embodiments for carrying out various embodiments of the present disclosure, the antibody or antigen-binding fragment comprises an antibody or antigen-binding fragment comprising six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or a combination thereof, where, as defined according to the Kabat numbering system, CDRH1 comprises SEQ ID NO: 201, CDRH2 comprises SEQ ID NO: 214, CDRH3 comprises SEQ ID NO: 215, CDRL1 comprises SEQ ID NO: 216, CDRL2 comprises SEQ ID NO: 217, and CDRL3 comprises any one of SEQ ID NOs: 218 or 224. In some embodiments, preferred antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or a combination thereof, where, as defined according to the Kabat numbering system, CDRH1 comprises SEQ ID NO: 201, CDRH2 comprises any one of SEQ ID NOs: 219 or 226, CDRH3 comprises SEQ ID NO: 220, CDRL1 comprises SEQ ID NO: 216, CDRL2 comprises SEQ ID NO: 222, and CDRL3 comprises any one of SEQ ID NOs: 223, 225, or 227.

[0177] In some embodiments, antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or a combination thereof, where, as defined according to the Chothia numbering system, CDRL1 comprises SEQ ID NO: 228, CDRL2 comprises SEQ ID NO: 229, CDRH3 comprises SEQ ID NO: 230, CDRH1 comprises SEQ ID NO: 231, CDRH2 comprises an ETS, and CDRL3 comprises SEQ ID NO: 233. In some embodiments, preferred antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or a combination thereof, where, as defined according to the Chothia numbering system, CDRL1 comprises SEQ ID NO: 234, CDRH2 comprises SEQ ID NO: 235, CDRH3 comprises SEQ ID NO: 236, CDRH1 comprises SEQ ID NO: 237, CDRL2 comprises an EVS, and CDRL3 comprises any one of SEQ ID NOs: 239 or 240.

[0178] In some embodiments, antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or a combination thereof, where, as defined according to the IMGT numbering system, CDRH1 comprises SEQ ID NO: 250, CDRH2 comprises SEQ ID NO: 251, CDRH3 comprises SEQ ID NO: 252, CDRL1 comprises SEQ ID NO: 253, CDRL2 comprises an ETS, and CDRL3 comprises SEQ ID NO: 255 or 264. In some embodiments, preferred antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or a combination thereof, where, as defined according to the IMGT numbering system, CDRL1 comprises SEQ ID NO: 250, CDRH2 comprises SEQ ID NO: 256, CDRH3 comprises SEQ ID NO: 257, CDRH1 comprises SEQ ID NO: 258, CDRL2 comprises an EVS, and CDRL3 comprises any one of SEQ ID NOs: 260, 261, or 263.

[0179] In some embodiments, an antibody or antigen-binding fragment for practicing various embodiments of the present disclosure comprises an antibody or antigen-binding fragment comprising a heavy chain variable domain comprising any one of SEQ ID NOs: 400, 402, 409, 420, and a light chain variable domain comprising any one of SEQ ID NOs: 410, 412, 419, 421, 422.

[0180] In some embodiments, an antibody or antigen-binding fragment for practicing various embodiments of the present disclosure comprises an antibody or antigen-binding fragment comprising a heavy chain variable domain comprising SEQ ID NO: 400 and a light chain variable domain comprising SEQ ID NO: 410. In some embodiments, a preferred antibody or antigen-binding fragment for practicing various embodiments of the present disclosure comprises an antibody or antigen-binding fragment comprising a heavy chain variable domain comprising any one of SEQ ID NOs: 402, 409, or 420 and a light chain variable domain comprising any one of SEQ ID NOs: 412, 419, or 421. In some embodiments, a preferred antibody or antigen-binding fragment for practicing various embodiments of the present disclosure comprises an antibody or antigen-binding fragment comprising a heavy chain variable domain comprising SEQ ID NO: 402 and a light chain variable domain comprising SEQ ID NO: 412. In some embodiments, a preferred antibody or antigen-binding fragment for practicing various embodiments of the present disclosure comprises an antibody or antigen-binding fragment comprising a heavy chain variable domain comprising SEQ ID NO: 409 and a light chain variable domain comprising SEQ ID NO: 419. In some embodiments, a preferred antibody or antigen-binding fragment for practicing various embodiments of the present disclosure comprises an antibody or antigen-binding fragment comprising a heavy chain variable domain comprising SEQ ID NO: 420 and a light chain variable domain comprising SEQ ID NO: 421. In some embodiments, a preferred antibody or antigen-binding fragment for practicing various embodiments of the present disclosure comprises an antibody or antigen-binding fragment comprising a heavy chain variable domain comprising SEQ ID NO: 420 and a light chain variable domain comprising SEQ ID NO: 422.

[0181] In some embodiments, a preferred antibody or antigen-binding fragment for practicing various embodiments of the present disclosure is Ab102, Ab109, Ab130, Ab132, Ab133, or Ab141. In some embodiments, a preferred antibody or antigen-binding fragment for practicing various embodiments of the present disclosure is Ab109, Ab133, or Ab141.

[0182] In some embodiments, an anti-pro / latent myostatin antibody, or antigen-binding portion thereof, comprises a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of any one of the antibodies shown in Tables 2a-f. The present disclosure also includes any nucleic acid sequence encoding a molecule comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3 provided for any one of the antibodies shown in Tables 2a-f.

[0183] In some embodiments, the consensus CDR sequences provided in Table 2a are based on antibodies Ab109, Ab132, and Ab133. In some embodiments, the consensus CDR sequences provided in Table 2a are based on antibodies Ab102 and Ab130. In some embodiments, the consensus CDR sequences shown in Table 2a are based on all of the antibodies shown in Table 2d.

[0184] In some embodiments, the consensus CDR sequences provided in Tables 2b and 2c are based on antibodies 102, 109, 130, 132, and 133. In some embodiments, the consensus CDR sequences provided in Tables 2b and 2c are based on antibodies Ab109, Ab133, and Ab141.

[0185] [Table 3]

[0186] [Table 4]

[0187] [Table 5]

[0188] [Table 6]

[0189] [Table 7]

[0190] [Table 8]

[0191] [Table 9]

[0192] [Table 10]

[0193] [Table 11]

[0194] [Table 12]

[0195] [Table 13]

[0196] [Table 14]

[0197] In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding portions thereof suitable for implementing various embodiments of the present disclosure include, as defined by the Kabat numbering system, a CDRH1 comprising the sequence set forth in SEQ ID NO: 201, a CDRH2 comprising the sequence set forth in SEQ ID NO: 214, a CDRH3 comprising the sequence set forth in SEQ ID NO: 215, a CDRL1 comprising the sequence set forth in SEQ ID NO: 216, a CDRL2 comprising the sequence set forth in SEQ ID NO: 217, and a CDRL3 comprising the sequence set forth in SEQ ID NO: 218 or 224.

[0198] In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding portions thereof suitable for implementing various embodiments of the present disclosure include, as defined by the Chothia numbering system, a CDRH1 comprising the sequence set forth in SEQ ID NO: 228, a CDRH2 comprising the sequence set forth in SEQ ID NO: 229, a CDRH3 comprising the sequence set forth in SEQ ID NO: 230, a CDRL1 comprising the sequence set forth in SEQ ID NO: 231, a CDRL2 comprising the amino acid sequence ETS, and a CDRL3 comprising the sequence set forth in SEQ ID NO: 233 or 224.

[0199] In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding portions thereof suitable for implementing various embodiments of the present disclosure include a CDRH1 comprising the sequence set forth in SEQ ID NO: 201, a CDRH2 comprising the sequence set forth in SEQ ID NO: 219 or 226, a CDRH3 comprising the sequence set forth in SEQ ID NO: 220, a CDRL1 comprising the sequence set forth in SEQ ID NO: 216, a CDRL2 comprising the sequence set forth in SEQ ID NO: 222, and a CDRL3 comprising the sequence set forth in SEQ ID NO: 223, 227, or 298, as defined by the Kabat numbering system.

[0200] In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding portions thereof suitable for implementing various embodiments of the present disclosure include, as defined by the Chothia numbering system, a CDRH1 comprising the sequence set forth in SEQ ID NO: 234, a CDRH2 comprising the sequence set forth in SEQ ID NO: 235, a CDRH3 comprising the sequence set forth in SEQ ID NO: 236, a CDRL1 comprising the sequence set forth in SEQ ID NO: 237, a CDRL2 comprising the amino acid sequence EVS, and a CDRL3 comprising the sequence set forth in SEQ ID NO: 239 or 240.

[0201] In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding portions thereof suitable for implementing various embodiments of the present disclosure include, as defined by the IMGT numbering system, a CDRH1 comprising the sequence set forth in SEQ ID NO: 250, a CDRH2 comprising the sequence set forth in SEQ ID NO: 256 or 262, a CDRH3 comprising the sequence set forth in SEQ ID NO: 257, a CDRL1 comprising the sequence set forth in SEQ ID NO: 258, a CDRL2 comprising the sequence set forth in EVS, and a CDRL3 comprising the sequence set forth in SEQ ID NO: 260, 262, or 290.

[0202] In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding portions thereof suitable for implementing various embodiments of the present disclosure include, as defined by the Kabat or IMGT numbering system, a CDRH1 comprising the sequence set forth in SEQ ID NO: 201, a CDRH2 comprising the sequence set forth in SEQ ID NO: 520, a CDRH3 comprising the sequence set forth in SEQ ID NO: 521, a CDRL1 comprising the sequence set forth in SEQ ID NO: 522, a CDRL2 comprising the sequence set forth in SEQ ID NO: 523, and a CDRL3 comprising the sequence set forth in SEQ ID NO: 524 or 647.

[0203] In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding portions thereof suitable for implementing various embodiments of the present disclosure include, as defined by the Chothia numbering system, CDRH1 comprising the sequence set forth in SEQ ID NO: 648, CDRH2 comprising the sequence set forth in SEQ ID NO: 654, CDRH3 comprising the sequence set forth in SEQ ID NO: 655, CDRL1 comprising the sequence set forth in SEQ ID NO: 656, CDRL2 comprising the amino acid sequence AAS, and CDRL3 comprising the sequence set forth in SEQ ID NO: 657.

[0204] In some embodiments, the disclosure includes anti-pro / latent myostatin antibodies or antigen-binding portions thereof having one or more CDR sequences that include up to 5, 4, 3, 2, or 1 mutations (e.g., 1, 2, or 3 substitutions, insertions, and / or deletions) in amino acid residues compared to the corresponding CDR regions in any one of the SEQ ID NOs set forth in Tables 2a-f. In some embodiments, the disclosure includes anti-pro / latent myostatin antibodies or antigen-binding portions thereof that include one or more CDR sequences, e.g., the set of CDRs identified in any one of the SEQ ID NOs set forth in Tables 2a-f, e.g., the set of all six CDRs corresponding to the set of SEQ ID NOs from the table identified for a particular antibody in the table.

[0205] [Table 15]

[0206] [Table 16]

[0207] [Table 17]

[0208] [Table 18]

[0209] [Table 19]

[0210] [Table 20]

[0211] [Table 21]

[0212] [Table 22]

[0213] [Table 23]

[0214] [Table 24]

[0215] [Table 25]

[0216] [Table 26]

[0217] [Table 27]

[0218] [Table 28]

[0219] In some examples, the disclosure includes an anti-pro / latent myostatin antibody or antigen-binding portion thereof comprising a heavy chain variable domain, a light chain variable domain, or paired heavy and light chain variable domains from Table 3 above.

[0220] In various embodiments, the anti-pro / latent myostatin antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 400 or a sequence at least 95% identical thereto, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 410 or a sequence at least 95% identical thereto.

[0221] In various embodiments, the anti-pro / latent myostatin antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 402 or a sequence at least 95% identical thereto, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 412 or a sequence at least 95% identical thereto.

[0222] In various embodiments, the anti-pro / latent myostatin antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 409 or a sequence at least 95% identical thereto, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 419 or a sequence at least 95% identical thereto.

[0223] In various embodiments, the anti-pro / latent myostatin antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 420 or a sequence at least 95% identical thereto, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 421 or a sequence at least 95% identical thereto.

[0224] In various embodiments, the anti-pro / latent myostatin antibody or antigen-binding fragment thereof is Ab 102 or Ab 130. In various embodiments, the anti-pro / latent myostatin antibody or antigen-binding fragment thereof is Ab 109, Ab 132, or Ab 133.

[0225] In one embodiment, the anti-pro / latent myostatin antibody or antigen-binding fragment thereof comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 501 or a sequence at least 95% identical thereto, and / or a light chain region comprising the amino acid sequence of SEQ ID NO: 502 or a sequence at least 95% identical thereto.

[0226] In another embodiment, the anti-pro / latent myostatin antibody or antigen-binding fragment thereof comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 503 or a sequence at least 95% identical thereto, and / or a light chain region comprising the amino acid sequence of SEQ ID NO: 504 or a sequence at least 95% identical thereto.

[0227] In another embodiment, the anti-pro / latent myostatin antibody or antigen-binding fragment thereof comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 505 or a sequence at least 95% identical thereto, and / or a light chain region comprising the amino acid sequence of SEQ ID NO: 506 or a sequence at least 95% identical thereto.

[0228] In another embodiment, the anti-pro / latent myostatin antibody or antigen-binding fragment thereof comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 507 or a sequence at least 95% identical thereto, and / or a light chain region comprising the amino acid sequence of SEQ ID NO: 508 or a sequence at least 95% identical thereto.

[0229] In yet another embodiment, the anti-pro / latent myostatin antibody or antigen-binding fragment thereof comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 509 or a sequence at least 95% identical thereto, and / or a light chain region comprising the amino acid sequence of SEQ ID NO: 510 or a sequence at least 95% identical thereto.

[0230] In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding fragments thereof of the present disclosure include any antibody or antigen-binding fragment thereof comprising a heavy chain variable domain of any one of SEQ ID NOs: 400-409, 420 and a light chain variable domain of any one of SEQ ID NOs: 410-419, 421. In some embodiments, anti-pro / latent myostatin antibodies or antigen-binding fragments thereof of the present disclosure include any antibody comprising a heavy chain variable domain and a light chain variable domain of SEQ ID NOs: 400 and 410; 401 and 411; 402 and 412; 403 and 413; 404 and 414; 405 and 415; 406 and 416; 407 and 417; 408 and 418; 409 and 419; 420 and 421.

[0231] In some embodiments, the disclosure encompasses anti-pro / latent myostatin antibodies or antigen-binding fragments thereof comprising a heavy chain variable domain and / or a light chain variable domain comprising an amino acid sequence homologous to any one of the sequences described herein. In some embodiments, an anti-pro / latent myostatin antibody or antigen-binding fragment thereof comprises a heavy chain variable domain sequence that is at least 80%, 85%, or 90% identical to the heavy chain variable domain sequence of any one of SEQ ID NOs: 400-409, 420. In some embodiments, an anti-pro / latent myostatin antibody or antigen-binding fragment thereof comprises a light chain variable domain sequence that is at least 80%, 85%, or 90% identical to the light chain variable sequence of any one of SEQ ID NOs: 410-419, 421. In some embodiments, a heavy chain variable domain that is at least 90% identical does not contain any mutations in any of the CDR sequences provided herein. In some embodiments, a light chain variable domain that is at least 90% identical does not contain any mutations in any of the CDR sequences provided herein. For example, in some embodiments, the sequence variation in a heavy or light chain variable domain (eg, 90%, 95%, 98%, or 99%) occurs outside of the CDR sequences.

[0232] In some embodiments, the disclosure encompasses anti-pro / latent myostatin antibodies or antigen-binding fragments thereof comprising a variable domain sequence (i.e., the combined heavy and light chain variable domains) that is less than 70% identical to the variable domain sequence of Ab2 provided in PCT / US2015 / 059468. In some embodiments, the disclosure encompasses anti-pro / latent myostatin antibodies comprising a heavy chain sequence that is less than 70% identical to the heavy chain of Ab2 provided in PCT / US2015 / 059468. In some embodiments, these antibodies also comprise a heavy chain variable domain that is at least 80%, 85%, or 90% identical to the heavy chain variable domain sequence of any one of SEQ ID NOs: 400-409, 420. In some embodiments, an anti-pro / latent myostatin antibody or antigen-binding fragment thereof comprises a light chain variable domain sequence that is at least 80%, 85%, or 90% identical to the light chain variable sequence of any one of SEQ ID NOs: 410-419, 421.

[0233] In some embodiments, antibodies encompassed by the present disclosure comprise any one of the heavy chain variable domain sequences and / or any one of the light chain variable domain sequences provided in Table 3 and any IgG constant domain sequence. In some embodiments, the antibody comprises an IgG1 constant domain subtype or an IgG4 subtype. In the latter case, in some embodiments, the antibody comprises the Adair mutation (S228P).

[0234] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise an IgG constant domain or mutations in the constant domain that confer desirable properties. For example, to avoid potential problems due to Fab arm exchange, which is known to occur in native IgG4, the antibodies or antigen-binding fragments thereof provided herein may comprise an IgG1 constant domain, or, for example, a stabilizing "Adair" mutation in native IgG4 (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody," Mol Immunol 30, 105-108; 1993), in which serine 228 (EU numbering; Kabat numbering of residue 241) is converted to proline, resulting in an IgG1-like (CPPCP (SEQ ID NO: 58)) hinge sequence. Thus, any of the antibodies may comprise the stabilizing "Adair" mutation or the amino acid sequence CPPCP (SEQ ID NO: 58).

[0235] In some embodiments, the anti-pro / latent myostatin antibodies or antigen-binding portions thereof of the present disclosure may comprise antibody constant regions or portions thereof. For example, a VL domain may be attached at its C-terminus to a light chain constant domain such as Cκ or Cλ. Similarly, a VH domain or portion thereof may be attached to all or part of a heavy chain such as IgA, IgD, IgE, IgG, and IgM, and any isotype subclass. The antibody may comprise a suitable constant region (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within the scope of the present disclosure may comprise VH and VL domains, or antigen-binding portions thereof, combined with any suitable constant region.

[0236] In certain embodiments, the VH and / or VL domains can be reverted to their germline sequences, e.g., the framework regions (FR) of these domains are mutated using conventional molecular biology techniques to match those produced by germline cells. For example, the VH and / or VL domains can be reverted to the germline sequences of IgHV3-30 (SEQ ID NO: 36) and / or IgLV1-44 (SEQ ID NO: 37), respectively. It should be understood that either the VH and / or VL domains can be reverted to any suitable germline sequence. In other embodiments, the FR sequences remain deviant from the consensus germline sequences.

[0237] In some embodiments, the anti-pro / latent myostatin antibody or antigen-binding fragment may or may not include the framework regions of the antibodies set forth in SEQ ID NOs: 400 to 421. In some embodiments, the anti-pro-latent myostatin antibody is a murine antibody and includes murine framework region sequences.

[0238] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein specifically bind to pro / latent myostatin. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein bind to or near the toroid cleavage site or toroid docking site of pro / latent myostatin. In some embodiments, an antibody binds near the toroid cleavage site or toroid docking site if it binds within 15 or fewer amino acid residues of the toroid cleavage site or toroid docking site. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein bind within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the toroid cleavage site or toroid docking site. In some embodiments, the antibody binds to or near the toroid cleavage site of GDF8. For example, the antibody may bind to the amino acid sequence set forth in SEQ ID NO: 62 PKAPPLRELIDQYDVQRDDSSDGSLEDDDYHAT (SEQ ID NO: 62). In other embodiments, the antibodies or antigen-binding fragments thereof provided herein may bind at or near the proprotein convertase cleavage site or at or near the proprotein convertase docking site of pro / latent myostatin. In some embodiments, an antibody or antigen-binding fragment thereof binds near the proprotein convertase cleavage site or near the proprotein convertase docking site if it binds within 15 or fewer amino acid residues of the proprotein convertase cleavage site or proprotein convertase docking site. In some embodiments, any of the antibodies provided herein, or antigen-binding fragments thereof, bind within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the proprotein convertase cleavage site or proprotein convertase docking site. In some embodiments, the antibody binds at or near the proprotein convertase cleavage site of GDF8. For example, the antibody may bind to the amino acid sequence set forth in SEQ ID NO: 63 (GLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRC).

[0239] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein bind to an epitope comprising at least one amino acid residue of KALDEN (SEQ ID NO: 118) and / or FVQILRLIKPMKDGTRYTGIRSLK (SEQ ID NO: 57).

[0240] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein bind to an epitope comprising one or more amino acid residues F147, Q149, L151, Y163, R167, S168, K170, K205, L207, E209, and N210 based on the numbering of the human pro-GDF8 sequence set forth in SEQ ID NO: 52, which corresponds to the numbering of 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 some embodiments, such antibodies or antigen-binding fragments bind to an epitope within the prodomain of human myostatin, the epitope comprising one or more (e.g., all) amino acid residues F147, Q149, L151, Y186, S168, Q149, L151, Y163, S168, K170, K205, and / or L207, when numbered according to SEQ ID NO: 52 disclosed herein. In some embodiments, such antibodies or antigen-binding fragments bind to an epitope within the prodomain of human myostatin, the epitope comprising one or more (e.g., all) amino acid residues F147, Q149, L151, Y186, K170, K205, and / or L207, when numbered according to SEQ ID NO: 52. In some embodiments, such antibodies bind to an epitope comprising 7 or more, 6 or more, 5 or more, 4 or more, or 3 or more of the amino acid residues set forth above. In some embodiments, the antibody is Ab102 or Ab130. In some embodiments, the antibody is Ab109, Ab132, or Ab133. In some embodiments, the antibody is Ab109, Ab133, or Ab141.

[0241] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein specifically bind to pro / latent myostatin relative to other forms of myostatin and / or other members of the TGFβ family of growth factors, including, but not limited to, AMH, ARTN, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDNF, INHA, INHBA, INHBB, INHBC, INHBE, LEFTY1, LEFTY2, NODAL, NRTN, PSPN, TGFβ1, TGFβ2, and TGFβ3 proteins. In some embodiments, the antibodies or antigen-binding fragments thereof bind to pro / latent myostatin with at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1,000-fold greater binding affinity than other members of the TGFβ family of growth factors. In some embodiments, the antibodies or antigen-binding fragments thereof bind to pro / latent myostatin with at least 1,000-fold greater affinity than other members of the TGFβ family of growth factors. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to pro / latent myostatin with at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1,000-fold greater binding affinity than one or more forms of GDF11 or mature myostatin. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to pro / latent myostatin with at least 1,000-fold greater affinity than to one or more forms of GDF11 (e.g., proGDF11, latent GDF11, or mature GDF11) or mature myostatin.In some embodiments, the antibodies or antigen-binding fragments thereof provided herein exhibit at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1,000-fold greater inhibitory activity against proteolytic cleavage (by proprotein convertases or thrombin proteases) of pro / latent myostatin compared to other members of the TGFβ family, such as pro / latent GDF11. In another embodiment, the antibodies or antigen-binding fragments thereof disclosed herein do not bind to GDF11. Without being bound by theory, the antibodies or antigen-binding fragments thereof provided herein have an improved safety profile due to reduced toxicity associated with cross-reactivity with other TGFβ family members (e.g., compared to antibodies that cross-react with both myostatin and GDF11). One such potential toxicity is the loss of bone strength associated with GDF11 inhibition, as reported in Suh et al. Proceedings of the National Academy of Sciences Mar. 2020, 117 (9) 4910-4920, the contents of which are incorporated herein in their entirety.

[0242] Sweeping antibody Certain embodiments of the present disclosure relate to sweeping antibodies. As used herein, a "sweeping antibody" or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof that has both pH-sensitive antigen binding and at least a threshold level of binding to cell-surface neonatal Fc receptor (FcRn) at neutral or physiological pH. In some embodiments, a sweeping antibody, or antigen-binding portion thereof, binds to neonatal Fc receptor FcRn at neutral pH. For example, a sweeping antibody may bind to FcRn at a pH ranging from 7.0 to 7.6. In some embodiments, a sweeping antibody or antigen-binding portion thereof may bind to an antigen at the antigen-binding site and bind to cellular FcRn via the Fc portion of the antibody. In some embodiments, the sweeping antibody, or antigen-binding portion thereof, may then be internalized, releasing the antigen into acidic endosomes, which may be degraded. In some embodiments, a sweeping antibody or antigen-binding portion thereof that is no longer bound to an antigen may then be released by the cell (e.g., by exocytosis) and returned to the serum.

[0243] In some embodiments, FcRn in the vascular endothelium (e.g., of a subject) extends the half-life of the sweeping antibody, or antigen-binding portion thereof. In some embodiments, vascular endothelial cells internalize the sweeping antibody, or antigen-binding portion thereof, which, in some embodiments, is bound to an antigen, such as myostatin (e.g., promyostatin, latent myostatin, or primed myostatin). In some embodiments, the sweeping antibody, or antigen-binding portion thereof, is recycled back into the bloodstream. In some embodiments, the sweeping antibody, or antigen-binding portion thereof, has an increased half-life (e.g., in the serum of a subject) compared to its conventional counterpart. In some embodiments, the conventional counterpart of a sweeping antibody refers to the antibody, or antigen-binding portion thereof, from which the sweeping antibody, or antigen-binding portion thereof, is derived (e.g., before the Fc portion of the conventional antibody was engineered to bind to FcRn with higher affinity at pH 7). In some embodiments, the sweeping antibody or antigen-binding portion thereof has a serum half-life in a subject that is at least 1%, 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 100%, 150%, 200%, or 250% longer than its conventional counterpart.

[0244] In some embodiments, the Fc portion of the sweeping antibody binds to FcRn. In some embodiments, the Fc portion of the sweeping antibody binds to FcRn. -3 M~10 -8 K in the M range D and binds to FcRn at a pH of 7.4. In some embodiments, the sweeping antibody is -3 M~10 -7 M, 10 -3 M~10 -6 M, 10 -3 M~10 -5 M, 10 -3 M~10 -4 M, 10 -4 M~10 -8 M, 10 -4 M~10 -7 M, 10 -4 M~10 -6 M, 10 -4 M~10 -5 M, 10-5 M~10 -8 M, 10 -5 M~10 -7 M, 10 -5 M~10 -6 M, 10 -6 M~10 -8 M, 10 -6 M~10 -7 M or 10 -7 M~10 -8 K in the M range D and binds to FcRn at a pH of 7.4. In some embodiments, FcRn binds to the CH2-CH3 hinge region of the sweeping antibody. In some embodiments, FcRn binds to the same region as Protein A or Protein G. In some embodiments, FcRn binds to a binding site different from FcγR. In some embodiments, amino acid residue AA of the sweeping antibody Fc region is required for binding to FcRn. In some embodiments, amino acid residue AA of the sweeping antibody Fc region affects binding to FcRn.

[0245] In some embodiments, any of the antibodies, or antigen-binding fragments thereof, provided herein are engineered to bind to FcRn with higher affinity. In some embodiments, any of the antibodies, or antigen-binding fragments thereof, provided herein are engineered to bind to FcRn with higher affinity at pH 7.4. In some embodiments, the affinity of the antibodies, or antigen-binding fragments thereof, for FcRn is increased to enhance their pharmacokinetic (PK) properties compared to their conventional counterparts. For example, in some embodiments, the sweeping antibody induces fewer adverse reactions due to its efficacy at lower doses. In some embodiments, the sweeping antibody, or antigen-binding portion thereof, is administered less frequently. In some embodiments, the transcytosis of the sweeping antibody, or antigen-binding portion thereof, to specific tissue types is increased. In some embodiments, the sweeping antibody, or antigen-binding portion thereof, improves the efficiency of transplacental delivery. In some embodiments, the sweeping antibody, or antigen-binding portion thereof, is inexpensive to produce.

[0246] In some embodiments, any of the antibodies, or antigen-binding fragments thereof, provided herein are engineered to bind to FcRn with lower affinity. In some embodiments, any of the antibodies, or antigen-binding fragments thereof, provided herein are engineered to bind to FcRn with lower affinity at pH 7.4. In some embodiments, the affinity of the sweeping antibodies, or antigen-binding portions thereof, for FcRn is reduced to shorten their pharmacokinetic (PK) characteristics compared to their conventional counterparts. For example, in some embodiments, the sweeping antibodies, or antigen-binding portions thereof, are cleared more rapidly for imaging and / or radioimmunotherapy. In some embodiments, the sweeping antibodies, or antigen-binding portions thereof, promote the clearance of endogenous pathogenic antibodies as a treatment for autoimmune diseases. In some embodiments, the sweeping antibodies, or antigen-binding portions thereof, reduce the risk of adverse pregnancy outcomes that may be caused by transplacental transport of material fetal-specific antibodies.

[0247] In some embodiments, the sweeping antibody, or antigen-binding portion thereof, has reduced affinity for the antigen at low pH compared to neutral or physiological pH (e.g., pH 7.4). In some embodiments, the sweeping antibody, or antigen-binding portion thereof, has reduced affinity for the antigen at acidic pH (e.g., a pH in the range of 5.5 to 6.5) compared to physiological pH (e.g., pH 7.4).

[0248] It should be understood that any of the antibodies, or antigen-binding fragments thereof, provided herein can be engineered to dissociate from an antigen in response to a change in pH (e.g., a pH-sensitive antibody). In some embodiments, the sweeping antibodies, or antigen-binding portions thereof, provided herein are engineered to bind to an antigen in a pH-dependent manner. In some embodiments, the sweeping antibodies, or antigen-binding portions thereof, provided herein are engineered to bind to FcRn in a pH-dependent manner. In some embodiments, the sweeping antibodies, or antigen-binding portions thereof, provided herein are internalized by endocytosis. In some embodiments, the sweeping antibodies, or antigen-binding portions thereof, provided herein are internalized by FcRn binding. In some embodiments, the endocytosed sweeping antibodies, or antigen-binding portions thereof, release the antigen in an endosome. In some embodiments, the sweeping antibodies, or antigen-binding portions thereof, are recycled back to the cell surface. In some embodiments, the sweeping antibodies remain attached to the cells. In some embodiments, the endocytosed sweeping antibodies, or antigen-binding portions thereof, are recycled back to plasma. It should be understood that the Fc portion of any of the antibodies or antigen-binding fragments thereof provided herein can be engineered to have different FcRn-binding activities. In some embodiments, the FcRn-binding activity affects the clearance time of an antigen by a sweeping antibody. In some embodiments, the sweeping antibody can be a long-acting or rapid-acting sweeping antibody.

[0249] In some embodiments, converting a conventional therapeutic antibody or antigen-binding portion thereof to a sweeping antibody or antigen-binding portion thereof reduces the effective dose. In some embodiments, converting a conventional therapeutic antibody or antigen-binding portion thereof to a sweeping antibody or antigen-binding portion thereof reduces the effective dose by at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, converting a conventional therapeutic antibody or antigen-binding portion thereof to a sweeping antibody or antigen-binding portion thereof reduces the effective dose by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 15-fold, 20-fold, 50-fold, or 100-fold.

[0250] In some embodiments, selection of an appropriate dose of a sweeping antibody or antigen-binding portion thereof for treatment can be performed empirically. In some embodiments, a high dose of the sweeping antibody or antigen-binding portion thereof can saturate FcRn, resulting in an antibody that stabilizes the antigen in serum without internalization. In some embodiments, the sweeping antibody or antigen-binding portion thereof is administered once daily, once per week, once per 2 weeks, once per 3 weeks, once per 4 weeks, once per 6 weeks, once per 8 weeks, once per 10 weeks, once per 12 weeks, once per 16 weeks, once per 20 weeks, or once per 24 weeks.

[0251] In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein can be modified or engineered to become sweeping antibodies. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein can be converted into sweeping antibodies using any suitable method. For example, suitable methods for generating sweeping antibodies or antigen-binding portions thereof have been previously described in Igawa et al., (2013) "Engineered Monoclonal Antibody with Novel Antigen-Sweeping Activity In Vivo," PLoS ONE 8(5):e63236; and Igawa et al., "pH-dependent antigen-binding antibodies as a novel therapeutic modality," Biochimica et Biophysica Acta 1844 (2014) 1943-1950, the contents of each of which are incorporated herein by reference. However, it should be understood that the methods for generating sweeping antibodies or antigen-binding portions thereof provided herein are not intended to be limiting. Accordingly, additional methods for generating sweeping antibodies or antigen-binding portions thereof are within the scope of this disclosure.

[0252] Some aspects of the present disclosure relate to the affinity (e.g., K D This is based on the recognition that the K (expressed as K) of binding to pro / latent myostatin is sensitive to changes in pH. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein exhibit an increased K of binding to pro / latent myostatin at relatively low pH (e.g., a pH in the range of 4.0 to 6.5, e.g., pH 5.5) compared to relatively high pH (e.g., a pH in the range of 7.0 to 7.6, e.g., pH 7.4). D In some embodiments, the antibodies, or antigen-binding fragments thereof, provided herein have a pH of 10 at a pH of 4.0 to 6.5 (e.g., pH 5.5). -3 M, 10-4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 K of binding to pro / latent myostatin in the M range D In some embodiments, the antibodies, or antigen-binding fragments thereof, provided herein have a pH of 10 at a pH of 7.0 to 7.6 (e.g., pH 7.4). -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 K of binding to pro / latent myostatin in the M range D In some embodiments, the antibodies, or antigen-binding fragments thereof, provided herein have at least 2-fold, at least 10-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 200-fold, at least 250-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, or at least 10,000-fold greater activity at a pH of 4.0 to 6.5 (e.g., pH 5.5) compared to a pH of 7.0 to 7.6 (e.g., pH 7.4).

[0253] Antibodies and antigen-binding fragments that compete for antigen binding with novel anti-pro / latent myostatin antibodies or antigen-binding fragments thereof Certain embodiments of the present disclosure relate to antibodies and antigen-binding fragments thereof that compete or cross-compete for antigen binding with any of the antibodies or antigen-binding fragments thereof provided herein. Preferably, the antigen is human latent myostatin.

[0254] In some embodiments, an antibody, or antigen-binding portion thereof, binds at or near the same epitope as any of the antibodies provided herein. In some embodiments, an antibody, or antigen-binding portion thereof, binds near an epitope if it binds within 15 or fewer amino acid residues of the epitope. In some embodiments, any of the antibodies, or antigen-binding fragments thereof, provided herein bind within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the epitope bound by any of the antibodies or antigen-binding fragments thereof provided herein. In preferred embodiments, such an antibody or antigen-binding fragment cross-competes with Ab2 or apitegromab for binding to human pro / latent myostatin. The antibody or antigen-binding fragment may cross-compete with Ab2, as described herein, for binding to human pro / latent myostatin. In some embodiments, such antibodies or antigen-binding fragments bind to an epitope within the prodomain of human myostatin, wherein the epitope comprises one or more (e.g., all) amino acid residues F147, Q149, L151, Y186, S168, Q149, L151, Y163, S168, K170, K205, and / or L207 when numbered according to SEQ ID NO: 52 disclosed herein. In some embodiments, such antibodies or antigen-binding fragments bind to an epitope within the prodomain of human myostatin, wherein the epitope comprises one or more (e.g., all) amino acid residues F147, Q149, L151, Y186, K170, K205, and / or L207 when numbered according to SEQ ID NO: 52. In some embodiments, such antibodies or antigen-binding fragments comprise an HCDR3 paratope containing up to two amino acid differences compared to SEQ ID NO: 220. In some embodiments, such antibodies or antigen-binding fragments comprise an HCDR3 sequence comprising a leucine at amino acid position 3 and a tryptophan at amino acid position 9 when numbered according to SEQ ID NO:220.In some embodiments, such antibodies or antigen-binding fragments comprise an HCDR3 sequence comprising a leucine at amino acid position 3, a valine or isoleucine at amino acid position 4, a leucine at amino acid position 7, a glutamic acid at amino acid position 8, and / or a tryptophan at amino acid position 9, when numbered according to SEQ ID NO: 220.

[0255] In another embodiment, the antibody, or antigen-binding portion thereof, -8 The equilibrium dissociation constant K between the antibody and the protein is less than M D In another embodiment, the antibody, or antigen-binding portion thereof, is 10 -11 M~10 -8 K in the M range D In a preferred embodiment, the antibody has a bivalent K of less than 1 nM as measured by an SPR-based in vitro binding assay such as Biacore™. D It has.

[0256] Certain embodiments of the present disclosure relate to antibodies or antigen-binding portions thereof that compete for binding to pro / latent myostatin with any of the antibodies or antigen-binding fragments thereof provided herein. In some embodiments, the antibody or antigen-binding portion thereof binds to pro / latent myostatin at the same epitope as any of the antibodies or antigen-binding portions thereof provided herein. In other embodiments, the antibody or antigen-binding portion thereof binds to pro / latent myostatin at the same epitope as any of the antibodies or antigen-binding portions thereof provided herein. -6 an equilibrium dissociation constant, K, between the antibody or antigen-binding portion thereof and pro / latent myostatin that is less than M D In other embodiments, an antibody or antigen-binding portion thereof that competes with any of the antibodies or antigen-binding fragments thereof provided herein competes for binding to pro / latent myostatin at 10 -11 M~10 -8 K in the M range D It binds to pro- and latent myostatin.

[0257] Any of the antibodies or antigen-binding fragments thereof provided herein can be characterized using any suitable method. For example, one method is identifying 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, competitive assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, "Using Antibodies," a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In a further example, epitope mapping can be used to determine the sequence to which an antibody or its antigen-binding portion binds. The epitope can be a linear epitope, i.e., an epitope contained in a single stretch of amino acids, or a conformational epitope formed by three-dimensional interactions of amino acids that may not necessarily be contained in a single stretch (primary structure linear sequence). Peptides of various lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used in antibody binding assays. In another example, the epitope bound by an antibody, or its antigen-binding portion, can be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody, or its antigen-binding portion. According to gene fragment expression assays, the open reading frame encoding the target antigen is fragmented randomly or by specific gene construction, and the reactivity of the expressed fragments of the antigen with the antibody being tested is determined. Gene fragments can be produced, for example, by PCR, and then transcribed and translated into protein in vitro in the presence of radioactive amino acids. Binding of the antibody, or its antigen-binding portion, to the radiolabeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Specific epitopes can also be identified by using large libraries of random peptide sequences (phage library fragments) displayed on the surface of phage particles.Alternatively, defined library fragments of overlapping peptide fragments can be tested for binding to a test antibody or antigen-binding portion thereof in a simple binding assay. In a further example, mutagenesis of the antigen-binding domain, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and / or necessary for epitope binding. For example, domain swapping experiments can be performed using mutants of the target antigen in which various fragments of the pro / latent myostatin polypeptide are replaced (exchanged) with sequences from a closely related but antigenically distinct protein, such as another member of the TGFβ protein family (e.g., GDF11). By assessing the binding of the antibody or antigen-binding portion thereof to mutant pro / latent myostatin, the importance of binding of a particular antigen fragment to the antibody or antigen-binding portion thereof can be assessed.

[0258] Alternatively, a competition assay can be performed using another antibody ("reference antibody") known to bind to the same antigen to determine whether an antibody or antigen-binding portion thereof ("test antibody") binds to the same epitope as the other antibody or antigen-binding portion thereof. Competition assays are well known to those skilled in the art. If the test antibody blocks the reference antibody from binding to the antigen and the reference antibody blocks the test antibody from binding to the antigen, the reference antibody and the test antibody are said to cross-block or cross-compete with each other for antigen binding. It is well understood in the art that such antibodies bind to the same or overlapping epitopes within an antigen.

[0259] Suitable methods, such as any of the epitope mapping methods described herein, can be applied to determine whether an anti-pro / latent myostatin antibody or antigen-binding portion thereof binds to one or more of the specific residues / segments in pro / latent myostatin described herein. Furthermore, the interaction of an antibody or antigen-binding portion thereof with one or more of these defined residues in pro / latent myostatin can be determined by conventional techniques. For example, a crystal structure can be determined, and the distance between a residue in pro / latent myostatin and one or more residues in the antibody or antigen-binding portion thereof can be determined accordingly. Based on such distances, it can be determined whether a specific residue in pro / latent myostatin interacts with one or more residues in the antibody or antigen-binding portion thereof. Furthermore, suitable methods, such as competition assays and targeted mutagenesis assays, can be applied to determine the preferential binding of a candidate anti-pro / latent myostatin antibody or antigen-binding portion thereof to pro / latent myostatin compared to another target, such as mutant pro / latent myostatin.

[0260] In one embodiment, the disclosure encompasses a method for identifying antibodies or antigen-binding fragments thereof for competitive binding with one or more of the antibodies provided herein, the method comprising screening for antibodies or antigen-binding fragments thereof that compete or cross-compete with one or more of the antibodies provided herein (e.g., any of Ab101-141, e.g., any of Ab102, Ab109, Ab130, Ab132, or Ab133), and that have the following characteristic: a K of less than 5 nM (e.g., less than 4 nM, 3 nM, 2 nM, 1 nM, or 0.5 nM as measured by an SPR-based in vitro binding assay such as Biacore™). DThe methods include assaying for antibodies or antigen-binding fragments thereof that exhibit one or more (e.g., all) of the following: binding to pro / latent myostatin at pH 5.5 / 7.4, high pH-sensitive binding to pro / latent myostatin (e.g., greater than 10-fold as measured by comparing dissociation rates at pH 5.5 / 7.4), and / or a 2:1 Fab:promyostatin homodimer binding stoichiometry. In some embodiments, the present disclosure encompasses antibodies identified and / or prepared according to the methods of the present disclosure.

[0261] qualification In some embodiments, the antibodies and antigen-binding fragments of the present disclosure can be modified, for example, by conjugation to a therapeutic agent (e.g., as a bispecific antibody or antibody-drug conjugate) or a detectable agent, such as a detectable label. Such labels include, but are not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, non-radioactive paramagnetic metal ions, and affinity labels for detection and isolation of pro / latent myostatin. Detectable substances can be coupled or conjugated directly to the polypeptides of the present disclosure or indirectly via an intermediate (e.g., a linker) using suitable techniques. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, or acetylcholinesterase; non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; non-limiting examples of suitable fluorescent materials include biotin, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; non-limiting examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include radioactive metal ions, such as α-radiation emitters. Projectiles or other radioactive isotopes, such as iodine (I, I, I, I), carbon (C), sulfur (S), tritium (H), indium (In, In, In, In), and technetium (Tc, Tc), thallium (Ti), gallium (Ga, Ga), palladium (Pd), molybdenum (Mo ), xenon (133Xe), fluorine (18F), 153Sm, Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 86R, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, and tin (113Sn, 117Sn).A detectable substance can be coupled or conjugated directly to an anti-pro / latent myostatin antibody or antigen-binding portion thereof of the present disclosure, or indirectly via an intermediate (e.g., a linker, etc.) using suitable techniques. Anti-pro / latent myostatin antibodies, or antigen-binding portions thereof, conjugated to a detectable substance can be used in diagnostic assays such as those described herein.

[0262] As demonstrated herein, the present disclosure includes novel antibodies and antigen-binding fragments thereof that can selectively inhibit myostatin activation. However, unlike the inhibitors previously described in PCT / US2016 / 052014, at least some of the novel antibodies disclosed herein (e.g., Ab109, Ab105, Ab130, and Ab133) bind to latent myostatin with sufficiently high monovalent affinity so that one arm of the antibody (e.g., the Fab) can interact with the antigen. Without wishing to be bound by theory, it is believed that such high monovalent binding affinity allows flexibility for designing myostatin inhibitors that incorporate the six CDRs, variable domains (VH and / or VL), or the corresponding Fab domain into engineered constructs, such as bispecific antibodies and other modalities, including the antigen-binding portion of an antibody. Such recombinantly engineered constructs are encompassed by the present disclosure.

[0263] Pharmaceutical Composition The antibodies or antigen-binding fragments thereof described herein can be formulated into pharmaceutical compositions suitable for administration to human or non-human subjects. Such pharmaceutical compositions can be intended for therapeutic or prophylactic use. In some embodiments, the pharmaceutical compositions are suitable for subcutaneous administration. One or more myostatin inhibitors, e.g., anti-pro / latent myostatin antibodies, can be mixed with a pharmaceutically acceptable carrier (excipient), including a buffer, to form a pharmaceutical composition for administration to a patient who may benefit from reduced myostatin signaling in vivo. By "pharmaceutically acceptable," it is meant that the carrier is compatible with the active ingredient of the composition (preferably, capable of stabilizing the active ingredient) and is not harmful to the subject being treated. Examples of pharmaceutically acceptable excipients (carriers), including buffers, will be apparent to those skilled in the art and have been previously described. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K.E. Hoover.Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); polypeptides of low molecular weight (less than about 10 residues); serum albumin, gelatin, etc. Pharmaceutically acceptable excipients may include proteins such as antibodies, antibodies, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0264] In one example, the pharmaceutical compositions described herein contain two or more myostatin inhibitors, e.g., two or more anti-pro / latent myostatin antibodies, or antigen-binding portions thereof, that recognize different epitopes / residues of a target antigen.

[0265] In some examples, the pharmaceutical compositions described herein include emulsion-based or lipid-based formulations, such as liposomes, containing a myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody or an antigen-binding portion thereof, which can be prepared by any suitable method, such as those described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be produced by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

[0266] The anti-pro / latent myostatin antibody or antigen-binding portion thereof can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Exemplary techniques have been previously described; see, e.g., Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing (2000).

[0267] In other examples, the pharmaceutical compositions described herein can be formulated in a sustained-release format. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody or antigen-binding portion thereof, which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0268] Pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0269] The pharmaceutical compositions described herein may be in unit dosage form such as tablets, pills, capsules, powders, granules, solutions or suspensions for oral, parenteral or rectal administration, or administration by inhalation or insufflation, or suppositories.

[0270] Compositions, preferably in sterile, pharmaceutically acceptable solvents, may be nebulized by the use of gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.

[0271] kit The present disclosure also provides kits for use in alleviating myopathy or metabolic disorders, such as diseases / disorders associated with diabetes, obesity, or metabolic syndrome. Such kits may include one or more containers containing any of the anti-pro / latent myostatin antibodies or antigen-binding fragments thereof disclosed herein, e.g., Ab102, Ab109, Ab130, Ab132, Ab133, or Ab141. In some embodiments, such kits may further include one or more additional therapeutic reagents, such as one or more GLP-1 receptor agonists. In some embodiments, such kits may further include one or more diagnostic reagents.

[0272] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include instructions for administering any of the anti-pro / latent myostatin antibodies or antigen-binding fragments thereof disclosed herein, e.g., Ab102, Ab109, Ab130, Ab132, Ab133, or Ab141, to treat, delay the onset of, or alleviate a target disease, such as those described herein. The kit may further include instructions for selecting an individual suitable for treatment based on identifying whether the individual has the target disease. In yet other embodiments, the instructions include instructions for administering the antibody to an individual at risk for the target disease.

[0273] Instructions for using any of the anti-pro / latent myostatin antibodies, or antigen-binding fragments thereof, disclosed herein, e.g., Ab102, Ab109, Ab130, Ab132, Ab133, or Ab141, for treatment generally include information regarding the dosage, administration schedule, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., a multi-dose package), or subunit dose. Instructions provided in kits of the present disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions written on a magnetic or optical storage disk) are also acceptable.

[0274] The label or package insert indicates that the composition is used for treating, delaying the onset of, and / or alleviating a muscle disorder, e.g., a disease or disorder associated with myopathy, or a metabolic disorder, e.g., a disease or disorder associated with diabetes, obesity, and / or metabolic syndrome. Instructions for practicing any of the methods described herein can be provided.

[0275] The kits of the present disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Packaging for use in combination with a specific device, such as an inhaler, a nasal administration device (e.g., an atomizer), or an infusion device such as a minipump, is also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an anti-pro / latent myostatin antibody, or an antigen-binding fragment thereof, as described herein.

[0276] Kits may optionally provide additional components such as buffers and interpretive information. Typically, kits include a container and a label or package insert on or associated with the container. In some embodiments, the disclosure provides an article of manufacture comprising the contents of the above-described kit.

[0277] Production of anti-pro / latent myostatin antibodies or antigen-binding fragments thereof Numerous methods can be used to obtain the antibodies of the present disclosure, or antigen-binding fragments thereof. For example, antibodies and antigen-binding fragments thereof can be produced using recombinant DNA techniques. Monoclonal antibodies and antigen-binding fragments thereof can also be produced by generating hybridomas according to known methods (see, e.g., Kohler and Milstein (1975) Nature, 256:495-499). The hybridomas thus formed are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and BLI or SPR (e.g., Octet® or Biacore™) analysis, to identify one or more hybridomas that produce antibodies or antigen-binding portions thereof that specifically bind to a particular antigen. Any form of a particular antigen can be used as an immunogen, for example, recombinant antigens, native forms, any variants or fragments thereof, and antigenic peptides thereof (e.g., epitopes described herein either as linear epitopes or within scaffolds as conformational epitopes). One exemplary method for producing antibodies and antigen-binding portions thereof includes screening protein expression libraries, such as phage or ribosome display libraries, which express antibodies or fragments thereof (eg, scFvs). Phage display is described, for example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222:581-597; WO 92 / 18619; WO 91 / 17271; WO 92 / 20791; WO 92 / 15679; WO 93 / 01288; WO 92 / 01047; WO 92 / 09690; and WO 90 / 02809.

[0278] In addition to the use of display libraries, a particular antigen (e.g., promyostatin) can be used to immunize a non-human animal, e.g., a rodent, e.g., a mouse, hamster, or rat. In one embodiment, the non-human animal is a mouse.

[0279] In another embodiment, monoclonal antibodies are obtained from non-human animals, and then modified, for example, chimeric, using suitable recombinant DNA technology.Various methods for producing chimeric antibodies have been described.See, for example, Morrison et al., Proc.Natl.Acad.Sci.USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., EP 171496; EP 0173494, GB 2177096B.

[0280] For further antibody production techniques, see Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988. The present disclosure is not necessarily limited to any particular source, method of production, or other particular characteristics of the antibodies.

[0281] Some aspects of the present disclosure relate to host cells transformed with a polynucleotide or vector. The host cell can be a prokaryotic or eukaryotic cell. The polynucleotide or vector present in the host cell can be integrated into the genome of the host cell or maintained extrachromosomally. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal, or human cell. In some embodiments, fungal cells are, for example, those of the genus Saccharomyces, particularly those of the species S. cerevisiae. The term "prokaryote" includes all bacteria that can be transformed or transfected with DNA or RNA molecules to express antibodies or corresponding immunoglobulin chains. Prokaryotic hosts can include Gram-negative and Gram-positive bacteria, such as E. coli, S. typhimurium, Serratia marcescens, and Bacillus subtilis. The term "eukaryote" includes yeast, higher plants, insects, and vertebrate cells, e.g., mammalian cells such as NS0 and CHO cells. Depending on the host used in a recombinant production procedure, antibodies or immunoglobulin chains encoded by a polynucleotide may be glycosylated or non-glycosylated. The antibody or corresponding immunoglobulin chain may also include an initial methionine amino acid residue. In some embodiments, host cells, e.g., NS0 and CHO cells, can be transformed with a polynucleotide or vector encoding any one of the antibodies (e.g., Ab101-Ab141) or antigen-binding fragments thereof disclosed herein, or portions thereof (e.g., a first cell can be transformed with a polynucleotide or vector encoding the heavy chain of any one of Ab101-Ab141 or antigen-binding fragments thereof, and a second cell can be transformed with a polynucleotide or vector encoding the light chain of any one of Ab101-Ab141 or antigen-binding fragments thereof).

[0282] In some embodiments, once the vector has been incorporated into a suitable host, the host can be maintained under conditions suitable for high-level expression of the nucleotide sequence, followed, if desired, by collection and purification of immunoglobulin light chains, heavy chains, light / heavy chain dimers, or intact antibodies, antigen-binding fragments, or other immunoglobulin forms; see Beychok, Cells of Immunoglobulin Synthesis, Academic Press, NY (1979). Thus, the polynucleotide or vector is introduced into a cell, which then produces the antibody or antigen-binding fragment. For example, host cells, e.g., NSO or CHO cells, containing or harboring a portion of a polynucleotide or vector encoding any one of the antibodies (e.g., Ab101-Ab141) or antigen-binding fragments thereof disclosed herein can express the antibody or antigen-binding fragment (or the heavy or light chain of the antibody). Furthermore, transgenic animals, preferably mammals, containing the aforementioned host cells can be used for large-scale production of antibodies or antibody fragments. Large scale production typically refers to bioreactors (e.g., cell cultures) of 250 liters or more, for example, 250 L, 500 L, 1000 L, 1500 L, 2000 L, 3000 L, 4000 L, 5000 L, 6000 L, or more.

[0283] Transformed host cells can be grown in fermentors and cultured using any suitable technique to achieve optimal cell growth. Once expressed, whole antibodies, their dimers, individual light and heavy chains, other immunoglobulin forms, or antigen-binding fragments can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like; see Scopes, "Protein Purification," Springer Verlag, NY (1982). The antibodies or antigen-binding fragments can then be isolated from the growth medium, cell lysates, or cell membrane fractions. Isolation and purification of antibodies or antigen-binding fragments expressed, for example, in microorganisms, can be by any conventional means, such as preparative chromatographic separations and immunological separations, e.g., involving the use of monoclonal or polyclonal antibodies against the constant regions of the antibodies. Any one of Ab101-Ab141 or an antigen-binding fragment thereof produced by a host cell may be harvested, or the heavy and light chains of the antibody or antigen-binding fragment may be harvested from separate cells and then combined to form a complete antibody or antigen-binding fragment thereof.

[0284] Aspects of the present disclosure relate to hybridomas that provide an indefinitely sustainable source of monoclonal antibodies. As an alternative to obtaining immunoglobulins directly from hybridoma cultures, immortalized hybridoma cells can be used as a source of rearranged heavy and light chain loci for subsequent expression and / or genetic manipulation. Rearranged antibody genes can be reverse transcribed from the appropriate mRNA to produce cDNA. In some embodiments, the heavy chain constant region can be replaced with one of a different isotype or removed entirely. Variable regions can be linked to encode a single-chain Fv region. Multiple Fv regions can be linked to confer binding capabilities to more than one target, or chimeric heavy and light chain combinations can be used. Any suitable method can be used to clone antibody variable regions and generate recombinant antibodies and antigen-binding portions thereof.

[0285] In some embodiments, suitable nucleic acids encoding the heavy and / or light chain variable regions are obtained and inserted into expression vectors that can be transfected into standard recombinant host cells. A variety of such host cells can be used. In some embodiments, mammalian host cells can be advantageous for efficient processing and production. Exemplary mammalian cell lines useful for this purpose include CHO cells, 293 cells, or NSO cells. Antibodies or antigen-binding fragments can be produced by culturing the modified recombinant host under culture conditions appropriate for host cell growth and expression of the coding sequences. Antibodies or antigen-binding fragments can be recovered by isolating them from the culture. Expression systems can be designed to include a signal peptide so that the resulting antibody is secreted into the culture medium, although intracellular production is also possible.

[0286] The present disclosure also includes polynucleotides encoding at least the variable region of an immunoglobulin chain of any one of the antibodies described herein, e.g., Ab101-Ab141, or an antigen-binding fragment thereof. In some embodiments, the variable region encoded by the polynucleotide comprises at least one complementarity-determining region (CDR) of the VH and / or VL variable region of an antibody produced by any one of the above hybridomas.

[0287] The polynucleotide encoding the antibody or antigen-binding fragment can be, for example, DNA, cDNA, RNA, or synthetically produced DNA or RNA, or a recombinantly produced chimeric nucleic acid molecule comprising any of these polynucleotides, either alone or in combination. In some embodiments, the polynucleotide is part of a vector. Such vectors may contain additional genes, such as marker genes, that allow for selection of the vector in a suitable host cell and under suitable conditions.

[0288] In some embodiments, the polynucleotide is operably linked to an expression control sequence that allows expression in prokaryotic or eukaryotic cells. Expression of the polynucleotide includes transcription of the polynucleotide into translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They may include regulatory sequences that promote transcription initiation and, optionally, a polyA signal that promotes transcription termination and transcript stabilization. Additional regulatory elements may include transcriptional and translational enhancers and / or naturally associated or heterologous promoter regions. Possible regulatory elements that allow expression in prokaryotic host cells include, for example, the PL, Lac, Trp, or Tac promoters in Escherichia coli (E. coli). Examples of regulatory elements that allow expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast, or the CMV promoter, SV40 promoter, RSV promoter (Rous sarcoma virus), CMV enhancer (cytomegalovirus), SV40 enhancer, or globin introns in mammalian and other animal cells.

[0289] In addition to elements involved in transcription initiation, such regulatory elements may also include transcription termination signals downstream of the polynucleotide, such as an SV40 poly-A site or a tk poly-A site. Furthermore, depending on the expression system used, leader sequences capable of directing the polypeptide into a cellular compartment or secreting the polypeptide into the culture medium may be added to the coding sequence of the polynucleotide, as previously described. The leader sequence is assembled in an appropriate stage with translation, initiation and termination sequences, and preferably with a leader sequence capable of directing secretion of the translated protein or a portion thereof, for example, into the extracellular medium. Optionally, a heterologous polynucleotide sequence may be used that encodes a fusion protein containing a C- or N-terminal identification peptide that confers a desired property, such as stabilization or simplified purification of the expressed recombinant product.

[0290] In some embodiments, the polynucleotide encoding at least the light and / or heavy chain variable domain may encode the variable domains of both immunoglobulin chains or only one of them. Similarly, the polynucleotides may be under the control of the same promoter or may be separately controlled for expression. Furthermore, some aspects relate to vectors conventionally used in genetic engineering, in particular plasmids, cosmids, viruses and bacteriophages, comprising polynucleotides encoding the variable domains of the immunoglobulin chains of an antibody or antigen-binding fragment, optionally in combination with polynucleotides encoding the variable domains of other immunoglobulin chains of the antibody.

[0291] In some embodiments, expression control sequences are provided as eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, although control sequences for prokaryotic hosts can also be used. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpesvirus, or bovine papillomavirus can be used to deliver polynucleotides or vectors to target cell populations (e.g., to engineer cells to express antibodies or antigen-binding fragments). A variety of suitable methods can be used to construct recombinant viral vectors. In some embodiments, polynucleotides and vectors can be reconstituted into liposomes for delivery to target cells. Vectors containing polynucleotides (e.g., heavy and / or light chain variable domains of immunoglobulin chain-encoding sequences and expression control sequences) can be transferred into host cells by suitable methods, which vary depending on the type of cellular host.

[0292] Myostatin In some embodiments, the human pro / latent myostatin is wild-type human pro / latent myostatin (promyostatin) with a human kappa signal peptide and an N-terminal His6 tag (SEQ ID NO: 941) that are cleaved upon expression. Promyostatin undergoes incomplete furin cleavage during expression. In some embodiments, the human pro / latent myostatin has the amino acid sequence of SEQ ID NO: 135, as shown below: [ka]

[0293] In some embodiments, the sequence encoding the human immunoglobulin kappa signal peptide is MDMRVPAQLLGLLLWFSGVLG (SEQ ID NO: 136).

[0294] In some embodiments, the His6 tag of human promyostatin (SEQ ID NO: 941) has the underlined sequence (HHHHHN (SEQ ID NO: 942)) in SEQ ID NO: 135.

[0295] In some embodiments, the sequence encoding the human myostatin prodomain is SEQ ID NO: 137, shown below: [ka]

[0296] In some embodiments, the RSRR furin cleavage site of human promyostatin (sequence number 943) is the bolded sequence in sequence number 135.

[0297] In some embodiments, the sequence encoding the mature human myostatin growth factor is SEQ ID NO: 138, shown below: DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS (SEQ ID NO: 138).

[0298] In some embodiments, the DNA sequence encoding human promyostatin is SEQ ID NO: 139, shown below: [ka]

[0299] In some embodiments, the signal peptide is cleaved from the rest of the molecule when the protein is expressed.

[0300] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein bind to an epitope on promyostatin. In some embodiments, the antibodies or antigen-binding fragments disclosed herein compete with Ab2 for antigen binding. The variable domain and full-length antibody sequence of Ab2 are disclosed in PCT / US2015 / 059468 and PCT / US2016 / 052014.

[0301] The role of myostatin in muscle homeostasis and metabolic regulation Skeletal muscle is a dynamic organ that accounts for approximately 40% of body weight and turns over at a rate of 1–2% per day. Myostatin is thought to play a critical role in maintaining muscle homeostasis in both healthy and disease states. Myostatin can induce muscle atrophy through inhibition of myoblast proliferation, increased ubiquitin-proteasome activity, and downregulation of the IGF-Akt pathway. These well-recognized effects are seen in multiple conditions that cause atrophy, including injury, diseases such as cachexia, disuse, and spaceflight, demonstrating the importance of the myostatin signaling mechanism. Based on this central role, considerable research has been conducted to inhibit the action of myostatin in vivo. Indeed, inhibition of the myostatin pathway has been shown to promote muscle growth and maintain muscle mass.

[0302] Furthermore, muscle is known to be the body's major protein reservoir and thus contributes to amino acid homeostasis and metabolism. Along with glucose (primarily produced and stored as glycogen in the liver and muscles) and lipids (stored in adipose tissue), muscle protein can act as an energy source (i.e., broken down to generate energy). Deficiencies or imbalances in the utilization or mobilization of these energy sinks within the body can contribute, at least in part, to various types of metabolic dysregulation. Therefore, myostatin may play a direct role in regulating metabolism by adjusting the balance between the breakdown and synthesis / storage of glucose, fat, and / or muscle within the body (e.g., enhancing "metabolic adaptability"), such as adaptability or flexibility to fuel sources, and the ability to change metabolism to meet the needs of the organism or tissue based on exercise status or available fuel, thereby contributing to overall metabolic regulation within the body, e.g., energy expenditure. Indeed, since its discovery in 1997, myostatin has primarily been considered a key regulator of muscle metabolism, although more recent findings have demonstrated its effects as a metabolic regulator. See, e.g., PCT / US2018 / 012686. Accordingly, it is contemplated herein that myostatin inhibition, e.g., by the antibodies or antigen-binding fragments disclosed herein, may be useful in certain embodiments for treating various conditions associated with muscle and / or metabolic dysregulation, as described in more detail below.

[0303] therapeutic use In various embodiments, pharmaceutical compositions comprising the antibodies or antigen-binding fragments disclosed herein, e.g., compositions comprising any one of Ab101-Ab141 or antigen-binding fragments thereof, are suitable for administration to human patients for the treatment or prevention of diseases and conditions associated with myostatin signaling, e.g., conditions in which reduced myostatin signaling is desirable. In some embodiments, the antibodies and antigen-binding fragments disclosed herein can be used to improve a patient's metabolic health. Suitable diseases and conditions include, for example, muscle disorders such as muscle atrophy and myopathy, metabolic disorders such as obesity and diabetes, bone and connective tissue disorders or conditions, e.g., bone loss, anterior cruciate ligament (ACL) repair, and osteogenesis imperfecta (OI), cardiovascular diseases, e.g., heart disease (e.g., heart failure in patients with type 2 diabetes or obesity), and chronic inflammation and inflammatory diseases, e.g., chronic kidney disease (CKD), idiopathic pulmonary fibrosis (IPF), and fatty liver disease (e.g., NAFLD, NASH). Accordingly, the present disclosure encompasses therapeutic uses of such antibodies and antigen-binding fragments thereof (e.g., engineered constructs incorporating such fragments) to treat one or more of these diseases and conditions. In some embodiments, the myostatin-selective inhibitor is an antibody or antigen-binding fragment thereof that comprises all six CDRs of any one of Ab101-Ab141, e.g., the set of SEQ ID NOs identified for the particular antibodies in Tables 2d-f. In some embodiments, the myostatin-selective inhibitor is an antibody or antigen-binding fragment thereof that comprises the heavy and light chain variable domains of any one of Ab101-Ab141, e.g., the pair of SEQ ID NOs identified for the particular antibodies in Table 3. In some embodiments, the myostatin-selective inhibitor is an antibody that comprises the heavy and light chains of any one of Ab101-Ab141, e.g., the pair of SEQ ID NOs identified for the particular antibodies in Table 4. In some embodiments, the antibody sequence is that of Ab109, Ab133, or Ab141, or an antigen-binding fragment thereof.In some embodiments, the antibody is Ab101, Ab102, Ab103, Ab104, Ab105, Ab106, Ab107, Ab108, Ab109, Ab110, Ab111, Ab112, Ab113, Ab114, Ab115, Ab116, Ab117, Ab118, Ab119, Ab120, Ab121 The antibody or antigen-binding fragment thereof is selected from Ab109, Ab133, and Ab141, or an antigen-binding fragment thereof. In a preferred embodiment, the antibody is selected from Ab109, Ab133, and Ab141. The antibody or antigen-binding fragment may also be administered in combination with one or more additional agents disclosed herein, such as a GLP-1 pathway activator (e.g., a GLP-1 receptor agonist) or a biguanide (e.g., metformin), to treat any of the diseases or disorders.

[0304] Diseases and conditions encompassed by the present disclosure include, but are not limited to, metabolic disorders such as metabolic syndrome, obesity, type 2 diabetes mellitus (T2DM) (e.g., adult-onset diabetes), prediabetes, and obesity-related T2DM. T2DM is a chronic and progressive metabolic disease characterized by elevated blood glucose levels, which, if left untreated, can damage various organs and lead to complications such as cardiovascular disease, neuropathy, nephropathy (e.g., diabetic nephropathy), and retinopathy (e.g., diabetic retinopathy). Obesity is a risk factor for many serious medical conditions, including cardiovascular disease, prediabetes, T2DM, NAFLD, NASH, some types of cancer, and Alzheimer's disease. Therefore, treating obesity can treat or ameliorate these conditions. In some embodiments of the present disclosure, the patient is not treated with a TGFβ inhibitor, such as a TGFβ1 inhibitor. In some embodiments, the patient has a metabolic liver disorder, optionally the metabolic liver disorder comprises NAFLD, and further optionally the metabolic liver disorder has not progressed to NASH, optionally the metabolic liver disease has not progressed to fibrosis, or optionally the metabolic liver disease has not progressed to cirrhosis.

[0305] In some embodiments, a preferred myostatin inhibitor for therapeutic use is a myostatin-selective inhibitor, such as any one of the myostatin-selective antibodies or antigen-binding fragments disclosed herein, e.g., an antibody or antigen-binding fragment that selectively binds to pro- or latent myostatin, e.g., any one of Ab101-Ab141. Without wishing to be bound by theory, it may be beneficial to use a myostatin-selective inhibitor to provide a desired therapeutic effect while avoiding undesirable adverse effects, such as those associated with non-selective myostatin inhibition. In some embodiments, the desired therapeutic effect includes at least one, e.g., all of the following: preferential reduction of fat mass relative to lean mass; maintenance of fat mass loss; prevention of muscle mass loss; increase in muscle mass; increased endurance; reduction in fatigue; prevention of bone loss; improvement in blood glucose levels; and / or improvement in liver health. In some embodiments, it may be beneficial to avoid inhibition of GDF11, which has been reported to promote metabolic health but is also associated with adverse effects. See, for example, Frohlich et al. (Cell Prolif. 2022 Oct;55(10):e13310); Lu et al. (J Transl Med. 2019;17:422); Walker et al. (Sci Rep 10,4561(2020); and Muramatsu et al. (Sci Rep. 2021 Jan 25;11(1):2160).

[0306] In some embodiments, combining a selective inhibitor of myostatin activation with a GLP-1 pathway activator, a GIP activator or antagonist, or a glucagon modulator may provide additive or synergistic effects on a subject's metabolic health, e.g., by preventing muscle loss during weight loss, greater amounts of fat, including visceral fat, may be lost. Combining a selective inhibitor of myostatin activation with a GLP-1 pathway activator (e.g., a GLP-1 receptor agonist) may also increase the durability of the therapeutic effect, e.g., maintaining muscle mass may promote the durability of metabolic benefits.

[0307] Exemplary conditions under which the compositions and methods of the present disclosure may be useful are further described below.

[0308] Metabolic Disorders and Diseases In various embodiments, disclosed herein are methods for treating or preventing metabolic diseases in a subject. Metabolic diseases (also referred to as metabolic disorders or metabolic conditions) are generally associated with abnormal glucose, lipid / fat and / or protein / nitrogen metabolism, or osmoregulation dysregulation, and have pathological consequences resulting from such conditions. Some metabolic disorders of the present disclosure share certain characteristics, such as being associated with a loss of lean or non-lean muscle mass, excess body fat mass, a lower metabolic rate, insulin resistance, an inability to regulate blood glucose, weight gain, and / or an increased body mass index. In some cases, such metabolic diseases or disorders may be induced or exacerbated by drug therapy received by the patient.

[0309] The present disclosure is based, at least in part, on the discovery that administration of a myostatin inhibitor described herein (e.g., an anti-myostatin antibody or antigen-binding fragment thereof disclosed herein) to a subject with a metabolic disease, e.g., via a subcutaneous route, can improve both the physiological and functional characteristics of the subject.

[0310] Further examples of metabolic disorders that can be treated or prevented by the methods of the present disclosure include, but are not limited to, prediabetes and diabetes (e.g., type 1 or type 2 diabetes, or obesity-related diabetes), obesity (e.g., adult-onset obesity, diet-induced obesity, childhood obesity, etc.), obesity syndromes (e.g., diet-related or diet-induced obesity), insulin resistance, insulin insufficiency, hyperinsulinemia, impaired glucose tolerance (IGT), abnormal glycogen metabolism, hyperlipidemia, hypoalbuminemia, hypertriglyceridemia, renal disease, e.g., chronic kidney disease, syndrome X, fatty liver disease, and metabolic bone disease. Spinal cord injury (SCI) (e.g., complete or incomplete / partial SCI), hypometabolic states, double diabetes, and Cushing's disease (also referred to as Cushing's syndrome). In some embodiments, metabolic disorders include disorders involving impaired neural signaling or partial denervation. In some embodiments, metabolic disorders include conditions (e.g., side effects) induced by or associated with certain drug therapies.

[0311] Additional diseases or conditions associated with metabolic disorders and / or body composition will be apparent to one of skill in the art and are within the scope of the present disclosure.

[0312] As described in more detail herein, metabolic disorders can occur secondary to muscle pathologies or disorders, or can occur as a result of muscle pathologies or disorders. Because muscle homeostasis is correlated with amino acid / protein metabolism, it is further believed that myostatin inhibition can then regulate nitrogen metabolism and nitrogen mobilization in the body. In muscle catabolism, muscle tissue breaks down into its constituent amino acids, which can be considered the main reservoir (and therefore source) of nitrogen. Nitrogen is an element of ammonia, which is highly toxic to the body and is excreted in the form of urea in humans. When nitrogen metabolism becomes dysregulated, possible consequences include an imbalance in fluid retention, which can manifest as generalized or localized edema (e.g., congestion or fluid overload).

[0313] Diabetes is a common metabolic disorder that refers to a group of metabolic disorders characterized by high blood sugar (glucose) levels due to defective insulin secretion or action, or both. Insulin is a hormone released by the pancreas in response to elevated blood sugar (glucose) levels in the blood. There are two most common types of diabetes: type 1 diabetes and type 2 diabetes, both of which result from the body's inability to regulate insulin.

[0314] In type 2 diabetes mellitus (T2DM) (also known as non-insulin-dependent diabetes mellitus, or NDDM), the pancreas continues to produce insulin, sometimes even at higher-than-normal levels. However, the body develops resistance to its effects, resulting in relative insulin deficiency. As the pancreas continues to produce insulin and the body becomes insulin resistant, the insulin-producing cells in the pancreatic islets of Langerhans become exhausted, limiting or potentially eliminating the ability to continue insulin production. Type 2 diabetes can develop in children and adolescents, but it usually begins after age 30 and becomes increasingly common with age: approximately 15% of people over 70 have type 2 diabetes. Obesity is a risk factor for type 2 diabetes; 80 to 90 percent of people with this disorder are obese.

[0315] In some embodiments, diabetes includes prediabetes. "Prediabetes" refers to one or more early diabetic conditions including impaired glucose utilization, abnormal or impaired fasting blood glucose levels, impaired glucose tolerance, impaired insulin sensitivity, and insulin resistance. Prediabetes is a major risk factor for the development of type 2 diabetes, cardiovascular disease, and mortality. Much focus has been placed on developing therapeutic interventions to effectively treat prediabetes and thereby prevent the onset or progression of type 2 diabetes.

[0316] In some embodiments, diabetes includes double diabetes, which is a combination of type 1 diabetes with features of insulin resistance and type 2 diabetes.

[0317] Diabetes and prediabetes can be diagnosed by performing a glucose tolerance test, which can be determined by drawing venous blood from a subject in a fasting or non-fasting state. They can also be diagnosed by measuring blood levels of hemoglobin A1C (A1C), a glycosylated form of hemoglobin that reflects the average amount of blood glucose over the past two to three months. Normal, prediabetic, and diabetic A1C levels are known in the art and may vary with age. In some embodiments, the expected value for a normal fasting blood glucose concentration can be about 70 mg / dL (3.9 mmol / L) to about 100 mg / dL (5.6 mmol / L). In some embodiments, prediabetes is associated with hemoglobin A1C levels of about 100 mg / dL to about 125 mg / dL, or about 5.6 mmol / L to about 6.9 mmol / L. In some embodiments, diabetes is associated with fasting blood glucose levels greater than about 125 mg / dL or hemoglobin A1C levels greater than about 6.9 mmol / L.

[0318] Clinically, diabetes is often divided into several basic categories. Primary examples of these categories include autoimmune, non-insulin-dependent diabetes mellitus (NDDM type 1), insulin-dependent diabetes mellitus (IDDM type 2), non-autoimmune, non-insulin-dependent diabetes mellitus (NIDDM type 2), and maturity-onset diabetes of the young (MODY). A further classification, often referred to as secondary, refers to diabetes caused by some identifiable pathology that causes or enables the development of a diabetic syndrome. Examples of secondary categories include diabetes caused by pancreatic disease, hormonal abnormalities, drug- or chemical-induced diabetes, diabetes caused by insulin receptor abnormalities, diabetes associated with genetic syndromes, and diabetes of other causes. (See, e.g., Harrison's (1996) 14th ed., New York, McGraw-Hill.)

[0319] Obesity is another common metabolic disorder that can be treated or prevented by the methods of the present disclosure. "Obesity" refers to a chronic condition defined by an excessive amount of body fat. Normal amounts of body fat (expressed as a percentage of body weight) are 25-30% for women and 18-23% for men. Women with 30% or more body fat and men with 25% or more body fat are considered obese. Obesity can be defined using any clinically relevant definition. For example, in adults, body mass index (BMI, kg / m²) is frequently used as a measure of overweight and obesity, with overweight defined as a BMI of 25-29.9 kg / m², obesity defined as a BMI of 30 kg / m² or greater, and morbid obesity defined as a BMI greater than 40 kg / m². Obesity can also be defined in adults by central adiposity as measured by waist circumference, with a high waist circumference defined as 102 cm or greater in men and 88 cm or greater in women.

[0320] Subjects with obesity may exhibit other symptoms, such as increased fasting plasma glucose, impaired glucose tolerance, heart failure, hypertension, insulin resistance, increased fasting plasma triglycerides, decreased fasting high-density lipoprotein (HDL) levels, prediabetes, elevated blood pressure, stroke, heart failure, obstructive sleep apnea, reproductive hormone disorders, obstructive sleep apnea, osteoarthritis, gallstones, gastroesophageal reflux, or renal disease. Obesity can also lead to various orthopedic problems, skin disorders, and swelling of the feet and ankles. Serious complications of obesity include a much higher risk of coronary artery disease, as well as its major risk factors, type II diabetes, hyperlipidemia, and hypertension. Many of the obesity-related pathologies are associated with type II diabetes, as poorly controlled diabetes and obesity together lead to a constellation of symptoms known as syndrome X or metabolic syndrome. In some embodiments, the obesity is sarcopenic obesity. In some embodiments, the obese subject is undergoing a calorie restriction regimen.

[0321] In one aspect, the methods of the present disclosure are suitable for treating all forms of obesity, including diabetes-associated obesity, metabolic syndrome-associated obesity, monogenic disease-associated obesity, antipsychotic drug use-associated obesity, glucocorticoid-associated obesity, and hypothalamic obesity.

[0322] In another aspect, the methods of the present disclosure are suitable for treating or preventing metabolic disorders such as obesity syndrome. The term "obesity syndrome" refers to any disorder or condition that causes a subject to become significantly obese or overweight. Similar to other metabolic disorders, individuals with obesity syndrome typically have a loss of lean or non-lean muscle mass, excess body fat, a lower metabolic rate, insulin resistance, an inability to regulate blood sugar, weight gain, and an increased body mass index. In some embodiments, the obesity syndrome is selected from the group consisting of Prader-Willi syndrome, obesity syndrome associated with a genetic disorder, and obesity syndrome associated with a hypothalamic disorder.

[0323] The methods of the present disclosure are also suitable for treating or preventing metabolic diseases such as metabolic syndrome. As used herein, "metabolic syndrome" refers to the concept of a collection of metabolic risk factors that come together in a single individual and result in a high risk of developing diabetes and / or cardiovascular disease. Key features of metabolic syndrome include insulin resistance, hypertension (high blood pressure), cholesterol abnormalities, dyslipidemia, triglyceride abnormalities, an increased risk of clotting, especially in the abdominal region, and excess weight or obesity. In some embodiments, metabolic syndrome can be diagnosed by the presence of three or more of the following components: (1) high waist circumference (men, 40 inches (102 cm) or greater; women, 35 inches (88 cm) or greater); (2) high triglycerides (150 mg / dL or greater); (3) low high-density lipoprotein cholesterol (HDL) (men, less than 40 mg / dL; women, less than 50 mg / dL); (4) high blood pressure (130 / 85 mmHg or greater); and (5) high fasting blood glucose (100 mg / dL or greater).

[0324] Body composition can be measured by a variety of methods, including dual-energy X-ray absorptiometry (DEXA). A whole-body scan using DEXA generally provides an accurate and precise measurement of body composition, including bone mineral content, bone density, lean tissue mass, adipose tissue mass, and fat contribution.

[0325] Obesity is a risk factor for the development of cardiovascular disease. Obese individuals experience cardiovascular disease events at an earlier age, spend a larger portion of their lives with cardiovascular disease, and have a shorter life expectancy than normal-weight individuals. Obesity directly contributes to cardiovascular risk factors, including dyslipidemia, type 2 diabetes, hypertension, and sleep disorders. Obesity accelerates atherosclerotic changes through multiple mechanisms, including insulin resistance and inflammation. Obesity also contributes to the development of cardiovascular disease and mortality, independent of other cardiovascular risk factors. Visceral adiposity promotes systemic and vascular inflammation, which is essential for the atherosclerotic process. Obesity-induced inflammation increases the likelihood of LDL oxidation, thereby promoting atherogenesis. Insulin resistance is associated with dyslipidemia and metabolic syndrome, both of which are associated with atherosclerosis. For example, impaired endothelial function in obesity, due to reduced nitric oxide bioavailability in the context of inflammation and oxidative stress, also contributes to the progression of atherosclerosis. Obesity is also associated with abnormalities in the coronary microvasculature and epicardial coronary vessels. Another aspect of the present disclosure includes a method for treating a subject with an age-related metabolic disease or condition. Exemplary age-related diseases and conditions include, but are not limited to, sarcopenia (age-related muscle loss), frailty, and androgen deficiency.

[0326] Therefore, the methods of the present disclosure are suitable for treating or preventing metabolic diseases, such as cardiovascular diseases, e.g., cardiovascular diseases associated with metabolic syndrome. The term "cardiovascular disease" refers to any disease of the heart or blood vessels. Examples of cardiovascular diseases or heart diseases include, but are not limited to, angina pectoris, arrhythmias, coronary artery disease (CAD), coronary heart disease, cardiomyopathies (including dilated cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and diabetic cardiomyopathy), heart attack (myocardial infarction), heart failure (e.g., acute heart failure (AHF), chronic heart failure (CHF), or heart failure with preserved ejection fraction (HfpEF)), hypertrophic cardiomyopathy, mitral valve regurgitation, mitral valve prolapse, pulmonary valve stenosis, etc. Examples of vascular diseases include, but are not limited to, peripheral vascular disease, arterial disease, carotid artery disease, deep vein thrombosis, venous disease, and atherosclerosis. In some embodiments, the subject with heart failure is resistant to diuretic therapy. In another embodiment, subjects with heart failure respond poorly to diuretic therapy. It is believed herein that myostatin inhibitors may provide beneficial effects in reducing the risk of major cardiovascular events, particularly in patients with metabolic disorders such as obesity and diabetes. Such effects may be synergistic when used in combination with GLP-1 pathway activators, such as GLP-1 receptor agonists. In a preferred embodiment, the myostatin inhibitor is a selective myostatin inhibitor. In a particularly preferred embodiment, the myostatin inhibitor is selected from any one of the novel antibodies disclosed herein, e.g., Ab101-Ab141. In a most preferred embodiment, the myostatin inhibitor is selected from Ab109, Ab133, and Ab141.

[0327] For example, pulmonary edema and renal congestion are frequently observed in patients with heart failure associated with reduced cardiac output. Pulmonary congestion is, in fact, the most common cause of hospitalization in this clinical setting and correlates with poor prognosis. Similarly, in pathological conditions involving impaired osmoregulation, affected individuals may be particularly sensitive to salt intake, which can cause or exacerbate fluid overload. Therefore, for subjects with fluid retention or fluid overload, such as those with impaired osmoregulation and heart failure, e.g., chronic heart failure, current guidelines suggest that diuretic therapy should be attempted for decongestion (see, e.g., Regolisti et al., Nephrology@Point of Care 2016;2(1):e73-e87). However, in many cases, diuretic therapy is ineffective or subjects are refractory to diuretic therapy. Myostatin inhibition according to the present disclosure may provide clinical benefit to such patients. In particular, the methods of the present disclosure are suitable for increasing the responsiveness of subjects who are resistant to or have a low response to diuretic therapy. For example, administration of a myostatin inhibitor reduces the required diuretic dose and / or provides improved control of symptoms, such as CHF symptoms, improves cardiac function, and / or chronically prevents pathological cardiac remodeling or other deterioration of cardiac function. Myostatin inhibition using the inhibitors described herein also reduces the risk of CHF exacerbations, such as the development of acute pulmonary edema.

[0328] For subjects at high risk of developing acute pulmonary edema, such as those receiving intravenous (IV) fluid infusion, blood transfusion, or fluid transfer, the myostatin inhibitors disclosed herein can be administered prophylactically. For example, a subject with congestive heart failure who requires a blood transfusion can be administered a myostatin inhibitor prophylactically during the transfusion to prevent the development of acute pulmonary edema during the transfusion. In subjects with CHF and / or other fluid-overload conditions who develop hyponatremia due to the fluid overload itself or due to the diuretics used to treat the fluid overload, the myostatin inhibitors disclosed herein can be administered to treat hyponatremia and / or allow the use of higher doses of diuretics when diuretic administration is limited by hyponatremia as a side effect. Generally speaking, however, the myostatin inhibitors disclosed herein can be used to treat hyponatremia regardless of the underlying disease.

[0329] For other fluid overload conditions, such as renal failure or liver disease requiring high doses of diuretics, the myostatin inhibitors disclosed herein reduce the required diuretic dose; provide improved control of symptoms such as peripheral edema or internal congestion (including pleural effusion, ascites, hepatic congestion, or intraocular fluid overload, which can lead to retinal detachment), and / or reduce the risk of pulmonary edema.

[0330] Metabolic disorders and diseases for treatment by the methods provided herein also include metabolic conditions affecting the liver. Nonalcoholic fatty liver disease (NAFLD) is a group of liver diseases associated with metabolic and cardiovascular disorders and is strongly associated with metabolic syndrome (Godoy-Matos (2020) Diabetes Metab Synd 12:50). Metabolic conditions affecting the liver include nonalcoholic steatohepatitis (NASH), NAFLD, hereditary hemochromatosis, alpha-1 antitrypsin deficiency, and Wilson's disease. Fibrosis, a thickening of connective tissue, occurs in the later stages of both NASH and NAFLD as excessive tissue deposition leads to fibrous scarring. Fibroblast growth factor 21 (FGF21) and FGF19 have been observed to reduce hepatic steatosis. Therefore, in some embodiments, a subject is administered an FGF21 or FGF19 receptor agonist and a myostatin inhibitor of the present invention. Zhao et al., Signal Transduction and Targeted Therapy (2022) 7:206.In some embodiments, the subject is also receiving or has received one or more of the following therapeutic agents to treat liver disease: hydronidone, BIO89-100, effluxifermin, pegbelfermin, aldafermin, MK-3655, PRI-724, selonsertib, CC-90001, Epeleuton, elafibranor, saroglitazar, lanifibranor, pemafibrate, ZSP0678, obeticholic acid, cilofexor, nidufexor exor), TERN-101, bonafexor, EDP-305, tropifexor, JKB-121, JKB-122, semaglutide, tirzepatide, cotadutide, HM-15211, resmetirom, VK2809, cenicriviroc, belapectin, GB1211, azemiglitazone potassium, deuterium-stabilized l-pioglitazone, aramchol, PF-05221304, firsocostat, ZSP1601, eperayton, PXL-770, ALS-L1023, namodenoson, TVB-2640, LPCN 1144, HepaStem, BMS-986263, foralamuumab, elobixibat, aparalenone, PF-06835919, ARO-HSD, and CB4211.

[0331] The compositions and methods of the present disclosure are also suitable for treating or preventing metabolic disorders associated with hypometabolic conditions. The term "hypometabolic condition" refers to a state of reduced metabolism or metabolic activity when the body is not producing enough energy. Patients with hypometabolic conditions generally have a lower metabolic rate, reduced lean or non-lean muscle mass, excessive fat mass, insulin resistance, an inability to regulate blood glucose, weight gain, and an increased body mass index. In some embodiments, the hypometabolic condition is selected from the group consisting of conditions associated with prolonged immobilization, conditions associated with bed rest, conditions associated with casting, conditions associated with stroke, conditions associated with amputation, and post-surgical conditions. In some embodiments, the hypometabolic condition is a post-surgical condition, for example, paraspinal muscular atrophy after lumbar spinal surgery. In one embodiment, the paraspinal muscular atrophy is nerve injury-dependent muscular atrophy. In one embodiment, the surgery is spinal surgery. In one embodiment, the spinal surgery is a lumbar surgery or procedure, such as a lumbar fusion procedure, a lumbar non-fusion procedure, a posterior lumbar fusion procedure, an anterior lumbar fusion procedure, a minimally invasive (MIS) posterior lumbar decompression procedure, a minimally invasive (MIS) posterior lumbar fusion procedure, a non-MIS equivalent procedure, etc.

[0332] In some embodiments, myostatin inhibitors of the present invention can be used to attenuate spinal cord injury (SCI)-induced losses in sublesional muscle mass and overall body weight, while simultaneously reducing the mass of undesirable adipose tissue, such as white and visceral adipose tissue. Subjects treated with myostatin inhibitor therapy can also demonstrate significant improvements in their locomotor function, muscle strength, and motor coordination and balance skills.

[0333] In another aspect, the methods of the present disclosure are suitable for treating or preventing metabolic disorders such as Cushing's disease, also known as Cushing's syndrome or hyperadrenocorticism. The term "Cushing's disease" refers to a collection of signs and symptoms resulting from prolonged exposure to cortisol. In some embodiments, the Cushing's disease is selected from the group consisting of corticosteroid-induced Cushing's disease and tumor-induced Cushing's disease. In some embodiments, the antibodies or antigen-binding fragments disclosed herein may be useful as an alternative or additive treatment option to the current standard of care for patients suffering from Cushing's disease.

[0334] Thus, the present disclosure provides a method for treating or preventing a metabolic disease in a human subject. The method includes selecting a human subject suffering from a metabolic disease and administering an effective amount of a myostatin inhibitor (e.g., Ab102, Ab109, Ab130, Ab132, or Ab133) to the human subject, thereby treating or preventing the metabolic disease in the human subject. Preferably, the myostatin inhibitor is a myostatin-selective inhibitor. More preferably, the myostatin-selective inhibitor is an antibody, or antigen-binding fragment thereof, that specifically binds to pro / latent myostatin but does not bind to GDF11, e.g., Ab102, Ab109, Ab130, Ab132, or Ab133. An antibody that specifically recognizes pro / latent myostatin but not GDF11 is beneficial and avoids undesirable toxicity in a subject caused by off-target binding of the antibody to GDF11. In one embodiment, the subject is a pediatric subject. In one embodiment, the subject is between 2 and 19 years of age, inclusive. In one embodiment, the subject is 12 years of age or older (e.g., between 12 and 17 years of age), inclusive.

[0335] In one embodiment, the monoclonal antibody specifically binds to myostatin GDF8 and inhibits its activation process. In some embodiments, such antibodies bind to promyostatin and / or latent myostatin and inhibit the activation and subsequent release of mature myostatin, but do not bind to mature myostatin that is not associated with the latent (inactive) complex. In some embodiments, the antibody or fragment thereof binds to a tethered form (e.g., intramuscular) of inactive myostatin (e.g., promyostatin), which has the ability to act locally on tissue-associated myostatin within the disease niche. In some embodiments, the antibody or fragment thereof binds to a soluble form (e.g., in the circulation) of inactive myostatin (e.g., latent myostatin), which has the ability to act on circulating latent myostatin, which may have endocrine or systemic effects. In any of these embodiments, preferred inhibitors of myostatin for practicing the methods of the present disclosure are selective for myostatin and do not antagonize other members of the TGFβ superfamily of growth factors / cytokines, such as GDF11. Such selectivity is particularly advantageous in pediatric patient populations and / or patient populations requiring long-term care (e.g., chronic treatment), where inhibition of other pathways, such as GDF11, can result in harmful or undesirable side effects or adverse events. In any of such embodiments, preferred inhibitors of myostatin for practicing the methods of the present disclosure include Ab102, Ab109, Ab130, Ab132, or Ab133.

[0336] weight management Currently available obesity treatments, such as GLP-1 receptor agonists, focus almost exclusively on weight loss. In comparison, the present disclosure takes into account the quality of weight management beyond mere weight loss to achieve improved metabolic health. To this end, incorporating a myostatin inhibitor into a weight management regimen may achieve one or more of the following: preferentially reducing fat mass relative to lean mass; maintaining fat mass loss; preventing muscle mass loss; increasing muscle mass; increasing endurance; reducing fatigue; preventing bone loss; improving blood glucose levels; and / or improving liver health. Thus, myostatin inhibitors, such as the novel antibodies and antigen-binding fragments disclosed herein, may contribute to safe and sustainable weight management, particularly when used in conjunction with another therapy aimed at addressing metabolic dysregulation.

[0337] The present disclosure further provides methods for promoting consistent weight loss (e.g., reduction in body fat mass (e.g., weight of fat in the body) without a concomitant loss of lean muscle mass) in both healthy subjects, e.g., bodybuilders, or in subjects with a metabolic disease such as obesity, e.g., diet-induced obesity, metabolic syndrome, and / or type 2 diabetes mellitus (T2DM). Where weight loss occurs in both fat stores and muscle during dieting compared to diet alone (e.g., a calorie-restricted diet, a low-carbohydrate diet, a ketogenic diet, a vegan diet, etc.), a myostatin inhibitor disclosed herein (e.g., an anti-myostatin antibody or antigen-binding fragment thereof disclosed herein, e.g., Ab102, Ab109, Ab130, Ab132, Ab133, or Ab141) results in weight loss in fat stores while sparing muscle. In some embodiments, administration of a myostatin inhibitor results in a reduction in body fat mass of about 5%, about 10%, about 15%, or about 20% or more in a subject during the course of treatment.

[0338] Body fat mass increases with age in both men and women throughout middle age. In particular, abdominal fat is associated with a higher risk of cardiovascular disease, metabolic syndrome, hypertension, diabetes, or dyslipidemia in subjects compared to the risk in individuals without abdominal obesity. Body fat mass or abdominal body fat mass can be directly measured by dual-energy X-ray absorptiometry, ultrasound, computed tomography, or magnetic resonance imaging (e.g., qNMR). Clinically, abdominal obesity is defined as a waist circumference of 102 cm or more in men and 88 cm or more in women.

[0339] In some embodiments, administration of a myostatin inhibitor disclosed herein (e.g., an anti-myostatin antibody or antigen-binding fragment thereof disclosed herein, e.g., Ab102, Ab109, Ab130, Ab132, Ab133, or Ab141) may result in more consistent weight loss due to the maintenance of a higher metabolic rate; improved cardiometabolic benefits (e.g., lipid profile, glucose metabolism, cardiovascular risk, etc.); and greater fat reduction (e.g., total or visceral fat and / or other harmful fat levels) compared to administration of diet or another standard of care alone. Additionally, administration of a myostatin inhibitor treatment may prevent or reduce muscle atrophy and / or bone loss that may occur concomitantly with diets, such as calorie-restricted diets, low-carbohydrate diets, ketogenic diets, etc. Overall, administration of a myostatin inhibitor disclosed herein (e.g., an anti-myostatin antibody or antigen-binding fragment thereof disclosed herein, e.g., Ab102, Ab109, Ab130, Ab132, Ab133, or Ab141) can increase the muscle-to-fat ratio in a subject.

[0340] Administration of a myostatin inhibitor disclosed herein (e.g., an anti-myostatin antibody or antigen-binding fragment thereof disclosed herein, e.g., Ab102, Ab109, Ab130, Ab132, Ab133, or Ab141) to a subject with a metabolic disease, e.g., obesity, metabolic syndrome, and / or diabetes, e.g., T2DM, can prevent or mitigate a decline in metabolic rate and prevent or reduce lean muscle loss in the subject. Myostatin inhibitor treatment can be combined with another standard of care, e.g., a diet, e.g., a calorie-restricted diet. In some embodiments, a moderately calorie-restricted diet is recommended because it results in better patient compliance and better long-term outcomes because the subject does not have to adhere to a strict, stressful diet, e.g., a severely calorie-restricted diet.

[0341] Such treatment is particularly useful for subjects with physical activity limitations, such as those with orthopedic injuries, spinal cord injuries, musculoskeletal disorders, pulmonary disorders, cardiovascular disorders, neurological disorders, severe obesity, etc. In such subjects, administration of a myostatin inhibitor treatment prevents muscle atrophy and / or bone loss that are more pronounced in these subjects due to their limitations on physical activity. In some embodiments, such treatment allows subjects with physical activity limitations to adopt a healthier diet, e.g., a calorie-restricted diet, because they are no longer limited by concerns about muscle loss or bone loss due to administration of a myostatin inhibitor.

[0342] In some embodiments, the subject is on a diet but not an exercise regimen. In some embodiments, the subject is on an exercise regimen but not a diet regimen. In some embodiments, the subject is on a diet and an exercise regimen. Examples of dietary regimens include, but are not limited to, calorie restriction (e.g., reduced calorie intake or reduced calorie absorption), a nutritionally modified diet (e.g., high protein, low fat, low carbohydrate, keto, paleo, etc.), or altered timing of food intake (e.g., intermittent fasting, increased eating frequency, etc.), or a combination of altered timing, portion size, and nutritional composition (e.g., smaller, more frequent meals containing high protein, low fat, and / or other nutritional restrictions).

[0343] The availability of GLP-1 receptor agonists has had a significant impact on the treatment of obesity in recent years, but rapid weight loss is associated with decreased bone mineral density. The present applicant has previously shown that selective inhibition of myostatin can enhance bone (PCT / US2018 / 012686, the contents of which are incorporated herein by reference in their entirety). This, coupled with the observation that GDF11 can exert beneficial effects on bone, suggests that a myostatin-selective approach may offer advantages over non-selective approaches in preventing bone loss during obesity treatment. For example, Suh et al. have reported that GDF11 promotes bone formation, and follistatin increases muscle mass but weakens bone (Proc Natl Acad Sci US A. 2020 Mar 3;117(9):4910-4920;doi:10.1073 / pnas.1916034117). Therefore, it is believed that inhibition of GDF11 may be detrimental to bone health and that the selectivity of myostatin inhibition (e.g., over activin A or GDF11 inhibition) may be beneficial in preventing bone loss.

[0344] Thus, the present disclosure encompasses methods of using myostatin-selective inhibitors to prevent bone loss, for example, during weight loss and / or treatment of metabolic disorders. In some embodiments, the myostatin-selective inhibitor does not cause a decrease in bone mineral density compared to baseline as measured by dual-energy X-ray absorptiometry. In some embodiments, the myostatin-selective inhibitor is an antibody or antigen-binding fragment thereof that inhibits myostatin but not GDF11 or activin A. In some embodiments, the myostatin-selective inhibitor includes, but is not limited to, neutralizing antibodies that bind to mature myostatin, thereby preventing or interfering with receptor binding, and activating antibodies that bind to pro / latent myostatin and inhibit myostatin activation. In some embodiments, the myostatin-selective inhibitor is any one of the antibodies or antigen-binding fragments disclosed herein. In a preferred embodiment, the myostatin inhibitor is an inhibitor that selectively targets pro / latent myostatin, e.g., Ab109, Ab133, or Ab141. In a preferred embodiment, the myostatin inhibitor is Ab109.

[0345] Diseases associated with impaired neural signaling The antibodies and antigen-binding fragments disclosed herein may be useful for intervention in conditions involving defects in communication between muscles and their nerve neurons. The spinal cord houses the major nerves that control motor function. Thus, the present disclosure provides methods for treating or preventing diseases in a subject, e.g., a human subject, associated with impaired neural signaling between neurons and target tissues that express myostatin. The disorder may be, for example, injury-based (e.g., spinal cord injury) or genetic (e.g., resulting from a gene mutation, e.g., SMA).

[0346] In some embodiments, the methods include administering to a subject suffering from a disease associated with impaired neural signaling between neurons and a target tissue an effective amount of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof, that specifically binds to myostatin and inhibits myostatin signaling, thereby treating or preventing the disease associated with impaired neural signaling in the subject. Preferably, the antibody or antigen-binding fragment thereof specifically binds to pro / latent myostatin but does not bind to mature GDF11. In some embodiments, such an antibody or fragment does not bind to mature myostatin GDF8. In some embodiments, antibodies suitable for practicing these embodiments include Ab102, Ab109, Ab130, Ab132, Ab133, and Ab141.

[0347] As used herein, the term "disease with impaired neural signaling" refers to any disease or disorder caused by or associated with disruption or impaired transmission of signaling between neurons and their target tissue, e.g., muscle tissue, brain tissue, liver tissue, vascular tissue, or adipose tissue. In some embodiments, impaired neural signaling occurs due to damage to neuronal structure, which prevents the neuron from transmitting signals to its target. In other embodiments, the neuron's structure remains intact, but there is a functional disruption or defect, e.g., a blockage at the neuromuscular junction, such that the neuron's ability to transmit signals is affected.

[0348] In some embodiments, a "disease with impaired neural signaling" refers to a disease or condition associated with denervation, e.g., partial loss or disruption of the nerve supply or nerve input to its target, such as a muscle. In some embodiments, the denervation is induced by injury. In some embodiments, the denervation is associated with a disease, such as a genetic disease. In the case of a genetic disease, in some embodiments, a patient may be diagnosed with the genetic disease by genetic screening. In some embodiments, such genetic screening may be performed on fetal, neonatal, or pediatric subjects. Non-limiting examples of diseases with impaired neural signaling include, for example, vocal cord insufficiency / paralysis, spinal cord injury (SCI), myasthenia gravis, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, bulbar muscular atrophy, and spinal muscular atrophy (SMA).

[0349] Spinal cord injury The disclosed methods are suitable for treating or preventing conditions involving impaired neural signaling due to nerve injury. In some embodiments, such a condition is spinal cord injury (SCI). As used herein, the term "spinal cord injury" refers to damage to any portion of the spinal cord or nerves at the end of the spinal canal. Spinal cord injury often causes permanent changes in strength, sensation, and other bodily functions below the site of injury. There are currently no treatments in development aimed at improving or reducing muscle atrophy in SCI, representing a significant unmet need. Additionally, there is a need for therapies to treat metabolic conditions that develop as a result of SCI.

[0350] SCI patients are stratified based on the level (paraplegia vs. quadriplegia) and the completeness of the lesion (complete vs. incomplete). This stratification has been expanded into the American Spinal Injury Association (ASIA) Impairment Scale (Roberts, et al. (2017) Clin Orthop Relat Res 475:1499), which has two broad groups based on the level of paralysis: complete (AIS grade A / B) and incomplete (AIS grade C / D / E). There are seven cervical (neck), 12 thoracic (chest), five lumbar (back), and five sacral (tail) vertebrae. Lesions in SCI can occur anywhere along the vertebrae. In a complete spinal cord injury, the spinal cord cannot send signals below the level of the injury. As a result, the patient is paralyzed below the injury. In an incomplete injury, the patient has some movement and sensation below the injury.

[0351] There are multiple stages associated with spinal cord injury. A subject may be in the acute spinal cord injury phase, immediately after injury, when the diagnosis between complete and incomplete spinal cord injury is generally difficult, due in part to the trauma and associated inflammation. Typically, the acute phase is defined as the initial hospitalization period after the event / injury during which acute medical / surgical care is received, which generally lasts approximately two weeks. A subject may be in the subacute spinal cord injury phase, when the distinction between complete and incomplete spinal cord injury is made and recovery is possible through ongoing rehabilitation. Typically, the subacute phase comprises from about two weeks to up to about 18 months after injury (e.g., 3-6 months after injury). Additionally, a subject may be in the chronic spinal cord injury phase, generally beginning about 6-12 months after injury, when patients show a substantial decrease in recovery rate or have reached a plateau in rehabilitation efforts despite ongoing standard treatment attempts (e.g., plateau).

[0352] Muscle strength can be graded according to the maximum strength achieved, regardless of how briefly that strength is maintained during the test. Muscles are tested with the patient supine. Motor level is determined by the most caudal major muscle with a strength of 3 or greater but normal (=5) upper segments. Scoring for the motor index uses a score of 0 to 5 for each major muscle, with a total score of 25 per limb, for a total possible score of 100. The lower extremity motor score (LEM) uses the major muscles of the ASIA for both lower limbs, with a total possible score of 50 (i.e., a maximum score of 5 for each major muscle (L2, L3, L4, L5, and S1 per limb)). An LEM of 20 or less indicates that the patient is likely to have limited ambulation. An LEM of 30 or greater suggests that the individual is likely to be able to ambulate indoors and outdoors.

[0353] Monitoring the functional outcome and quality of life of SCI patients is a complex task, as selecting an appropriate functional measure depends on the completeness and level of injury. One common measure applicable to all patients is the Functional Independence Measure (FIM), a 7-point scale designed to quantify a patient's dependency on caregivers. An additional index for measuring quality of life that has recently gained attention is the SCI-QOL, which incorporates both a patient's functional skills and emotional well-being (Tulsky 2015, J Spinal Cord Med. 38(3):257-69). Many other functional assessment indices have been outlined by the SCIRE project.

[0354] In some embodiments, a significant clinical benefit achieved by administering an effective amount of a myostatin inhibitor described herein to an SCI patient may correspond to, for example, an increase of at least 6 points (≧6) from baseline in the ASIA total motor score at week 24. In some embodiments, a significant clinical benefit achieved by administering an effective amount of a myostatin inhibitor described herein to an SCI patient may correspond to a statistically significant difference in the mean total SCIM III score between the treated and untreated / control groups at day 112 (+ / - 7 days). In some embodiments, a significant clinical benefit achieved by administering an effective amount of a myostatin inhibitor described herein to an SCI patient may correspond to an increase of more than 4 points (>4) in the Functional Independence Measure for Locomotor Activity (FIM-L) score.

[0355] Individuals with spinal cord injury have an increased prevalence of abnormalities in carbohydrate and lipid metabolism associated with immobilization, muscle atrophy, and increased adiposity. Body composition is substantially altered, characterized by a rapid and prolonged loss of metabolically active muscle mass and bone, and a significant increase in central adiposity. The latter contributes to a maladaptive metabolic profile that promotes substantial weight gain, occ...

Claims

1. 1. An antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, wherein the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises the sequence SYGMS (SEQ ID NO:201) and CDRH2 comprises the sequence SFTGSGGGX. １ YYPDSVKG (SEQ ID NO: 202), １ is T or A, CDRH3 comprises the sequence DLLIRFLEWSHYYGMDV (SEQ ID NO:203), CDRL1 comprises the sequence RSSQSLLHSSGHNFLH (SEQ ID NO:204), CDRL2 comprises the sequence EVSNRVS (SEQ ID NO:205), and CDRL3 comprises the sequence X １ QQTQYPX ２ T (SEQ ID NO: 206), １ is M or Q, and X ２ is P or G, and the CDR sequences are numbered according to the Kabat numbering system.

2. 2. The antibody or antigen-binding fragment thereof of claim 1, comprising: a CDRH1 comprising the sequence of SEQ ID NO: 201; a CDRH2 comprising any one of the sequences of SEQ ID NO: 219 or 226; a CDRH3 comprising the sequence of SEQ ID NO: 220; a CDRL1 comprising the sequence of SEQ ID NO: 216; a CDRL2 comprising the sequence of SEQ ID NO: 222; and a CDRL3 comprising any one of the sequences of SEQ ID NO: 223, 225, 227, or 298, as defined according to the Kabat numbering system.

3. 3. The antibody or antigen-binding fragment thereof of claim 1 or claim 2, comprising, as defined by the Kabat numbering system, a CDRH1 comprising the sequence of SEQ ID NO: 201, a CDRH2 comprising the sequence of SEQ ID NO: 219, a CDRH3 comprising the sequence of SEQ ID NO: 220, a CDRL1 comprising the sequence of SEQ ID NO: 216, a CDRL2 comprising the sequence of SEQ ID NO: 222, and a CDRL3 comprising the sequence of SEQ ID NO:

223.

4. 3. The antibody or antigen-binding fragment thereof of claim 1 or claim 2, comprising, as defined by the Kabat numbering system, a CDRH1 comprising the sequence of SEQ ID NO: 201, a CDRH2 comprising the sequence of SEQ ID NO: 219, a CDRH3 comprising the sequence of SEQ ID NO: 220, a CDRL1 comprising the sequence of SEQ ID NO: 216, a CDRL2 comprising the sequence of SEQ ID NO: 222, and a CDRL3 comprising the sequence of SEQ ID NO:

225.

5. 3. The antibody or antigen-binding fragment thereof of claim 1 or claim 2, comprising, as defined by the Kabat numbering system, a CDRH1 comprising the sequence of SEQ ID NO: 201, a CDRH2 comprising the sequence of SEQ ID NO: 226, a CDRH3 comprising the sequence of SEQ ID NO: 220, a CDRL1 comprising the sequence of SEQ ID NO: 216, a CDRL2 comprising the sequence of SEQ ID NO: 222, and a CDRL3 comprising the sequence of SEQ ID NO:

227.

6. 3. The antibody or antigen-binding fragment thereof of claim 1 or claim 2, comprising, as defined by the Kabat numbering system, a CDRH1 comprising the sequence of SEQ ID NO: 201, a CDRH2 comprising the sequence of SEQ ID NO: 226, a CDRH3 comprising the sequence of SEQ ID NO: 220, a CDRL1 comprising the sequence of SEQ ID NO: 216, a CDRL2 comprising the sequence of SEQ ID NO: 222, and a CDRL3 comprising the sequence of SEQ ID NO:

298.

7. 3. The antibody or antigen-binding fragment thereof of claim 1 or claim 2, comprising a heavy chain variable domain sequence that is at least 90% identical to any one of SEQ ID NOs: 402, 409, or 420, and / or a light chain variable domain sequence that is at least 90% identical to any one of SEQ ID NOs: 412, 419, 421, or 422.

8. 8. The antibody or antigen-binding fragment of claim 7, comprising a pair of variable domain sequences comprising SEQ ID NOs: 402 and 412, SEQ ID NOs: 409 and 419, SEQ ID NOs: 420 and 421, or SEQ ID NOs: 420 and 422.

9. 3. The antibody or antigen-binding fragment thereof of claim 1 or claim 2, comprising a heavy chain sequence that is at least 70% identical to any one of SEQ ID NOs: 503, 507, or 509, and / or a light chain sequence that is at least 70% identical to any one of SEQ ID NOs: 504, 508, 510, or 511.

10. 10. The antibody or antigen-binding fragment thereof of claim 9, comprising a pair of heavy and light chain sequences comprising SEQ ID NOs: 503 and 504; SEQ ID NOs: 507 and 508; SEQ ID NOs: 509 and 510; or SEQ ID NOs: 509 and 511.

11. 1. An antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, wherein the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises the sequence SYGMS (SEQ ID NO:201), CDRH2 comprises the sequence SITGSGGETYYPDSVKG (SEQ ID NO:207), CDRH3 comprises the sequence DLLVRFLEWSHYYGMDV (SEQ ID NO:208), CDRL1 comprises the sequence RSSQSLLHSSGHNFLH (SEQ ID NO:204), CDRL2 comprises the sequence EVSNRVS (SEQ ID NO:205), and CDRL3 comprises the sequence X １ QATQFPRP (SEQ ID NO: 210), １ is M or Q, and the CDR sequences are numbered according to Kabat.

12. 12. The antibody or antigen-binding fragment thereof of claim 11, wherein, as defined by the Kabat numbering system, the CDRH1 comprises SEQ ID NO: 201, the CDRH2 comprises SEQ ID NO: 214, the CDRH3 comprises SEQ ID NO: 215, the CDRL1 comprises SEQ ID NO: 216, the CDRL2 comprises SEQ ID NO: 217, and the CDRL3 comprises SEQ ID NO: 218 or 224.

13. 13. The antibody or antigen-binding fragment thereof of claim 11 or 12, wherein, as defined by the Kabat numbering system, the CDRH1 comprises SEQ ID NO: 201, the CDRH2 comprises SEQ ID NO: 214, the CDRH3 comprises SEQ ID NO: 215, the CDRL1 comprises SEQ ID NO: 216, the CDRL2 comprises SEQ ID NO: 217, and the CDRL3 comprises SEQ ID NO:

218.

14. 13. The antibody or antigen-binding fragment thereof of claim 11 or 12, wherein, as defined by the Kabat numbering system, the CDRH1 comprises SEQ ID NO: 201, the CDRH2 comprises SEQ ID NO: 214, the CDRH3 comprises SEQ ID NO: 215, the CDRL1 comprises SEQ ID NO: 216, the CDRL2 comprises SEQ ID NO: 217, and the CDRL3 comprises SEQ ID NO:

224.

15. 13. The antibody or antigen-binding fragment thereof of claim 11 or 12, comprising a heavy chain variable domain sequence that is at least 90% identical to any one of SEQ ID NOs: 400 or 407, and / or a light chain variable domain sequence that is at least 90% identical to any one of SEQ ID NOs: 410 or 417.

16. 8. The antibody or antigen-binding fragment of claim 7, comprising a pair of variable domain sequences comprising SEQ ID NOs: 400 and 410, or SEQ ID NOs: 407 and 417.

17. 13. The antibody or antigen-binding fragment thereof of claim 11 or 12, comprising a heavy chain sequence that is at least 70% identical to any one of SEQ ID NOs: 501 or 505, and / or a light chain sequence that is at least 70% identical to any one of SEQ ID NOs: 502 or 506.

18. 10. The antibody or antigen-binding fragment thereof of claim 9, comprising a pair of heavy and light chain sequences comprising SEQ ID NOs: 501 and 502 or SEQ ID NOs: 505 and 506.

19. An antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, the antibody or antigen-binding fragment comprising six complementarity-determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3; CDRH1 comprises the sequence GFTFSSYG (SEQ ID NO:3); CDRH2 has the sequence FTGSGGX １ (SEQ ID NO: 291), １ is selected from T and A; CDRH3 comprises the sequence ARDLLIRFLEWSHYYGMDV (SEQ ID NO:257); CDRL1 comprises the sequence QSLLHSSGHNF (SEQ ID NO:258); CDRL2 comprises the sequence EVSNRVS (SEQ ID NO:289); CDRL3 is sequence X １ QQTQYPX ２ T (SEQ ID NO: 292), １ is M or Q, and X ２ is selected from P and G, and the CDR sequences are numbered according to IMGT.

20. 20. The antibody or antigen-binding fragment of claim 19, comprising a heavy chain variable domain sequence selected from the amino acid sequences of SEQ ID NO: 402 or 420.

21. 21. The antibody or antigen-binding fragment of claim 19 or 20, comprising a light chain variable domain sequence selected from the amino acid sequences of SEQ ID NO: 412, 421, or 422.

22. 22. The antibody or antigen-binding fragment of any one of claims 19 to 21, comprising a pair of heavy and light chain variable domain sequences comprising the amino acid sequences of SEQ ID NOs: 402 and 412.

23. 22. The antibody or antigen-binding fragment of any one of claims 19 to 21, comprising a pair of heavy and light chain variable domain sequences comprising the amino acid sequences of SEQ ID NOs: 420 and 421.

24. 22. The antibody or antigen-binding fragment of any one of claims 19 to 21, comprising a pair of heavy and light chain variable domain sequences comprising the amino acid sequences of SEQ ID NOs: 420 and 422.

25. 25. The antibody or antigen-binding fragment thereof of any one of claims 1 to 24, wherein the antibody or antigen-binding fragment has an equilibrium dissociation constant KD of less than 5 nM, and optionally the antibody or antigen-binding fragment has a KD of less than 1 nM (e.g., less than 0.7 nM, less than 0.5 nM, or less than 0.2 nM).

26. 26. The antibody or antigen-binding fragment thereof of any one of claims 1 to 25, wherein the antibody or antigen-binding fragment is capable of inhibiting mTLL-2-induced activation of myostatin with an IC50 of less than 1 nM as measured by functional ELISA.

27. For example, when the antibody and the antigen are mixed at 15 μM each and allowed to form an immune complex at neutral pH, the antibody or antigen-binding fragment has a Fab:promyostatin binding stoichiometry of 2:1, as measured by analytical size exclusion chromatography (SEC).

28. 28. The antibody or antigen-binding fragment thereof of any one of claims 1 to 27, wherein the antibody or antigen-binding fragment has at least 9-fold, such as at least 10-fold, increased pH-sensitive binding compared to an antibody having the heavy chain variable domain sequence of Ab2 and the light chain variable domain sequence of Ab2.

29. The antibody or antigen-binding fragment thereof of any one of claims 1 to 28, wherein the antibody or antigen-binding fragment is capable of reducing total serum myostatin levels compared to background levels.

30. The antibody or antigen-binding fragment thereof of any one of claims 1 to 29, wherein the antibody or antigen-binding fragment cross-competes with an antibody having the heavy chain variable domain sequence of Ab2 and the light chain variable domain sequence of Ab2 for binding to pro / latent myostatin.

31. An antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, the antibody or antigen-binding fragment comprising six complementarity-determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3; CDRH1 comprises the amino acid sequence SYGMS (SEQ ID NO:201); CDRH2 comprises the amino acid sequence SFTGSGGTYYPDSVKG (SEQ ID NO:219); CDRH3 comprises the amino acid sequence DLLIRFLEWSHYYGMDV (SEQ ID NO:220); CDRL1 comprises the amino acid sequence RSSQSLLHSSGHNFLH (SEQ ID NO:216); CDRL2 comprises the amino acid sequence EVSNRVS (SEQ ID NO:222); An antibody or antigen-binding fragment thereof, wherein CDRL3 comprises the amino acid sequence MQQTQYPPT (SEQ ID NO: 223), and said CDR sequences are numbered according to Kabat.

32. 32. The antibody or antigen-binding fragment of claim 31 , comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 402, or a sequence that is at least 95% identical thereto, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 412, or a sequence that is at least 95% identical thereto.

33. 33. The antibody or antigen-binding fragment of claim 31 or 32, comprising a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 503 or a sequence that is at least 95% identical thereto; and / or a light chain sequence comprising the amino acid sequence of SEQ ID NO: 504 or a sequence that is at least 95% identical thereto.

34. 34. The antibody or antigen-binding fragment of any one of claims 31 to 33, comprising the amino acid sequence of SEQ ID NO: 503 and the amino acid sequence of SEQ ID NO:

504.

35. an antibody or antigen-binding fragment thereof that competes with Ab2 for binding to the prodomain of human pro / latent myostatin or binds to a region of the prodomain of human pro / latent myostatin in an epitope comprising one or more amino acid residues of the sequence FVQILRLIKPMKDGTRYTGIRSLK (SEQ ID NO:57) (amino acid positions 147-170 of human promyostatin when numbered according to SEQ ID NO:52) and / or KALDEN (SEQ ID NO:118) (amino acid positions 205-210 of human promyostatin when numbered according to SEQ ID NO:52), wherein the antibody or antigen-binding fragment is not Ab2; the antibody binds to the antigen in a pH-dependent manner as measured by a BLI-based in vitro binding assay (e.g., Octet®); and / or The antibody, or antigen-binding fragment thereof, is capable of inhibiting myostatin activation with an IC50 of less than 1 nM (e.g., less than 0.5 nM) as measured by a functional ELISA, e.g., involving detecting the presence of mature myostatin in an assay mixture comprising an antibody-antigen immune complex and mTLL-2.

36. 36. The antibody or antigen-binding fragment thereof of claim 35, wherein i) the L-CDR1 of the antibody shares 20% or less sequence identity with the L-CDR1 of Ab2; ii) the L-CDR2 of the antibody shares 30% or less sequence identity with the L-CDR2 of Ab2; and / or iii) the L-CDR3 of the antibody shares 10% or less sequence identity with the L-CDR3 of Ab2, or the heavy and light chain variable domains of the antibody share less than 70% cumulative sequence identity with the heavy and light chain variable region sequences of Ab2.

37. 37. The antibody or antigen-binding fragment thereof of claim 35 or 36, wherein the antibody dissociates from the bound antigen at a higher rate in acidic conditions than in neutral conditions.

38. An antibody or antigen-binding fragment thereof described in any one of claims 35 to 37, wherein the antibody or antigen-binding fragment binds to pro / latent myostatin with a bivalent KD of less than 1 nM (e.g., less than 0.7 nM, less than 0.5 nM, or less than 0.2 nM) as measured by an SPR-based in vitro binding assay (e.g., Bicore™).

39. 39. The antibody or antigen-binding fragment of any one of claims 35 to 38, wherein when the antibody and the antigen are mixed at 15 μM each and allowed to form an immune complex at neutral pH, the antibody can bind to the antigen at an antibody-to-antigen stoichiometry of 1:2, wherein the binding stoichiometry is measured by analytical size exclusion chromatography (SEC).

40. 40. The antibody or antigen-binding fragment of any one of claims 35 to 39, comprising the antibody or antigen-binding fragment of any one of claims 1 to 24.

41. the antigen-binding fragment: a first monovalent arm capable of selectively binding to human latent myostatin and inhibiting myostatin activation; and a second monovalent arm that binds to a second target incorporated into an engineered construct comprising; 41. The antibody or antigen-binding fragment of any one of claims 1 to 40, optionally wherein the engineered construct is a bispecific antibody.

42. An antibody or antigen-binding fragment thereof that binds to the pro domain of human pro / latent myostatin, wherein the antibody or antigen-binding fragment: (a) binds to pro / latent myostatin with a bivalent KD of less than 1 nM (e.g., less than 0.7 nM, less than 0.5 nM, or less than 0.2 nM) as measured by an SPR-based in vitro binding assay (e.g., Biacore™); (b) is capable of inhibiting myostatin activation with an IC50 of less than 1 nM (e.g., less than 0.7 nM) as measured by functional ELISA, e.g., the measuring includes detecting the presence of mature myostatin in an assay mixture comprising an antibody-antigen immune complex and mTLL-2; (c) dissociates from bound antigen at a higher rate under acidic conditions than under neutral conditions, as measured by a BLI-based in vitro binding assay (e.g., Octet®); (d) For example, an antibody or antigen-binding fragment thereof that can bind to the antigen at an antibody-to-antigen stoichiometry of 1:2 when the antibody and the antigen are mixed at 15 μM each and allowed to form an immune complex at neutral pH, wherein the binding stoichiometry is measured by analytical size exclusion chromatography (SEC).

43. An antibody or antigen-binding fragment thereof that binds to the pro domain of human pro / latent myostatin, wherein the antibody or antigen-binding fragment: (a) binds to pro / latent myostatin with a bivalent KD of less than 1 nM (e.g., less than 0.7 nM, less than 0.5 nM, or less than 0.2 nM); (b) inhibiting myostatin activation; (c) dissociates from bound antigen at a higher rate under acidic conditions than under neutral conditions; (d) when the antibody and the antigen are mixed at higher concentrations (e.g., 5 μM each, 10 μM each, 15 μM each, or more) and allowed to form an immune complex at neutral pH, they are capable of binding to the antigen at an antibody-to-antigen stoichiometry of 1:2, as measured by analytical size exclusion chromatography (SEC); (e) An antibody or antigen-binding fragment thereof that is capable of binding to the antigen at a 1:1 antibody to antigen stoichiometry when the antibody:antigen complex is present at a lower concentration (e.g., 0.45 μM or less).

44. 44. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 43 and a pharmaceutically acceptable excipient, optionally further comprising a second agent, such as a GLP-1 pathway activator (e.g., a GLP-1R agonist or a GLP-1 analogue) and / or a biguanide (e.g., metformin).

45. 45. The antibody or antigen-binding fragment of any one of claims 1 to 43, or the pharmaceutical composition of claim 44, for use in the treatment or prevention of one or more of the following conditions in a human subject: muscle disorders (e.g., muscle atrophy or myopathy), metabolic disorders (e.g., obesity or diabetes), bone disorders (e.g., bone loss), cardiovascular diseases (e.g., heart failure), and chronic inflammation and inflammatory diseases (e.g., chronic kidney disease (CKD), idiopathic pulmonary fibrosis (IPF), or rheumatoid arthritis (RA)), or liver diseases (e.g., fatty liver disease, NAFLD, or NASH); 45. The antibody or antigen-binding fragment of any one of claims 1 to 43, or the pharmaceutical composition of claim 44, wherein the treatment comprises administering the antibody, antigen-binding fragment, or pharmaceutical composition in an amount effective to treat or prevent one or more conditions.

46. 46. ​​The antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to claim 45, wherein the one or more pathological conditions comprise a metabolic disorder, and optionally the metabolic disorder comprises diabetes and / or obesity.

47. 47. The antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to claim 46, wherein the obesity is childhood obesity, and optionally the antibody or antigen-binding fragment comprises Ab109 or an antigen-binding fragment thereof.

48. 46. ​​The antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to claim 45, wherein the antibody or antigen-binding fragment is administered in combination with a second agent suitable for treating diabetes or obesity.

49. 46. ​​The antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to claim 45, wherein the one or more pathological conditions comprise a metabolic disorder associated with impaired neural signaling, and optionally the metabolic disorder is associated with a muscle disorder, and optionally the muscle disorder is a spinal cord injury, a muscular dystrophy, or a muscle atrophy.

50. 50. The antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to claim 49, wherein the pathological condition associated with impaired nerve signal transduction is a neuromuscular disorder.

51. The antibody or antigen-binding fragment thereof or pharmaceutical composition for use described in claim 45, wherein the one or more pathological conditions include liver disease, optionally wherein the liver disease includes fatty liver disease, NAFLD, or NASH, and optionally wherein the subject is not being treated with a TGFβ inhibitor (e.g., a TGFβ1 inhibitor).

52. 46. ​​The antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to claim 45, wherein the one or more pathological conditions comprise a bone disorder, and optionally the bone disorder comprises bone loss.

53. 46. ​​The antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to claim 45, wherein the one or more pathological conditions comprise cardiovascular disease, and optionally the cardiovascular disease comprises heart failure.

54. 46. ​​The antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to claim 45, wherein the one or more pathological conditions comprise chronic inflammation or an inflammatory disease, and optionally the chronic inflammation or inflammatory disease comprises CKD, IPF, or RA.

55. 55. The antibody or antigen-binding fragment thereof, or pharmaceutical composition for use according to any one of claims 45 to 54, wherein the pharmaceutical composition, antibody, or antigen-binding fragment is administered in combination with a GLP-1 pathway activator, and optionally the subject is undergoing a diet and / or exercise regimen.

56. 56. The antibody or antigen-binding fragment thereof, or pharmaceutical composition for use according to claim 55, wherein the GLP-1 pathway activator is semaglutide, liraglutide, tirzepatide, or retatortide.

57. 57. The antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to claim 55 or 56, wherein the GLP-1 pathway activator is administered at a dose lower than the dose approved for use of the GLP-1 pathway activator as monotherapy.

58. 58. The antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to claim 57, wherein the second agent is a biguanide (e.g., metformin).

59. 59. The antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to any one of claims 45 to 58, wherein the treatment comprises administering an effective amount of the antibody, antigen-binding fragment thereof or pharmaceutical composition sufficient to slow fat accumulation in the subject by at least 10% compared to the rate of fat accumulation in the subject before receiving the treatment.

60. 60. The antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to any one of claims 45 to 59, wherein the treatment comprises administering an effective amount of the antibody, antigen-binding fragment thereof or pharmaceutical composition sufficient to reduce body fat mass in the subject by at least 5% (e.g., at least 10%, 15%, 20%, 25%, or more) compared to baseline.

61. 61. The antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to any one of claims 45 to 60, wherein the treatment comprises administering an effective amount of the antibody, antigen-binding fragment thereof or pharmaceutical composition sufficient to reduce visceral fat mass by at least 5% compared to baseline.

62. 62. The antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to any one of claims 46 to 61, wherein the treatment comprises administering an effective amount of the antibody, antigen-binding fragment thereof or pharmaceutical composition sufficient to reduce subcutaneous fat mass by at least 5% compared to baseline.

63. 63. The antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to any one of claims 45 to 62, wherein the treatment comprises administering an effective amount of the antibody, antigen-binding fragment thereof or pharmaceutical composition sufficient to prevent loss of lean mass in the subject.

64. 64. The antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to any one of claims 45 to 63, wherein the pharmaceutical composition, antibody, or antigen-binding fragment is formulated for subcutaneous administration.

65. 62. The antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to claim 61, wherein the subcutaneous formulation comprises about 100-200 mg / mL of the antibody or antigen-binding fragment.

66. 45. The antibody or antigen-binding fragment thereof of any one of claims 1 to 43, or the pharmaceutical composition of claim 44, and a second agent suitable for treating the metabolic disorder (e.g., a GLP-1 pathway activator and / or a biguanide such as metformin) for use in treating a metabolic disorder in a subject, wherein the treatment comprises administering the antibody or antigen-binding fragment, or the pharmaceutical composition and the second agent in amounts effective to treat the metabolic disorder; Optionally, the metabolic disorder is obesity, diabetes, or both; Further optionally, the effective amount of the second agent is less than a recommended monotherapy dose of the second agent, or the antibody or antigen-binding fragment thereof of any one of claims 1 to 43, or the pharmaceutical composition of claim 44, and a second agent.

67. 67. The antibody, antigen-binding fragment thereof, or pharmaceutical composition and second agent for use according to claim 66, wherein the use comprises subcutaneous administration.

68. 68. The antibody, antigen-binding fragment, or pharmaceutical composition for use of claim 66 or 67, and a second agent, wherein the metabolic disorder comprises adrenoleukodystrophy, type 1 diabetes, type 2 diabetes, Gaucher disease, glucose-galactose malabsorption, hereditary hemochromatosis, Lesch-Nyhan syndrome, maple syrup urine disease, Menkes syndrome, NAFLD, NASH, Niemann-Pick disease, obesity, pancreatic cancer, phenylketonuria, Pompe disease (glycogen storage disease type II), Prader-Willi syndrome, porphyria, Refsum disease, Tangier disease, Tay-Sachs disease, Wilson's disease, or Zellweger syndrome.

69. 1. A biguanide for use in treating obesity or improving body composition in a subject, said treatment comprising administering to said subject said biguanide in combination with a drug that is not a GLP-1 receptor agonist, optionally wherein said drug comprises a myostatin inhibitor.

70. A biguanide for use according to claim 69, wherein the myostatin inhibitor is a non-selective myostatin inhibitor or a selective myostatin inhibitor, and optionally the myostatin inhibitor comprises an antibody, or an antigen-binding fragment thereof, according to any one of claims 1 to 43.

71. 71. The biguanide for use according to claim 69 or 70, wherein the biguanide is metformin.

72. 1. A myostatin inhibitor for use in treating obesity or improving body composition in a subject, said treatment comprising administering said myostatin inhibitor in combination with a biguanide to said subject, said subject not receiving GLP-1 receptor agonist therapy at the time of said administering, said subject being overweight or obese but not having diabetes, and optionally said biguanide being metformin.

73. A biguanide and a myostatin inhibitor for use in treating obesity or improving body composition in a subject, said treatment comprising administering said biguanide and said myostatin inhibitor to said subject, said subject not receiving GLP-1 receptor agonist therapy at the time of said administering; Optionally, the biguanide is metformin; Further optionally, the subject is overweight or obese but not diabetic.

74. 1. A myostatin selective inhibitor and metformin for use in treating obesity or improving body composition in a subject, comprising administering to said subject a myostatin selective inhibitor and metformin, optionally wherein said subject is not receiving GLP-1 receptor agonist therapy at the time of said administering.

75. The object is i) have low tolerance to GLP-1 receptor agonists; ii) is a woman of childbearing potential; iii) diagnosed with or at risk of developing cancer; and / or iv) A myostatin inhibitor, a myostatin selective inhibitor, a biguanide (e.g. metformin) or a combination for use according to any one of claims 69 to 74, in patients at risk of suicidal thoughts, self-harm or depression.

76. The myostatin inhibitor is i) an antibody that binds to latent myostatin and inhibits myostatin activation; or ii) a neutralizing antibody that binds to mature myostatin but does not bind to activin A or GDF11, optionally wherein the neutralizing antibody is trevoglumab or a variant thereof; 76. The myostatin inhibitor or myostatin selective inhibitor, biguanide (e.g., metformin), or combination for use according to any one of claims 69 to 75, optionally wherein the myostatin inhibitor does not cause a decrease in bone mineral density compared to baseline as measured by dual-energy X-ray absorptiometry.

77. 77. The myostatin inhibitor, myostatin selective inhibitor, biguanide (e.g., metformin), or combination for use according to any one of claims 69 to 76, wherein the myostatin inhibitor is selected from the group consisting of an antibody or antigen-binding fragment thereof of any one of claims 1 to 43; apitegromab; and GYM329.

78. 1. A compound comprising metformin and a non-selective myostatin inhibitor for use in treating obesity or improving body composition in a subject, the compound comprising administering metformin and a non-selective myostatin inhibitor to said subject; said subject is not receiving GLP-1 receptor agonist therapy at the time of said administering; Optionally, the subject is not a female of childbearing potential; Further, optionally, the non-selective myostatin inhibitor comprises: i) an antibody that binds to ActRIIB or ActRIIA; ii) a neutralizing antibody that binds to myostatin and at least one additional structurally related ligand selected from GDF11 and activin A; and iii) a ligand trap capable of binding to mature myostatin metformin and a non-selective myostatin inhibitor.

79. 75. The metformin and non-selective myostatin inhibitor for use according to claim 74, wherein the body composition is measured by the ratio of fat mass to lean mass before and after the administration.

80. 80. The antibody or antigen-binding fragment thereof, pharmaceutical composition, or use of any one of claims 1 to 79, wherein the myostatin selective inhibitor does not cause a decrease in bone mineral density compared to baseline as measured by dual-energy X-ray absorptiometry.

81. A method of treating a subject for obesity or improving body composition, comprising administering a myostatin-selective inhibitor to a subject who has discontinued treatment with a GLP-1 receptor agonist.

82. 82. The method of claim 81, wherein the subject has been administered the GLP-1 receptor agonist for at least 12 weeks (e.g., at least 6 months).

83. 83. The method of claim 81 or 82, wherein the GLP-1 receptor agonist comprises semaglutide or tirazepatide.

84. 84. The method of any one of claims 81-83, wherein administering the myostatin selective inhibitor reduces body fat mass by at least 10% compared to a subject not administered the myostatin selective inhibitor after discontinuing the GLP-1 receptor agonist.

85. 85. The method of any one of claims 81-84, wherein administering the myostatin selective inhibitor prevents the recovery of body fat mass by more than 20% after discontinuing the GLP-1 receptor agonist but compared to a subject not administered the myostatin selective inhibitor, and wherein the myostatin selective inhibitor prevents the recovery of body fat mass for up to 6 months from the time of discontinuing the GLP-1 receptor agonist.

86. 86. The method of any one of claims 81-85, wherein administering the myostatin selective inhibitor prevents a decrease in the ratio of lean mass to fat mass of more than 20% after discontinuing the GLP-1 receptor agonist but compared to a subject not administered the myostatin selective inhibitor, and wherein the myostatin selective inhibitor prevents recovery of fat mass for up to 6 months from the time of discontinuing the GLP-1 receptor agonist.

87. 87. The method of any one of claims 81 to 86, wherein the subject is further administered metformin.

88. The method of any one of claims 81 to 87, wherein the myostatin selective inhibitor comprises an antibody or antigen-binding fragment of any one of claims 1 to 3.

89. A method for reducing body fat mass regain in a subject after discontinuing treatment with a GLP-1 receptor agonist, comprising administering to the subject an amount of a myostatin selective inhibitor (e.g., any one of the myostatin selective antibodies or antigen-binding fragments described in any one of claims 1 to 43) effective to reduce body fat mass gain compared to a subject who has discontinued treatment with a GLP-1 receptor agonist but has not been treated with the myostatin selective inhibitor.

90. 90. The method of claim 89, wherein said administration reduces regain of body fat mass following discontinuation of said GLP-1 receptor agonist but compared to a subject not administered a myostatin selective inhibitor, and wherein said myostatin selective inhibitor prevents regain of body fat mass for up to 6 months from the time of discontinuation of said GLP-1 receptor agonist.

91. 91. The method of claim 89 or claim 90, wherein the administration reduces body fat mass recovery by at least 10% (e.g., at least 10%, 20%, 25%, or more) compared to a subject following discontinuation of the GLP-1 receptor agonist but not administered a myostatin selective inhibitor.

92. 92. The method of any one of claims 89 to 91, wherein the myostatin selective inhibitor is administered prior to discontinuing the GLP-1 receptor agonist (e.g., in conjunction with the GLP-1 receptor agonist).

93. 93. The method of any one of claims 89 to 92, wherein the myostatin selective inhibitor is administered within 6 months of discontinuing the GLP-1 receptor agonist.

94. The method of any one of claims 89 to 93, wherein the myostatin selective inhibitor comprises an antibody or antigen-binding fragment of any one of claims 1 to 43.

95. A method for reducing liver fat content in a subject, e.g., an obese subject and / or a subject with fatty liver disease, comprising administering to the subject an amount of a selective myostatin inhibitor effective to reduce liver fat content, preferably in a subject receiving a GLP-1 agonist and / or metformin.

96. The method of claim 95, wherein administering the myostatin selective inhibitor reduces liver fat content by at least 10% (e.g., 10%, 20%, 25%, or more) compared to the subject's liver fat content before administering the myostatin selective inhibitor.

97. A method for improving bone strength and / or preventing bone loss in a subject (e.g., an obese subject), comprising administering to the subject a myostatin selective inhibitor in an amount effective to improve bone strength and / or prevent bone loss compared to a subject (e.g., an obese subject) not administered the myostatin selective inhibitor.

98. The method of claim 97, wherein administration of the myostatin selective inhibitor reduces fractures by at least 10% (e.g., 10%, 20%, 25%, or more) compared to a subject not administered the myostatin selective inhibitor.

99. 99. The method of any one of claims 95 to 98, wherein the subject is receiving or has been receiving a GLP-1 agonist and / or metformin, and optionally the GLP-1 agonist comprises semaglutide or tirzepatide.

100. A method for improving blood glucose or hemoglobin A1C (A1C) levels in a prediabetic or diabetic subject who is or has been administered a GLP-1 agonist, comprising administering to the subject an amount of a myostatin selective inhibitor effective to reduce blood glucose or A1C levels compared to the levels prior to administration of the myostatin selective inhibitor.

101. The method of claim 100, wherein the administration of the myostatin selective inhibitor reduces blood glucose or A1C levels by at least 10% (e.g., 10%, 20%, 25%, or more) compared to the subject's blood glucose or A1C levels before administration of the myostatin selective inhibitor, and optionally, the reduction in glucose is a reduction in fasting blood glucose.

102. 102. The method of claim 100 or claim 101, wherein the reduction in blood glucose or A1C levels is greater than the reduction in blood glucose or A1C levels achieved by administering the GLP-1 receptor agonist alone.

103. The method of any one of claims 100 to 102, wherein the GLP-1 agonist comprises semaglutide or tirzepatide.

104. 104. The method of any one of claims 100 to 103, wherein the subject is or has been administered metformin.

105. The method of any one of claims 95 to 104, wherein the myostatin selective inhibitor comprises an antibody or antigen-binding fragment of any one of claims 1 to 43.

106. 1. A myostatin selective inhibitor for use in treating obesity or improving body composition in a subject, said treatment comprising administering said myostatin selective inhibitor to said subject in combination with a GLP-1 receptor agonist and a biguanide, wherein said myostatin selective inhibitor, said GLP-1 receptor agonist and said biguanide are administered in amounts effective to treat obesity or improve body composition.

107. The myostatin selective inhibitor for use according to claim 106, wherein the GLP-1 receptor agonist comprises semaglutide or tirzepatide.

108. 108. The myostatin selective inhibitor for use according to claim 106 or claim 107, wherein the biguanide is metformin.

109. A myostatin selective inhibitor for use according to any one of claims 106 to 108, wherein the myostatin selective inhibitor comprises an antibody or antigen-binding fragment according to any one of claims 1 to 43.

110. A myostatin inhibitor for use in treating chronic inflammation in a subject, said treatment comprising administering to the subject an effective amount of the myostatin inhibitor to treat the chronic inflammation, optionally wherein the chronic inflammation is inflammation associated with a muscle disorder (e.g., Duchenne muscular dystrophy (DMD)), inflammation associated with chronic kidney disease (CKD), inflammation associated with non-alcoholic fatty liver disease (NAFLD), inflammation associated with non-alcoholic steatohepatitis (NASH), inflammation associated with idiopathic pulmonary fibrosis (IPF), inflammation associated with obesity, inflammation associated with pancreatitis, and / or inflammation associated with an autoimmune disease (e.g., rheumatoid arthritis (RA)).

111. The myostatin inhibitor for use according to claim 110, wherein the myostatin inhibitor is used in conjunction with additional therapy, optionally wherein the additional therapy comprises a GLP-1 receptor agonist, a TGFβ1 inhibitor (e.g., an LTBP-selective TGFβ1 inhibitor), and / or an iron enhancer (e.g., a HIF-PH inhibitor or an RGMc inhibitor).

112. A myostatin inhibitor for use according to claim 110 or claim 111, wherein the myostatin inhibitor is a myostatin selective inhibitor, optionally the myostatin selective inhibitor is an antibody or antigen-binding fragment of any one of claims 1 to 40, and further optionally the myostatin selective inhibitor is Ab109, Ab133, Ab141, apitegromab, trevoglumab, GYM329, or any variant thereof.

113. 112. The myostatin inhibitor for use according to claim 110 or claim 111, wherein the myostatin inhibitor is a non-selective inhibitor of myostatin, optionally wherein the non-selective inhibitor of myostatin is an anti-myostatin-Adnectin, an ActRII receptor antagonist (e.g., bimagrumab or a variant thereof), a follistatin-based drug (e.g., AAV-follistatin or a follistatin-based ligand trap), a soluble ActRII-based ligand trap, or an anti-myostatin antibody.

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