Selective and potent inhibitors of myostatin activation

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] 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 on December 22, 2022, U.S. Provisional Patent Application No. 63 / 477,552, filed on December 28, 2022, U.S. Provisional Patent Application No. 63 / 515,267, filed on July 24, 2023, and U.S. Provisional Patent Application No. 63 / 588,081, filed on October 5, 2023. Each of these applications is incorporated herein by reference in its entirety for all purposes. Technical Field

[0003] The present application generally relates to novel myostatin-inhibiting antibodies and their use for treating disorders, including metabolic and neuromuscular disorders. The disclosure further relates to new adjuvants and combination therapies for improving metabolic health. Background Art

[0004] Myostatin (also known as growth differentiation factor-8 or GDF8) is a member of the TGFβ superfamily of cytokines and is encoded by the MSTN gene in humans. Like other members of the TGFβ superfamily, myostatin is a homodimer that is initially expressed as an inactive precursor polypeptide (called promyostatin). In the overall structure of promyostatin, the mature growth factor is locked in the prodomain, which is a cage-like structure containing two α helices connected by a loop called the "latency lasso" (see, for example, PCT / US2014 / 036933). The amino acid sequence of the human myostatin polypeptide corresponds to UniProt accession number 014793; the mouse counterpart corresponds to UniProt accession number 008689. Myostatin activation involves two separate protease cleavage steps. The first cleavage event in myostatin activation involves furin cleavage of the promyostatin between the prodomain and the growth factor domain, generating a "latent myostatin" in which the mature myostatin remains non-covalently associated with the prodomain and is shielded by the prodomain from binding to its receptor. The second cleavage event is the BMP-1 / Tolloid family protease (such as mammalian tolloid-like 2 (mTLL-2)) that triggers activation, releasing the mature active myostatin growth factor from the latent complex. After activation, mature myostatin signals by binding to a complex of type I and type II cell surface receptors (Alk4 / 5 and ActRIIB), whose downstream signaling induces muscle breakdown and atrophy.

[0005] Myostatin has been implicated in muscle and metabolic disorders due to its primary role as a negative regulator of muscle mass and its involvement in metabolic regulation. However, clinical programs evaluating various myostatin inhibitors in a variety of muscle indications have failed and been discontinued, raising questions about their therapeutic potential. Myostatin inhibitors that have failed or been discontinued in the clinic to date include neutralizing monoclonal antibodies against mature myostatin, such as stamulumab / MYO-029 (evaluated in Becker Muscular Dystrophy (BMD), facioscapulohumeral muscular dystrophy (FSHD), and limb-girdle muscular dystrophy (LGMD)), domagrozumab / PF-06252616 (evaluated in Duchenne muscular dystrophy), and MYO-029 (evaluated in Becker Muscular Dystrophy (BMD), facioscapulohumeral muscular dystrophy (FSHD), and limb-girdle muscular dystrophy (LGMD)). Muscular Dystrophy (DMD)), landogrozumab / LY2495655 (evaluated for cachexia associated with pancreatic cancer and osteoarthritis after total hip replacement), trevogrumab / REGN1033 (evaluated for sporadic inclusion body myositis (sIBM)); soluble ActRIIB ligand traps such as ramatercept / ACE-031 (evaluated for sIBM); follistatin-Fc constructs such as ACE-083 (evaluated for FSHD and Charcot-Marie-Dupuytren disease (CMD)); ie-Tooth); anti-myostatin fibronectin (adnectin), such as BMS-986089 / RG6202 / RO-7239361 (evaluated for DMD); anti-ActRIIB antibodies, such as bimagrumab / BYM338 (evaluated for sIBM, etc.); follistatin gene therapy, such as AAAVI.CMV.F344 and rAAVI.CMV.huFollistatin334 (evaluated for BMD, sIBM and DMD); and anti-myostatin peptibody, such as AMG-745 (evaluated for age-related muscle loss).

[0006] For obese patients with type 2 diabetes, the anti-ActRIIB antibody bimalumab has been shown to reduce total body fat mass and increase lean body mass (Heymsfield et al., 2021). However, bimalumab antagonizes the ActRII receptor, which not only inhibits myostatin, but also inhibits other structurally similar ligands, including GDF11 and activin, which play a role in regulating follicle-stimulating hormone secretion. Therefore, it is unclear whether the observed effects are due to myostatin inhibition, other ligands, or a combination thereof. It is worth noting that Muramatsu et al. (Sci Rep. [Science Report] January 25, 2021; 11 (1): 2160) reported that in preclinical models, GDF11 inhibition had a negative effect on muscle strength, which may block common receptors, which is actually disadvantageous. Although myostatin inhibition and follistatin overexpression have been shown to increase muscle mass, follistatin overexpression in mice has been reported to cause bone structure changes and abnormal regulation of bone metabolism. See Suh et al. (Proc Natl Acad Sci US A. [Proceedings of the National Academy of Sciences of the United States] March 3, 2020; 117(9): 4910-4920) and Chang et al. (JBMR Plus. [Journal of Bone and Mineral Research Plus] 5(4): e10477). This may be due to the broad-spectrum inhibitory effect of follistatin on myostatin, activin, and GDF11. In addition, based on gene knockout studies, there are toxicity issues associated with the inhibition of GDF11 and activin A. For example, inhibition of GDF11 signaling can have a negative impact on bone (Suh et al., Proc Natl Acad Sci [Proceedings of the National Academy of Sciences of the United States] (2020) 117: 4910). Patients with nonsense, frameshift, or missense variants in GDF11 experience craniofacial, vertebral, neurological, cardiac, auditory, and connective tissue abnormalities (Ravenscroft et al., Genet Med [Genetic Medicine] (2021) 23: 1889). In addition, bimalumab has been shown to significantly reduce follicle-stimulating hormone (FSH) in women, and bimalumab clinical trials require women of childbearing age to use multiple forms of contraception (Garito et al., Diabetes Obes Metab. [Journal of Diabetes Obesity and Metabolism] 2018; 20(1): 94-102). Therefore, selectivity in targeting myostatin is beneficial for the ability to drive the efficacy of increasing or maintaining muscle mass while avoiding potential toxicity caused by inhibiting signaling of closely related factors.

[0007] Currently, apitemab remains the only selective myostatin inhibitor that has demonstrated efficacy and safety in a Phase 2 human clinical trial (TOPAZ; NCT03921528) recruiting patients with SMA. Apitemab is being studied in an ongoing Phase 3 trial (SAPPHIRE; NCT05156320) as an intravenous (iv) formulation, which is suitable for conditions such as SMA. However, the subcutaneous route of administration may be a more attractive option for adult and / or ambulatory patients or patients with certain other conditions. Therefore, there remains an unmet need for potent and selective myostatin inhibitors to treat these conditions. Summary of the Invention

[0008] The present disclosure provides, inter alia, novel antibodies and antigen-binding fragments thereof that are highly selective and potent inhibitors of myostatin activation, as well as therapeutic uses thereof. In some embodiments, the antibodies disclosed herein are suitable for subcutaneous administration, for example, at least in part due to higher efficacy. Further disclosed herein are novel adjuvants and combination therapies comprising selective myostatin inhibitors (e.g., new medical uses of selective myostatin inhibitors) for the treatment of metabolic disorders such as obesity and type 2 diabetes, for example, in combination with additional therapeutic agents and / or diet and exercise. Also disclosed are uses for the treatment of cardiovascular metabolic conditions (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).

[0009] The applicant has previously disclosed antibodies that selectively bind to latent myostatin, thereby preventing the activation step of myostatin. See, for example, PCT / US2015 / 059468 and PCT / US2016 / 052014, the contents of which are hereby incorporated in their entirety. The crystal structure of one such antibody bound to the antigen reveals that the two arms of the antibody interact with the homodimeric prodomain, forming a ring-shaped stable complex with a 1:1 binding stoichiometry. See Dagbay et al., J Biol Chem. [Journal of Biological Chemistry] April 17, 2020; 295(16): 5404-5418, the contents of which are hereby incorporated 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 to achieve inhibitory activity. The discovery of an epitope on the prodomain distal to the BMP-1 / Tolloid proteolytic cleavage site of the prodomain required for myostatin activation suggests that heterotopic antibody binding inhibits protease-dependent activation of latent myostatin. Indeed, despite relatively weak monovalent affinity, the antibody demonstrated robust in vivo efficacy in multiple preclinical models.

[0010] Identification of the inhibitory epitopes discussed above provides target regions for the development of additional antibodies, including those that compete for binding (e.g., cross-blocking) with the aforementioned antibodies. Applicants, therefore, sought to further discover novel inhibitory antibodies, including those that bind to the same region or substantially overlapping regions of the myostatin prodomain, i.e., cross-competing antibodies, and in particular, those that compete with Ab2 (disclosed in PCT / US2016 / 052014 and PCT / US2015 / 059468) for binding to promyostatin. Over 30 different antibodies were identified and subsequently demonstrated to exhibit greater inhibitory potency than Ab2 against protease-induced myostatin activation. Modifications to some of these antibody sequences were also evaluated for various properties as described herein. A small class of antibodies with unexpected characteristics has been identified. Surprisingly, in addition to having higher affinity, these novel antibodies also exhibit different properties, such as with respect to one or more of the following: binding stoichiometry, pH sensitivity, and serum myostatin clearance. In certain embodiments of the present disclosure, the novel antibodies or antigen-binding fragments thereof bind to the same or overlapping epitopes as the prior art reference antibodies discussed above. In some embodiments, the novel antibodies bind to the same region of promyostatin / latent myostatin as Ab2, but surprisingly, also do so with a single arm, while maintaining high affinity and inhibitory potency (e.g., an IC50 of less than 1 nM, as measured by a functional ELISA described in detail herein). These surprising features create the possibility that the novel antibodies / antigen-binding fragments disclosed herein can be used to engineer multispecific constructs (such as bispecific antibodies).

[0011] Thus, in some embodiments, the present disclosure encompasses antibodies or antigen-binding fragments thereof that bind to human promyostatin / latent myostatin, but not to mature myostatin or GDF11, wherein the binding is capable of inhibiting myostatin activation, wherein the antibody or antigen-binding fragment binds to the same epitope and / or competes for antigen binding as Ab2 provided in PCT / US2015 / 059468, and / or wherein the antibody or antigen-binding fragment binds to human promyostatin / latent myostatin at / or near amino acid positions 147-170 and / or amino acid positions 205-210, as numbered according to the proGDF8 sequence provided herein (SEQ ID NO: 52). In some embodiments, any of the antibodies or fragments discussed above may be characterized in that: i) the sum of the combined heavy and light chain variable domains (i.e., cumulative VH+VL) shares less than 70% sequence identity with the sum of Ab2; ii) the heavy chain sequence shares less than 90%, 80%, or 70% sequence identity with the heavy chain sequence 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 no more than 25% (e.g., no more than 20%) 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 no more than 20% (e.g., no more than 10%) sequence identity with the L-CDR3 of Ab2.

[0012] In some embodiments, the binding of the protein to the target cell is determined by a suitable in vitro binding assay such as surface plasmon resonance (SPR) (e.g., Biacore TM ), biolayer interferometry (BLI) (e.g. The antibodies or antigen-binding fragments disclosed herein bind to human promyostatin / 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 SPR-based assays such as Biacore TM )Determine KD.

[0013] In some embodiments, the antibody or antigen-binding fragment is capable of inhibiting protease-dependent activation of myostatin with an IC50 (mTLL2IC) of less than 1 nM (e.g., less than 0.5 nM) as measured by a functional ELISA (e.g., measuring the ability of the antibody or antigen-binding fragment to inhibit protease-dependent activation of myostatin as determined by ELISA detecting mature myostatin). 50 ) inhibits mammalian tolloid-like 2 (mTLL-2)-induced myostatin activation.

[0014] In some embodiments, the antibodies or antigen-binding fragments disclosed herein bind to human promyostatin / latent myostatin in a pH-dependent manner, wherein the pH dependence is optionally greater than 10x as determined by comparing the off-rates at pH 5.5 / 7.4, wherein the off-rates are determined by a BLI-based assay (e.g., )Measurement.

[0015] In some embodiments, the present disclosure provides antibodies or antigen-binding fragments that are capable of monovalently binding to an antigen (e.g., a latent myostatin complex) with a monovalent KD of 50 nM or less as measured by, for example, a BLI-based in vitro binding assay or an SPR-based in vitro binding assay. In some embodiments, the monoclonal antibodies of the present disclosure bind to human promyostatin / latent myostatin with a 1:2 antibody:antigen stoichiometry. In some embodiments, the monoclonal antibodies of the present disclosure bind to human promyostatin / latent myostatin with both a 1:2 antibody:antigen stoichiometry and a daisy-chain format. In some embodiments, the monoclonal antibodies of the present disclosure bind to human promyostatin / latent myostatin in a daisy-chain format. In some embodiments, the Fab fragments of the monoclonal antibodies of the present disclosure bind to human promyostatin / latent myostatin with a 2:1 Fab:antigen stoichiometry. In some embodiments, the Fab fragments of the monoclonal antibodies of the present disclosure bind to human promyostatin / latent myostatin with a 1:1 Fab:antigen stoichiometry. In some embodiments, the antibody binds to human promyostatin / latent myostatin with a 1:2 mAb:Ag binding stoichiometry as measured by analytical SEC-MALS, wherein the mAb and Ag are present in a 1:1, 2:1, or 3:1 ratio (i.e., a mAb:Ag mixture), wherein the total protein concentration is between about 3.5 mg / mL (e.g., about 15 μM each of mAb and Ag) and about 8 mg / mL (e.g., about 45 μM mAb and about 15 μM Ag), and is allowed to form immune complexes at room temperature at neutral pH for a suitable duration (such as 1 to 48 hours, preferably about 24 hours). In some embodiments, the mAb:Ag mixture further comprises oligomeric complexes, including 2:1 mAb:Ag complexes and / or 2:2 mAb:Ag complexes. In some embodiments, the mAb:Ag mixture does not comprise detectable levels of polydaisy chains as measured by analytical SEC-MALS. In some embodiments, the antibody is capable of binding to the antigen with a 1:2 antibody:antigen stoichiometry when the antibody and antigen are mixed at 15 μM each and allowed to form an immune complex at neutral pH, where the binding stoichiometry is measured by analytical size exclusion chromatography (SEC) (e.g., SEC-MALS).

[0016] In some embodiments, the antibodies or antigen-binding fragments disclosed herein (e.g., Ab109, Ab133, Ab141) do not cause accumulation of circulating myostatin (e.g., total myostatin or latent myostatin in serum). Although certain myostatin selective activation inhibitors of the prior art (such as Apitrema) cause latent myostatin (e.g., latent myostatin-antibody immune complex) to accumulate in serum (i.e., circulating myostatin), the antibodies disclosed herein (e.g., Ab109) are capable of reducing the total serum myostatin level (compared to background levels) of the subject in some embodiments. In some embodiments, the antibodies or antigen-binding fragments disclosed herein cause a rapid decrease in free latent myostatin in the circulation (i.e., serum). In some embodiments, when a single dose of 2-20 mg / kg of the antibody is administered to mice, free latent myostatin levels are reduced from background (e.g., about 50 ng / mL) to below detectable levels (e.g., within one day of administration) and remain undetectable or nearly undetectable (e.g., for at least 42 days).

[0017] In some embodiments, the antibody binds to human pro-myostatin / 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:antigen stoichiometry. In some embodiments, the antibodies disclosed herein (e.g., Ab109) are capable of reducing serum concentrations of total myostatin or latent myostatin. Without wishing to be bound by theory, it is contemplated that faster serum clearance may be associated with larger immune complexes (e.g., multiple daisy chains) formed in vivo and that larger immune complex formation (e.g., oligomers) may facilitate clearance, for example, by enhancing FcRn interactions. Advantageously, such enhanced clearance can be achieved or improved without engineering the antibody by introducing mutations into the Fc region (see, e.g., Muramatsu et al., Sci Rep. 2021; 11:2160), thereby minimizing the risk of unwanted immunogenicity.

[0018] In some embodiments, 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 SFTGSGGX1YYPDSVKG (SEQ ID NO: 202), wherein 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), wherein X1 is M or Q, and X2 is P or G, wherein the CDR sequences are numbered according to the Kabat numbering system.

[0019] In some embodiments, 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 X1QATQFPRP (SEQ ID NO: 210), wherein X1 is M or Q, and wherein 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, wherein 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); CDRL1 comprises the sequence RX1SQSX2LHSX3X4HNFLH (SEQ ID NO: 212), wherein 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), wherein X1 is A or V; X2 is R or L; X3 is V or A, and CDRL3 comprises the sequence QQX1TQYPPT (SEQ ID NO: 214). NO:214), wherein X1 is Q or Y, and wherein the CDR sequences are numbered according to Kabat.

[0021] In some embodiments, 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 SX1TGSGGX2TYYPDSVKG (SEQ ID NO: 275), wherein X1 is F or I, and X2 is E or A; CDRH3 comprises the sequence DLLX1RFLEWSHYYGMDV (SEQ ID NO: 272), wherein X1 is I or V; CDRL1 comprises the sequence RSSQSLLHSSGHNFLH (SEQ ID NO: 204); CDRL2 comprises the sequence ETSNRX1X2 (SEQ ID NO: 276), wherein X1 is V or A and X2 is P or S; and CDRL3 comprises the sequence X1QQX2TQX3PX4X5 (SEQ ID NO: NO: 277), wherein 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, wherein the CDR sequences are numbered according to the Kabat numbering system.

[0022] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises the sequence GFTFX1SY (SEQ ID NO: 278), wherein X is S or T; CDRH2 comprises the sequence TGSGG (SEQ ID NO: 279); CDRH3 comprises the sequence LLX1RFLEWSHYYGMD (SEQ ID NO: 280), wherein X1 is I or V; CDRL1 comprises the sequence SQSLLHSSGHNF (SEQ ID NO: 281); CDRL2 comprises the sequence EX1S, wherein X1 is T or V; and CDRL3 comprises the sequence XXXXX6, wherein 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; wherein the CDR sequences are numbered according to the Chothia numbering system. In some embodiments, CDRL3 comprises the sequence QTQYPX1 (SEQ ID NO: 293), wherein X1 is P or G.

[0023] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises the sequence GFTFTSSYG (SEQ ID NO: 284); CDRH2 comprises the sequence X1TGSGGX2T (SEQ ID NO: 285), wherein X1 is F or I and X2 is E, T, or A; CDRH3 comprises the sequence ARDLLVRFLEWSHYYGMDV (SEQ ID NO: 286); CDRL1 comprises the sequence QSLLHSSGHNF (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), wherein 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, wherein the CDR sequences are numbered according to the IMGT numbering system.

[0024] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 of SEQ ID NO: 201; HCDR2 of SEQ ID NO: 202, wherein X1 is T or A; HCDR3 of SEQ ID NO: 203; LCDR1 of SEQ ID NO: 204; LCDR2 of SEQ ID NO: 205; and LCDR3 of SEQ ID NO: 206, wherein X1 is M or Q and X2 is P or G, numbered according to the Kabat numbering system.

[0025] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 of SEQ ID NO: 293; HCDR2 of SEQ ID NO: 279; HCDR3 of SEQ ID NO: 296; LCDR1 of SEQ ID NO: 281; LCDR2 of EVS; and LCDR3 of SEQ ID NO: 297, wherein X1 is P or G and is numbered according to the Clothia numbering system.

[0026] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 of SEQ ID NO: 293; HCDR2 of SEQ ID NO: 294, wherein X1 is T or A; HCDR3 of SEQ ID NO: 257; LCDR1 of SEQ ID NO: 258; LCDR2 of EVS; and LCDR3 of SEQ ID NO: 292, wherein X1 is M or Q and X2 is P or G, numbered according to the IMGT numbering system.

[0027] In some embodiments, an anti-promyostatin / latentmyostatin antibody, or antigen-binding portion thereof, suitable for practicing various embodiments of the present disclosure comprises the following six CDRs: a CDRH1 comprising GFTFSSYG (SEQ ID NO: 3); a CDRH2 comprising FTGSGGX1 (SEQ ID NO: 291), wherein X1 is selected from the group consisting of T and A; a CDRH3 comprising ARDLLIRFLEWSHYYGMDV (SEQ ID NO: 257); a CDRL1 comprising QSLLHSSGHNF (SEQ ID NO: 258); a CDRL2 comprising EVSNRVS (SEQ ID NO: 289); and a CDRL3 comprising X1QQTQYPX2T (SEQ ID NO: 292), wherein X1 is selected from the group consisting of M and Q, and X2 is selected from the group consisting of 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).

[0028] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein 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, as defined by the Kabat numbering system. In some embodiments, preferred antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising the following six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or a combination thereof, wherein CDRH1 comprises SEQ ID NO: 201, CDRH2 comprises any one of SEQ ID NO: 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 NO: 223, 225, or 227, as defined according to the Kabat numbering system.

[0029] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein 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 SEQ ID NO: 218, 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: 214, a CDRH3 comprising the sequence of SEQ ID NO: 215, a CDRL1 comprising the sequence of SEQ ID NO: 216, a CDRL2 comprising the sequence of SEQ ID NO: 217, and a 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 the sequence of any one of SEQ ID NOs: 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 the sequence of any one of SEQ ID NOs: 223, 225, or 227, 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: 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 according to 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 according to the Kabat numbering system.

[0030] The novel antibodies and antigen-binding fragments thereof disclosed herein are suitable for therapeutic use in human patients for the treatment of one or more myostatin-related conditions. Myostatin-related diseases and conditions include, but are not limited to, muscle disorders (e.g., atrophy and neuromuscular disorders such as SMA) and cardiovascular metabolic disorders (e.g., obesity, diabetes, prediabetes, fatty liver, bone disorders, and heart failure). Such antibodies are particularly suitable for subcutaneous formulations due, in part, to their high efficacy and favorable developability. In some embodiments, a pharmaceutical composition comprising such an antibody (or an engineered construct comprising an antigen-binding fragment of such an antibody) formulated for subcutaneous administration is used to treat a metabolic disorder, wherein optionally, the metabolic disorder is obesity, metabolic syndrome, diabetes, and / or prediabetes. In some embodiments, the antibody is selected from Abl01, Abl02, Abl03, Abl04, Abl05, Abl06, Abl07, Abl08, Abl09, Abl10, Abl11, Abl12, Abl13, Abl14, Abl15, Abl16, Abl17, Abl18, Abl19, Abl20, Abl21, Abl22, Abl23, Abl24, Abl25, Abl26, Abl27, Abl28, Abl29, Abl30, Abl31, Abl32, Abl33, Abl34, Abl35, Abl36, Abl37, Abl38, Abl39, Abl40, and Abl41. In a preferred embodiment, the antibody is Abl09, Abl33, or Abl41.

[0031] Currently available obesity therapies, such as GLP-1 receptor agonists, focus primarily on weight loss. In contrast, the present disclosure considers the quality of weight management rather than pure 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 prioritizing fat mass over lean body mass loss; maintaining reduced fat mass; preventing muscle loss; increasing lean body 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, can contribute to safe and sustainable weight management, particularly when used in combination with another therapy aimed at addressing metabolic dysregulation.

[0032] Thus, 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 an amount effective to treat the metabolic disorder, the myostatin selective inhibitor alone or in combination with an additional agent, such as a GLP-1 pathway activator (e.g., a GLP-1 receptor agonist), 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 selected from Abl01, Abl02, Abl03, Abl04, Abl05, Abl06, Abl07, Abl08, Abl09, Abl10, Abl11, Abl12, Abl13, Abl14, Abl15, Abl16, Abl17, Abl18, Abl19, Abl20, Abl21, Abl22, Abl23, Abl24, Abl25, Abl26, Abl27, Abl28, Abl29, Abl30, Abl31, Abl32, Abl33, Abl34, Abl35, Abl36, Abl37, Abl38, Abl39, Abl40, and Abl41. 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 developed by Amgen), or danuglipron (an oral GLP-1 receptor agonist developed by Pfizer). In some embodiments, when used in combination with the myostatin selective inhibitors disclosed herein, the amount and / or frequency of administration of the GLP-1 receptor agonist effective for treating metabolic disorders can be reduced.

[0033] In some embodiments, the present disclosure provides a method of treating a metabolic disorder in a subject, comprising administering to the subject a selective inhibitor of myostatin (e.g., any of the antibodies or antigen-binding fragments described herein), wherein optionally, the subject is receiving or has received at least one dose of a GLP-1 receptor agonist and / or metformin. In some embodiments, metformin is administered to the subject without administering 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 selected from Abl01, Abl02, Abl03, Abl04, Abl05, Abl06, Abl07, Abl08, Abl09, Abl10, Abl11, Abl12, Abl13, Abl14, Abl15, Abl16, Abl17, Abl18, Abl19, Abl20, Abl21, Abl22, Abl23, Abl24, Abl25, Abl26, Abl27, Abl28, Abl29, Abl30, Abl31, Abl32, Abl33, Abl34, Abl35, Abl36, Abl37, Abl38, Abl39, Abl40, and Abl41. In some embodiments, the myostatin selective inhibitor comprises Abl09, Abl33, or Abl41. In some embodiments, the myostatin selective inhibitor is Ab 109. In some embodiments, the amount and / or frequency of administration of a 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 a method for treating obesity or improving body composition, comprising administering a myostatin selective inhibitor (e.g., any of the antibodies or antigen-binding fragments described herein) to a subject, wherein optionally, the subject is receiving or has received 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 a GLP-1 receptor agonist. In some embodiments, the GLP-1 receptor agonist comprises semaglutide. In some embodiments, the antibody is selected from Abl01, Abl02, Abl03, Abl04, Abl05, Abl06, Abl07, Abl08, Abl09, Abl10, Abl11, Abl12, Abl13, Abl14, Abl15, Abl16, Abl17, Abl18, Abl19, Abl20, Abl21, Abl22, Abl23, Abl24, Abl25, Abl26, Abl27, Abl28, Abl29, Abl30, Abl31, Abl32, Abl33, Abl34, Abl35, Abl36, Abl37, Abl38, Abl39, Abl40, and Abl41. In some embodiments, the myostatin selective inhibitor comprises Abl09, Abl33, or Abl41. In some embodiments, the myostatin selective inhibitor is Abl09. In some embodiments, the myostatin selective inhibitor is Abl09. In some embodiments, the amount and / or frequency of administration of a GLP-1 receptor agonist is reduced when used in combination with a myostatin selective inhibitor disclosed herein.

[0035] In some embodiments, the present disclosure provides a method of treating obesity or improving body composition comprising administering a myostatin selective inhibitor to a subject who has discontinued GLP-1 receptor agonist treatment. In some embodiments, the myostatin selective inhibitor is an antibody selected from Abl01, Abl02, Abl03, Abl04, Abl05, Abl06, Abl07, Abl08, Abl09, Abl10, Abl11, Abl12, Abl13, Abl14, Abl15, Abl16, Abl17, Abl18, Abl19, Abl20, Abl21, Abl22, Abl23, Abl24, Abl25, Abl26, Abl27, Abl28, Abl29, Abl30, Abl31, Abl32, Abl33, Abl34, Abl35, Abl36, Abl37, Abl38, Abl39, Abl40, and Abl41, or an antigen-binding fragment thereof. In a preferred embodiment, the antibody is Abl09, Abl33, or Abl41.

[0036] In some embodiments, the present disclosure provides a method for reducing fat mass rebound in a subject after discontinuing treatment with a GLP-1 receptor agonist, wherein the method comprises administering a myostatin inhibitor to the subject in an amount effective to reduce fat mass gain compared to a subject who has discontinued GLP-1 receptor agonist therapy but has not been treated with a myostatin inhibitor. In some embodiments, after discontinuation of GLP-1 receptor agonist therapy, myostatin inhibitor therapy reduces the degree of fat mass rebound. In some embodiments, after discontinuation of GLP-1 receptor agonist therapy, myostatin inhibitor therapy reduces the rate of fat mass rebound. In preferred embodiments, the myostatin inhibitor is a myostatin selective inhibitor, such as any one of the antibodies or antigenic fragments disclosed herein (such as Ab109, Ab133, or Ab141), trogolubumab (REGN1033), and GYM329 (RO7204239), which is an anti-latent myostatin Fc-engineered antibody discovered by Chugai Pharmaceutical Co., Ltd. and developed by Roche. In a preferred embodiment, the myostatin selective inhibitor is an antibody or antigen-binding fragment selective for promyostatin / latent myostatin, such as any one of Ab101 to 141. In a most preferred embodiment, the myostatin selective inhibitor is Ab109, Ab133 or Ab141.

[0037] In some embodiments, the present disclosure provides a method of reducing liver fat in a subject (e.g., an obese subject and / or a subject with fatty liver disease), the method comprising administering to the subject a myostatin selective inhibitor in an amount effective to reduce liver fat. In preferred embodiments, the myostatin selective inhibitor is used in combination with a GLP-1 receptor agonist for a duration sufficient to synergistically reduce relative liver weight (e.g., a duration greater than five weeks). In some embodiments, the myostatin selective inhibitor is an antibody selected from Abl01, Abl02, Abl03, Abl04, Abl05, Abl06, Abl07, Abl08, Abl09, Abl10, Abl11, Abl12, Abl13, Abl14, Abl15, Abl16, Abl17, Abl18, Abl19, Abl20, Abl21, Abl22, Abl23, Abl24, Abl25, Abl26, Abl27, Abl28, Abl29, Abl30, Abl31, Abl32, Abl33, Abl34, Abl35, Abl36, Abl37, Abl38, Abl39, Abl40, and Abl41, or an antigen-binding fragment thereof. In a preferred embodiment, the antibody is Abl09, Abl33, or Abl41.

[0038] 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 in combination with another 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 an antibody selected from Abl01, Abl02, Abl03, Abl04, Abl05, Abl06, Abl07, Abl08, Abl09, Abl10, Abl11, Abl12, Abl13, Abl14, Abl15, Abl16, Abl17, Abl18, Abl19, Abl20, Abl21, Abl22, Abl23, Abl24, Abl25, Abl26, Abl27, Abl28, Abl29, Abl30, Abl31, Abl32, Abl33, Abl34, Abl35, Abl36, Abl37, Abl38, Abl39, Abl40, and Abl41, or an antigen-binding fragment thereof. In a preferred embodiment, the antibody is Abl09, Abl33, or Abl41. In some embodiments, the amount and / or frequency of administration of additional agents suitable for treating metabolic disorders can be reduced when used in combination with the myostatin selective inhibitors disclosed herein.

[0039] In some embodiments, the present disclosure provides a method for improving bone strength and / or preventing bone loss in a subject (e.g., an obese subject), the method 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) to which a myostatin selective inhibitor has not been administered. In some embodiments, bone strength is measured according to bone mineral density and / or fracture frequency or severity. In some embodiments, the subject is receiving or has received GLP-1 receptor agonist therapy. In some embodiments, the subject is receiving weight loss therapy. In some embodiments, the subject is receiving or has received a therapy that causes GDF11 inhibition (e.g., a selective or non-selective inhibitor of GDF11). In some embodiments, GDF11 inhibitor therapy is replaced with a myostatin selective inhibitor therapy (such as a novel antibody disclosed herein). In some embodiments, the myostatin selective inhibitor is an antibody selected from Abl01, Abl02, Abl03, Abl04, Abl05, Abl06, Abl07, Abl08, Abl09, Abl10, Abl11, Abl12, Abl13, Abl14, Abl15, Abl16, Abl17, Abl18, Abl19, Abl20, Abl21, Abl22, Abl23, Abl24, Abl25, Abl26, Abl27, Abl28, Abl29, Abl30, Abl31, Abl32, Abl33, Abl34, Abl35, Abl36, Abl37, Abl38, Abl39, Abl40, and Abl41, or an antigen-binding fragment thereof. In a preferred embodiment, the antibody is Abl09, Abl33, or Abl41.

[0040] In some embodiments, the present disclosure provides a method of improving blood glucose or hemoglobin A1C (A1C) levels in a pre-diabetic or diabetic subject who is receiving or has received a GLP-1 receptor agonist, the method comprising administering to the subject a myostatin selective inhibitor in an amount effective to lower blood glucose (e.g., fasting glucose) or A1C levels compared to baseline (i.e., before administration of the myostatin selective inhibitor). In some embodiments, the myostatin selective inhibitor is an antibody selected from Abl01, Abl02, Abl03, Abl04, Abl05, Abl06, Abl07, Abl08, Abl09, Abl10, Abl11, Abl12, Abl13, Abl14, Abl15, Abl16, Abl17, Abl18, Abl19, Abl20, Abl21, Abl22, Abl23, Abl24, Abl25, Abl26, Abl27, Abl28, Abl29, Abl30, Abl31, Abl32, Abl33, Abl34, Abl35, Abl36, Abl37, Abl38, Abl39, Abl40, and Abl41, or an antigen-binding fragment thereof. In a preferred embodiment, the antibody is Abl09, Abl33, or Abl41.

[0041] In any embodiment disclosed herein, the GLP-1 receptor agonist may comprise semaglutide, tilportide, AMG-133 (a GLP-1 receptor agonist / GIP-1 receptor antagonist developed by Amgen), or danglitazone (an oral GLP-1 receptor agonist developed by Pfizer).

[0042] In any of the embodiments disclosed herein, the myostatin selective inhibitor used according to the present disclosure can be an antibody or antigen-binding fragment described herein, such as Abl09, Abl33, or Abl41, or an antigen-binding fragment thereof, trogelutumab (REGN1033), or GYM329 (RO7204239), an anti-latent myostatin Fc-engineered antibody discovered by Chugai Pharmaceutical Co., Ltd. and developed by Roche for SMA. In some embodiments, the myostatin selective inhibitor used according to the present disclosure is an antibody selected from Abl01, Abl02, Abl03, Abl04, Abl05, Abl06, Abl07, Abl08, Abl09, Abl10, Abl11, Abl12, Abl13, Abl14, Abl15, Abl16, Abl17, Abl18, Abl19, Abl20, Abl21, Abl22, Abl23, Abl24, Abl25, Abl26, Abl27, Abl28, Abl29, Abl30, Abl31, Abl32, Abl33, Abl34, Abl35, Abl36, Abl37, Abl38, Abl39, Abl40 and Abl41, or an antigen-binding fragment thereof. In preferred embodiments, the antibody is Abl09, Abl33, or Abl41. In some embodiments, the myostatin selective inhibitor used in accordance with the present disclosure is Abl09.

[0043] The present disclosure further encompasses combination or adjunctive (add-on) therapies comprising a myostatin inhibitor and a biguanide (e.g., metformin) without a GLP-1 receptor agonist. This combination or adjunctive (add-on) therapy can be used to treat a metabolic disorder in a patient, wherein optionally, the metabolic disorder is obesity, diabetes, prediabetes, and / or metabolic syndrome. In some embodiments, the patient responds poorly to GLP-1 receptor agonist therapy, has low tolerance to GLP-1 receptor therapy, and / or is at risk for developing depression, suicidal ideation, or cancer. In some embodiments, the myostatin inhibitor is a non-selective inhibitor, such as an agent that inhibits myostatin and GDF11 but does not inhibit activin A, or an agent that inhibits myostatin and activin A but does not inhibit GDF11. In some embodiments, the non-selective inhibitor is an ActRII receptor antagonist (such as bimalumab), an anti-myostatin (such as the peptide deluprobe alfa (taldefgrobepalfa), 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 antigenic fragments disclosed herein (such as Abl09, Abl33, and Abl41), trogoluzumab (REGN1033), and GYM329 (RO7204239), which is an anti-latent myostatin Fc-engineered antibody discovered by Chugai Pharmaceutical Co., Ltd. and developed by Roche for SMA. In some embodiments, according to the present disclosure The myostatin selective inhibitor used is an antibody selected from Abl01, Abl02, Abl03, Abl04, Abl05, Abl06, Abl07, Abl08, Abl09, Abl10, Abl11, Abl12, Abl13, Abl14, Abl15, Abl16, Abl17, Abl18, Abl19, Abl20, Abl21, Abl22, Abl23, Abl24, Abl25, Abl26, Abl27, Abl28, Abl29, Abl30, Abl31, Abl32, Abl33, Abl34, Abl35, Abl36, Abl37, Abl38, Abl39, Abl40 and Abl41, or an antigen-binding fragment thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1A and Figure 1B The antibodies of the present disclosure were shown to inhibit myostatin activation.

[0045] Figure 2A Binding of Abs 101 to 109, Abl35, Ab2, and controls to purified promyostatin from human, mouse, or cynomolgus monkey is shown. Figures 2B to 2D Antibodies Ab122-128 and Ab2 showed that they are effective against human promyostatin ( Figure 2B ), mouse pro-myostatin ( Figure 2C ) and cynomolgus monkey pro-myostatin ( Figure 2D ) combination.

[0046] Figure 3 The antibodies disclosed herein are shown to bind to human or mouse GDF-11.

[0047] Figures 4A to 4BThe pH-dependent dissociation of the antibodies disclosed herein is shown at pH 7.4 compared to pH 5.5. The values ​​to the right of the curves represent the calculated fold difference in the off-rate at the different pH values ​​tested.

[0048] Figures 5A to 5F The binding stoichiometry of antibody: promyostatin or Fab:myostatin is shown. The figures are simplified depictions and not drawn to scale.

[0049] Figures 6A to 6C The in vivo efficacy of Ab2, Ab102, Ab105, Ab121, and Ab123 in mice with dexamethasone-induced muscle atrophy was demonstrated. Figure 6A Shows percent weight change from baseline. Figure 6B Shown are the percent changes in lean body mass relative to baseline. Figure 6C Shown are the percent changes in gastrocnemius muscle mass relative to baseline. The dashed line in each graph shows the mean of 10 mg / kg Ab2 treatment. ****p<0.0001; ***p<0.005; **p<0.01; *p<0.05.

[0050] 7A to 7D The in vivo efficacy of Ab2, Ab130, and Ab109 in mice with dexamethasone-induced muscle atrophy was demonstrated. Figure 7A Shows weight changes. Figure 7B Shows changes in lean body mass. Figure 7C Showing changes in gastrocnemius muscle mass. Figure 7D Shown are changes in quadriceps muscle mass. ****p<0.0001; ***p<0.005; **p<0.01; *p<0.05.

[0051] Figure 8 Shown are serum total myostatin levels of mice treated with Ab2, Abl09, or Abl30. Statistical analysis was performed using one-way ANOVA (Dunnett's multiple comparison test).

[0052] 9A to 9D The in vivo efficacy of Ab2, Ab112, Ab109, and Ab127 in mice with dexamethasone-induced muscle atrophy was demonstrated. Figure 9A Shows weight changes. Figure 9B Shown are changes in lean body mass as measured by qNMR. Figure 9C Showing changes in gastrocnemius muscle mass. Figure 9D Changes in quadriceps muscle mass are shown. The dashed lines in each graph correspond to the mean values ​​of 10 mg / kg Ab2 treatment; ****p<0.0001; ***p<0.005; **p<0.01; *p<0.05.

[0053] 10A to 10F The in vivo efficacy of Ab2, Ab109, and Ab133 in mice with dexamethasone-induced muscle atrophy was demonstrated. Figure 10A Shows percent weight change from baseline. Figure 10B Shown are the percent changes in lean body mass relative to baseline. Figure 10C Shown are the percent changes in gastrocnemius muscle weight relative to control. Figure 10D The percent change in quadriceps muscle weight relative to control is shown. Figure 10E Gastrocnemius muscle weights are shown. Figure 10F Quadriceps weights are shown. The dashed lines in each graph correspond to the mean values ​​for 3 mg / kg Ab2 treatment. ****p<0.0001; ***p<0.005; **p<0.01; *p<0.05.

[0054] Figures 11A to 11H The in vivo efficacy of Ab2, Ab109, or Ab130 combined with liraglutide in treating DIO mice was shown. Figures 11A to 11C Shows weight during treatment. Figure 11D Shows percent weight change from baseline. Figure 11E Shown are the percent changes in lean body mass relative to baseline; ****p<0.0001; ***p<0.005; **p<0.01; *p<0.05. Figure 11F Shown are the percent changes in gastrocnemius muscle mass compared to liraglutide alone (left panel) or IgG control (right panel). Figure 11G Shown are the percent changes in fat mass from baseline from day 15 to day 1 (left panel) or from day 29 to day 1 (right panel). Figure 11H Serum exposure of Ab2, Abl09 and Abl30 is shown.

[0055] FIG. 12A to FIG. 12B The in vivo efficacy of Ab 109 combined with metformin treatment in mice fed a high fat diet was demonstrated. Figure 12A The efficacy of Abl09 and / or metformin on fat mass in mice fed a 60% high fat diet and switched to a 45% high fat diet was shown. Figure 12B The efficacy of Abl09 and / or metformin on lean body mass in mice fed a 60% high fat diet and switched to a 45% high fat diet is shown.

[0056] 13A to 13B The in vivo efficacy of semaglutide treatment in combination with IgG control, Abl09, or Abl41 in mice fed a high-fat diet is shown. Figure 13A Shows effects on subcutaneous adipose tissue weight. Figure 13B Effects on epididymal adipose tissue weight were shown.

[0057] Figures 14A to 14K The in vivo efficacy of semaglutide treatment in combination with IgG control or Ab109 in DIO mice fed a high-fat diet is shown. Figure 14A Shows effect on body weight. Figure 14B and Figure 14C Shows effects on lean body mass. Figure 14D Shows effect on fat mass. Figure 14E Effects on gastrocnemius muscle weight are shown. Figure 14F Effects on inguinal fat pad weight are shown. Figure 14G Effects on epididymal fat pad weight are shown. Figure 14H Shown are the effects of Ab109 and semaglutide on lean body mass (left panel) and fat mass (right panel), as measured by qNMR; statistical analysis was performed using one-way ANOVA (Duncan's multiple comparison test; ****p<0.0001; ***p<0.005; **p<0.01; *p<0.05). Figure 14I Shown are fasting serum glucose throughout the study and on days 18 and 64 following treatment with Ab109 alone or in combination with semaglutide. Figure 14J Shown are relative liver weight changes (expressed as % of liver weight relative to 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 Relative fat mass and circulating leptin of mice in this study are shown, ****p<0.0001; **p<0.01; *p<0.05. Statistical analysis was performed using one-way ANOVA (Duncan's multiple comparison test).

[0058] FIG. 15A to FIG. 15B Dexamethasone ( Figure 15A ) or liraglutide ( Figure 15B ) Serum total myostatin levels in mice treated in combination with Ab2, Abl09 or Abl30.

[0059] Figure 16A Dose-dependent serum exposure was shown for Abl09 and Abl41 given at 0.1 to 3 mg / kg alone or in combination with semaglutide. Figure 16B Shown are total myostatin following treatment with Abl09 or Abl41 given at 0.1 to 3 mg / kg, alone or in combination with semaglutide. Figure 16C Shown are levels of free latent myostatin (latent myostatin not bound by the antibody) following 22 days of treatment with Abl09 or Abl41 given at 0.1 to 3 mg / kg, alone or in combination with semaglutide.

[0060] Figure 17 Shown are free latent myostatin levels in mice treated with Abl09, Abl33, and Abl41 compared to a control mIgG antibody. Data for a single animal are shown in each graph.

[0061] 18A to 18B The body weight changes of mice treated with Ab109 and semaglutide ( Figure 18A ) and percentage of weight change ( Figure 18B The upper graph in each figure shows the efficacy of semaglutide at a dose of 0.04 mg / kg; the lower graph in each figure shows the efficacy of semaglutide at a dose of 0.01 mg / kg.

[0062] Figures 19A to 19B Shown are changes in fat mass in mice treated with Ab109 and semaglutide. Figure 19A The efficacy of 0.04 mg / kg semaglutide was demonstrated. Figure 19B The upper panel of each figure shows the absolute change in fat mass; the lower panel of each figure shows the percentage change in fat mass. Figure 19C The percentage changes in fat mass were compared between the semaglutide 0.04 mg / kg group and the semaglutide 0.01 mg / kg group.

[0063] FIG. 20A to FIG. 20B Shown are the changes in lean body mass and the percentage changes in lean body mass in mice treated with Ab109 and semaglutide. Figure 20A The efficacy of 0.04 mg / kg semaglutide was demonstrated. Figure 20B The efficacy of 0.01 mg / kg semaglutide was shown. Figure 20C The percentage changes in lean body mass were compared between the semaglutide 0.04 mg / kg group and the semaglutide 0.01 mg / kg group.

[0064] Figures 21A to 21B The effects of Ab109 and semaglutide treatment on the weight of certain muscle tissues are shown. Figure 21A Shown are the relative weight (left) and percent weight change (right) of the quadriceps muscles. Figure 21B Shown are the relative weight (left) and percentage change in weight (right) of the gastrocnemius muscle.

[0065] FIG. 22A to FIG. 22B The effect of Ab109 and semaglutide treatment on the weight of certain adipose tissues is shown. Figure 22A Shown are the relative weight (left) and percent weight change (right) of the perigonadal fat pad. Figure 22B Shown are the relative weight (left) and percent weight change (right) of the inguinal fat pad.

[0066] Figures 23A to 23CShown are the averages of negative stain electron microscopy 2D levels for each of the following: Ab2:promyostatin ( Figure 23A ), 1:2 input sample 1:1 complex Ab109: promyostatin ( Figure 23B ), and 1:1 complexed Ab133:promyostatin ( Figure 23C ).

[0067] Figure 24 Shown are mean serum concentrations of Ab109 in female cynomolgus monkeys up to 28 days after dosing. DETAILED DESCRIPTION

[0068] The present disclosure encompasses novel antibodies and antigen-binding fragments thereof that selectively bind to promyostatin / latent myostatin with high affinity, and these novel antibodies and antigen-binding fragments thereof are capable of inhibiting protease-dependent activation of myostatin with high potency (preferably as measured by a functional ELISA, e.g., an IC50 of less than 1 nM, e.g., an IC50 of less than 0.5 nM). Such antibodies and antigen-binding fragments thereof specifically bind to promyostatin / 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 promyostatin / latent myostatin complex, e.g., a region similar or identical to the region bound by Ab2, thereby conferring robust inhibitory efficacy. However, unlike the aforementioned inhibitors, in certain embodiments, the antibodies / fragments disclosed herein are capable of binding to an antigen (i.e., promyostatin / latent myostatin) with high monovalent affinity without compromising inhibitory efficacy. These features provide opportunities for engineering constructs such as bispecific antibodies comprising a first arm of an antibody that selectively binds to promyostatin / latent myostatin and inhibits its activation and a second arm of an antibody 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.

[0069] In some embodiments, the present disclosure also encompasses methods of treating or preventing conditions associated with myostatin dysregulation using a myostatin inhibitor disclosed herein (e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro-myostatin / latent myostatin and blocks myostatin activation) in an amount effective to treat or prevent such conditions. In some embodiments, the present disclosure provides a method for treating or preventing metabolic disorders (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-myostatin / latent myostatin and blocks myostatin activation. In some embodiments, the present disclosure encompasses the use of such an antibody or antigen-binding fragment as a monotherapy or in combination with at least one other therapy for treating or preventing 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, such as muscle disorders, such as various types of malnutrition, spinal cord injury, or spinal muscular atrophy.

[0070] The present disclosure provides antibodies and antigen-binding fragments that can selectively inhibit myostatin, and this selective inhibition is achieved by binding to promyostatin / latent myostatin with high potency and specificity. Such highly potent antibodies and antigen-binding fragments that specifically bind to promyostatin / latent myostatin can not only effectively treat conditions associated with myostatin signaling, but also provide an improved treatment profile (including enhanced safety and improved tolerability) and / or promote ease of administration (e.g., at concentrations suitable for subcutaneous administration). Myostatin antagonists described elsewhere have been observed to lack specificity and may pose a greater risk to certain patient populations due to off-target effects. For example, because mature myostatin proteins have high homology to other members of the TGFβ superfamily (e.g., activin A or GDF11), myostatin inhibitors that also bind to mature myostatin can block other biological pathways in addition to the myostatin signaling pathway. Thus, such off-target effects can potentially limit the patient population that can safely undergo treatment due to unacceptable side 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 wound healing and reproductive biology, and inhibition of activin A would therefore limit use in patients who have recently undergone surgery or injury or in women of childbearing age. Such increased risk of side effects or toxicity can be particularly concerning where i) the patient population requires long-term treatment (such as a chronic condition); and / or ii) the patient population is or includes pediatric patients who may be susceptible to such side effects and / or toxicity. Thus, the present disclosure provides improved myostatin inhibitors that specifically target promyostatin / latent myostatin with high potency, thereby providing a potentially greater safety profile.

[0071] In addition, the antibodies or antigen-binding fragments thereof disclosed herein may provide other surprising improvements compared to antibodies known in the art. In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein may provide one or more (e.g., all) of the following effects: increased affinity to facilitate reduced administration concentrations, increased myostatin binding stoichiometry, increased serum myostatin clearance, reduced circulating latent myostatin levels, and higher antigen binding pH sensitivity (i.e., binding to promyostatin / latent myostatin with greater affinity at physiological pH compared to acidic pH), preventing muscle atrophy and preserving 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% bioavailability or greater, compared to intravenous administration, as measured by serum exposure levels in animals (e.g., monkeys) receiving equivalent doses of the antibody administered via intravenous and subcutaneous routes.

[0072] definition

[0073] As used herein, the articles "a" and "an" refer to one or more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0074] Except in the operating examples or when otherwise indicated, all numbers used herein expressing the amount of ingredients or reaction conditions should be understood to be modified by the term "about" in all cases. When used in conjunction with percentages, the term "about" can mean ±1%. In addition, the term "about" can mean within ±1% of a numerical value.

[0075] Adjuvant therapy: The terms "adjuvant 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 an adjuvant therapy) is administered to a subject who is receiving, has received, or will receive treatment with a first agent (e.g., background therapy). The terms "in combination with" and "complementary to" are used interchangeably herein and refer to therapies that are used together, whether concurrently or partially overlapping in time.

[0076] Administration (Administer / Administration): The term "administer," "administering," or "administration" includes any method or act of delivering a pharmacological agent (e.g., a drug) to a predetermined subject (e.g., a patient). The pharmacological agent can be any suitable therapeutic agent, such as a biological 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 a modified analog thereof), or a low molecular weight agent (e.g., a structurally defined small molecule or chemical entity). Administration can be systemic or local. In some embodiments, administration can involve the administration of one or more agents concurrently, simultaneously, or sequentially.

[0077] Affinity: Affinity (or "binding affinity") is the binding force of a molecule (such as an antibody) to its ligand (such as an antigen). It is typically measured and reported in terms of the equilibrium dissociation constant (KD). In the context of antibody-antigen interactions, KD is the ratio of the antibody dissociation rate ("dissociation rate" or Koff) to the antibody association rate ("association rate" or Kon) of the antibody. Koff is how quickly the antibody dissociates from the antigen to which it binds, and Kon is how quickly the antibody binds to its antigen. For example, an antibody with an affinity ≤ 5 nM has a KD value of 5 nM or less (i.e., 5 nM or greater affinity) as determined by a suitable in vitro binding assay. Suitable in vitro binding assays can be used to measure the KD value of an antibody for its antigen. Suitable assays include, but are not limited to, assays based on biolayer interferometry (BLI) (such as ), surface plasmon resonance (SPR)-based assays (such as Biacore TM ), MesoScale Discovery (MSD) immunoassays (such as MSD-solution equilibrium titration or MSD-SET). In some embodiments, the BLI-based assay (such as ) to determine KD. In a preferred embodiment, the KD is determined by an SPR-based assay (such as Biacore TM )Determine KD.

[0078] Antibodies: 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 interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, namely CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, namely CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The antibodies provided herein include human antibodies and humanized antibodies.

[0079] Antigen-binding fragment: The terms "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 that retain the ability to specifically bind to an antigen (e.g., promyostatin / latent myostatin). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge located in 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 (Ward et al., (1989) Nature 341:544-546), consisting of the VH domain; (vi) isolated complementarity determining regions (CDRs); and (vii) fibronectin. In addition, although the two domains of the Fv fragment (VL and VH) are encoded by separate genes, they can be joined using recombinant methods through a synthetic linker that enables them to be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called 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 within the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and creating 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 of the novel antibodies disclosed herein that allows for single-arm binding to pro / latent myostatin.

[0080] Biolayer Interferometry (BLI): BLI is a label-free technique used to optically measure biomolecular interactions, such as the interaction between a ligand immobilized on the surface of a biosensor tip and an analyte in solution. BLI provides the ability to monitor binding specificity, association and dissociation rates, and / or concentration. BLI platform instruments are commercially available, for example, from Pall / ForteBio, and are commonly referred to as Unless explicitly specified otherwise, BLI-based assays were performed according to manufacturer's instructions (eg, binding was analyzed at ambient / room temperature, eg, at about 20°C to 25°C).

[0081] Body composition: The term "body composition" refers to the relative components that make up the body, including fat mass, muscle (lean body) mass, bone, and water, among others. In particular, in the context of weight management, body composition refers to the ratio of muscle mass to fat mass in the body. Unless expressly stated otherwise, body composition refers to the body composition of the entire body. 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), skin folds (using calipers), ultrasound, quantitative magnetic resonance (QMR), and measurement of circumference (e.g., at the waist).

[0082] Body Mass Index (BMI): The term "body mass index" or "BMI" is a numerical value derived from a person's mass and height and is defined as weight in kilograms divided by the square of height in meters (kg / m 2 BMI provides a general weight-to-height relationship that can be used to classify a person as underweight, normal weight, overweight, obese, or extremely obese based on tissue mass (muscle, fat, and bone) relative to height.

[0083] Combination therapy: As used herein, "combination therapy" refers to a treatment regimen involving the administration of two or more active agents (e.g., two or more pharmacological agents) to treat a predetermined indication and / or a condition associated therewith. The two or more agents can be formulated as separate compositions (e.g., formulations) or as a single composition (formulation). "Combination therapy" encompasses therapies that are used in conjunction with each other and that complement each other.

[0084] Competition: As used herein, the term "compete" or "block" with respect to antigen binding by an antibody or antigen binding fragment, refers 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 that results in a detectable reduction in the binding of the first antibody or antigen binding fragment to its epitope in the presence of the second antibody or antigen binding fragment, as compared to the binding of the first antibody or antigen binding fragment in the absence of the second antibody or antigen binding fragment. The alternative scenario where the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody may, but need not, be the case. That is, the first antibody may inhibit the binding of the second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its corresponding epitope. Competition between antibodies can be determined using any method known in the art, including techniques based on biolayer interferometry (BLI), such as ) or enzyme-linked immunosorbent assay (ELISA). In some embodiments, epitope binning experiments can be used to assess competitive binding between antibodies or antigen-binding fragments.

[0085] Cross-competition: As used herein, the term "cross-competition" or "cross-blocking" with respect to antigen binding by an antibody or antigen-binding fragment refers 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 that each antibody detectably inhibits the binding of the other antibody to its epitope or ligand, whether to the same, greater, or lesser extent. For example, if a first antibody measurably inhibits the binding of a second antibody to the antigen, then the first antibody cross-competes with the second antibody, and vice versa. This is different from the situation where a first antibody competes with a second antibody, but does not cross-compete, in which case the first antibody inhibits the second antibody from binding to the antigen, but the second antibody does not necessarily inhibit the first antibody from binding to the antigen. Cross-competition between antibodies can be determined using any method known in the art, including techniques based on biolayer interferometry (BLI), such as ) or enzyme-linked immunosorbent assay (ELISA). In some embodiments, epitope binning experiments can be used to assess competitive binding between antibodies or antigen-binding fragments.

[0086] Competition and cross-competition antibody are all within the scope of present disclosure.No matter how the mechanism of such competition or cross-competition occurs (for example, steric hindrance, conformational change or with common epitope or its part), those skilled in the art will understand that such competition and / or cross-competition antibody is contained and can be used for method and / or composition provided herein.In certain embodiments, competition or cross-blocking (cross-competition) are determined using the mensuration based on biolayer interferometry (BLI).In certain embodiments, the first antibody or Fab are fixed on biosensor and use premixed complex to determine the combination between the first antibody or Fab and the antigen, and this complex comprises the second antibody or Fab that is combined with antigen.In certain embodiments, the first antibody or Fab are fixed on biosensor, measure the progressive combination of antigen and the second antibody or Fab subsequently.

[0087] Reduce / reduce: As used herein, the term "reduce" or "reduce" in the context of disease symptoms refers to a statistically significant reduction in such levels. The reduction 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 reduction can also be, for example, about 1%-10%, 10%-20%, 1%-30%, 20%-50%, 30%-60%, 40%-70%, 50%-80%, or 60%-90%. In certain embodiments, the efficacy reduction level achievable by an individual with a disease is similar to that of an individual without such a disease or is within the normal range of efficacy for an individual without such a disease.

[0088] Dieting (diet) regimen: In the context of the present disclosure, certain diets may be incorporated as part of weight management (e.g., obesity treatment including pharmacological interventions). Dieting may include caloric restriction (i.e., reducing caloric intake or reducing caloric absorption) as well as changes in the types of food consumed (e.g., high-protein, lower-fat, and / or lower-carbohydrate regimens), and / or strictly controlled timing / schedules of food intake (e.g., intermittent fasting). Thus, when a patient incorporates a diet into an overall treatment regimen (e.g., as part of weight management) or is instructed by a physician or equivalent to incorporate a diet into an overall treatment regimen, the patient is receiving a "diet or calorie reduction regimen."

[0089] 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 elicit a desired biological or pharmaceutical effect in a tissue or subject. For example, in certain embodiments of the present disclosure, the intended purpose may be to inhibit the activation of myostatin in vivo, achieving a clinically meaningful outcome associated with myostatin inhibition. For any particular 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, on combination with other 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 specific agent being used; the specific composition being used; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and / or rate of excretion or metabolism of the specific agent being used; the duration of treatment; and factors well known in the medical field. In some embodiments, an effective amount can refer to an amount that, when administered according to a particular regimen, produces a positive physiological or clinical outcome with a reasonable, acceptable level of side effects (e.g., toxicity), resulting in side effects (if any) being tolerable enough to continue the experiment or tolerable enough for the patient to continue with the treatment regimen, and the therapeutic benefit outweighs the toxicity. One skilled in the art will appreciate that in some embodiments of the present disclosure, an administered amount may be considered an effective amount if it contains an amount suitable for administration and associated with a positive outcome.

[0090] Epitope: As used herein, the term "epitope" refers to the region of an antigen to which an antibody or fragment thereof binds. It includes any polypeptide determinant capable of specific binding to an antibody or fragment. In certain embodiments, epitope determinants include molecules classified by chemically active surfaces, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, they 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 when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. An epitope can be a linear epitope or a conformational epitope. Epitopes can be determined, for example, by crystallography of complexes of antigen and antibody. Antibodies are said to "bind to the same or similar epitope" if they cross-compete with each other.

[0091] Epitope binning: The term "epitope binning" (sometimes referred to as antibody binning or epitope mapping) refers to a method of sorting a group of monoclonal antibodies (e.g., a "library") that are raised against a target protein or protein complex (i.e., an antigen) based on competitive binding to a target. The antibodies in the library are tested in pairs to assess whether they block / cross-block the binding of another antibody to the antigen. Closely related binning profiles indicate that the antibodies have the same or closely related (e.g., overlapping) epitopes and are "binned" together. Since the biological activity (e.g., intervention; efficacy) achieved by an antibody binding to its target is likely to carry over to another antibody in the same bin, binning can provide applicable structure-function profiles for antibodies that share similar binding regions within the same antigen. Therefore, among antibodies within the same epitope bin, those with higher affinity (lower KD) typically have greater efficacy.

[0092] Exercise / exercise regimen: As used herein, the term "exercise" includes any physical activity. The term "exercise regimen" refers to a therapeutic regimen that incorporates physical activity as a component.

[0093] Fc variant: As used herein, an "Fc variant" of a reference antibody refers to an antibody that comprises one or more mutations within the Fc region as compared to the reference antibody. In some embodiments, the Fc variant antibody retains the same CDR sequences as the reference antibody. Fc variants can be generated that have altered (e.g., increased) affinity for an Fc receptor (FcR), such as the neonatal Fc receptor (FcRn). In some embodiments, antibodies that bind to FcRn with increased affinity can extend the serum half-life of the Fc variant compared to a reference antibody that does not have the Fc mutations.

[0094] GLP-1 analogs: As used herein, the term "GLP-1 analogs" or "incretin mimetics" refers to peptides or modified peptides that have structural similarity to naturally occurring GLP-1 and are capable of binding to and activating the GLP-1 receptor. GLP-1 analogs can be extensin-based therapies, DPP-IV resistance analogs. Non-limiting examples of GLP-1 analogs include albiglutide, beinaglutide, cotadutide, danuglipron, dulaglutide, exenatide, exenatide ER, liraglutide, lixisenatide, PEG-loxenatide, mazdutide, MEDI0382, noiiglutide, orforglipron, pemvidutide, PF-07081532, retatrutide, semaglutide, taspoglutide, telpotide, and XW003. GLP-1 analogs include human GLP-1 analogs conjugated to substances that slow renal excretion (e.g., fatty acids, albumin, α-aminoisobutyric acid, etc.); these may be acylated. GLP-1 analogs include peptides or modified peptides comprising the amino acid sequence EGTFTSD (SEQ ID NO: 116). GLP-1 analogs may also include peptides or modified peptides comprising the amino acid sequence HXXGXFTXD (SEQ ID NO: 117), where X is any amino acid residue.

[0095] GLP-1 receptor agonist: As used herein, the term "GLP-1 receptor agonist" or "GLP-1R agonist" or "GLP-1RA" refers to an agent that is capable of binding to and activating the GLP-1 receptor. GLP-1 is a naturally occurring GLP-1 receptor agonist. GLP-1 receptor agonists encompass GLP-1 analogs. The GLP-1 receptor agonist can be a small molecule GLP-1 receptor agonist. In some embodiments, the GLP-1 receptor agonist is a long-acting small molecule GLP-1 receptor agonist. In some embodiments, the GLP-1 receptor agonist is a GLP-1 analog.

[0096] GLP-1 pathway activator: The terms "GLP-1 pathway activator" and "activator of the GLP-1 signaling pathway" are used interchangeably herein and encompass any agent that increases or enhances the activity of the GLP-1 signaling pathway, regardless of the mechanism of action. Increased or enhanced activity of the GLP-1 signaling pathway may be the result of, for example, a greater degree of activity, a longer duration of activity, increased availability of one or more components of the signaling pathway, etc. In some embodiments, GLP-1 pathway activators include agents that modulate upstream regulatory elements 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 GLP-1 production or secretion; GLP-1 stabilizers). In some embodiments, GLP-1 pathway activators include agents that increase activation of the GLP-1 receptor (GLP-1R). In some embodiments, agents that increase GLP-1R activation include GLP-1 agonists, including GLP-1 analogs. In some embodiments, GLP-1 pathway activators include agents that activate GLP-1R downstream signaling (e.g., activators of PI3K, PKC, cAMP, etc.). In some embodiments, GLP-1 pathway activators may include agents that modulate receptor GLP-1R expression and / or migration (e.g., GLP-1R internalization inhibitors; see, e.g., Jones et al., Nat. Comm. (2018) 9:1602). GLP-1 pathway activators encompass activators of GLP-1R. In preferred embodiments, 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 binders and multifunctional molecules comprising GLP-1 analogs), peptides, GLP-1 gene therapy, and small molecules.

[0097] Human antibody: As used herein, the term "human antibody" refers to antibodies whose variable and constant regions are derived from human germline immunoglobulin sequences and fragments thereof.

[0098] 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.

[0099] Inhibit or inhibition of: As used herein, the term "inhibit" or "inhibition of" means to reduce by a measurable amount, and may include but does not require complete prevention or inhibition.

[0100] Insulin sensitivity; insulin resistance: The term "insulin sensitivity" refers to the metabolic effects of insulin on glucose disposal in a subject. A subject is said to have increased sensitivity to insulin if it requires a smaller amount of insulin to lower blood glucose levels than the average for the human population. Conversely, a subject is said to have decreased sensitivity to insulin if it requires a higher amount of insulin to lower blood glucose levels. A subject is said to have "insulin resistance" if the amount of exogenous or endogenous insulin required to increase glucose uptake and utilization is significantly higher than that of a healthy subject. For example, a subject is said to have "insulin resistance" if the amount of exogenous or endogenous insulin required to increase glucose uptake and utilization is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more compared to a healthy subject.

[0101] Circulating latent myostatin: As used herein, the phrase "circulating latent myostatin" or "circulating latent myostatin" refers to latent myostatin in blood, plasma, or serum.

[0102] Lean / Lean Body Mass: As used herein, "lean" mass or tissue refers to muscle mass or muscle tissue, as opposed to fat mass or adipose tissue (eg, fat).

[0103] Mature myostatin: The term "mature myostatin" refers to a dimeric growth factor, also known as GDF8, and is released by the latent myostatin complex. Mature myostatin is a soluble and biologically active ligand that binds to and activates its receptor. Unless otherwise expressly stated, the term "mature myostatin" refers to a fully processed, biologically active form of myostatin, or a full-length mature myostatin fragment that retains biological activity. The wild-type sequence (SEQ ID NO: 134) of the mature myostatin polypeptide sequence (i.e., single chain) is provided below. In some cases, mature myostatin may contain one or more mutations that may exhibit altered structure / function or stability.

[0104] DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS (SEQ ID NO: 134).

[0105] Metabolic disorder: The term "metabolic disorder" is used interchangeably with the term "metabolic disease" or "metabolic condition" and encompasses any condition involving dysregulation of the body's metabolic functions, resulting in a disturbance in the normal physiological homeostasis state due to changes in metabolism (anabolism and / or catabolic). Metabolic disorders may be inherited or acquired. Non-limiting examples of metabolic disorders include obesity or being overweight, type 2 diabetes, type 2 diabetes associated with obesity, and metabolic syndrome.

[0106] Metabolic rate: The term "metabolic rate" refers to the amount of energy expended in 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.

[0107] Metabolism: The term "metabolism" refers to the processes involved in the biosynthesis and breakdown of components that make up the body, such as fat (e.g., adipose tissue), muscle, and bone. Thus, "fat metabolism" means the biosynthesis and breakdown of fat.

[0108] Myostatin: In the context of the present disclosure, unless explicitly defined otherwise, the term "myostatin" may refer to any form of myostatin, such as promyostatin, latent myostatin and mature myostatin, each of which exists as a dimer in vivo.

[0109] 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 form, such as large molecules (biologics, such as antibodies and engineered protein constructs) and small molecules (e.g., structurally defined low molecular weight chemical entities). The term myostatin inhibitor encompasses both selective inhibitors of myostatin and non-selective inhibitors of myostatin. The myostatin inhibitor may be an anti-myostatin antibody or antigen-binding fragment thereof that binds to promyostatin and / or latent myostatin and / or mature myostatin. In some embodiments, the myostatin inhibitor may be an anti-promyostatin / latent myostatin antibody or antigen-binding fragment thereof that preferentially (e.g., selectively) binds to promyostatin and / or latent myostatin relative to mature myostatin. In various embodiments, the myostatin inhibitor can be an antibody (e.g., a neutralizing antibody), an activation inhibitor (e.g., an antibody that inhibits activation of pro-myostatin and / or latent myostatin), fibronectin, 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 is a gene therapy.

[0110] Myostatin selective inhibitor: The term "myostatin selective inhibitor" can be used interchangeably with "selective myostatin inhibitor" and refers to a myostatin inhibitor that inhibits myostatin but does not inhibit other members of the TGFβ superfamily (e.g., GDF11 or activin A). In some embodiments, the myostatin selective inhibitor inhibits at least one activity of myostatin signaling (e.g., inhibits myostatin activation and / or inhibits or prevents subsequent downstream signaling of myostatin) with a potency (e.g., affinity) for myostatin that is at least 100-fold, 200-fold, 500-fold, 1,000-fold, or greater than that for 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 a functional ELISA). In preferred embodiments, the myostatin selective inhibitor does not exhibit 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, myostatin selective inhibitors are antibodies that bind to promyostatin / latent myostatin and inhibit the activation step of myostatin. In some embodiments, myostatin selective inhibitors are antibodies or antigen-binding fragments provided herein (e.g., any one of Abl01-Ab141). In some embodiments, myostatin selective inhibitors are antibodies or antigen-binding fragments thereof comprising all six CDRs of any one of Abl01-Ab141 (e.g., a group of SEQ ID NOs identified for the specific antibodies in Table 2d-Table 2f). In some embodiments, myostatin selective inhibitors are antibodies or antigen-binding fragments thereof comprising the heavy and light chain variable domains of any one of Abl01-Ab141 (e.g., a pair of SEQ ID NOs identified for the specific antibodies in Table 3). In some embodiments, myostatin selective inhibitors are antibodies or antigen-binding fragments thereof comprising the heavy and light chain variable domains of any one of Abl01-Ab141 (e.g., a pair of SEQ ID NOs identified for the specific antibodies in Table 4). In some embodiments, the aforementioned antibody sequences are those of Abl09, Abl33, or Abl41, or antigen-binding fragments thereof.

[0111] Overweight / Obesity: A person is described as overweight or obese if their weight is above the normal weight adjusted for height. Using BMI-based classifications, for human adults (20 years and older), a BMI of 18.5 to 24.9 is considered normal weight; a BMI of 25 to 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 (2-19 years), a BMI at or above the 85th percentile on the CDC growth charts is considered overweight or obese; a BMI at or above the 95th percentile on the CDC growth charts is considered obese (including extreme obesity); and a BMI at or above 120% of the 95th percentile on the CDC growth charts is considered extremely obese.

[0112] Percent identity: As used herein, the term "percent identity" refers to the degree of similarity between two amino acid sequences or between 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 full 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 has been incorporated into the BLASTN and BLASTX programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. BLAST protein alignments can be performed using the BLASTX program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecule of interest. In the event that there is a gap between the 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 each program (e.g., XBLAST and NBLAST) can be used. In embodiments where a cumulative or total percent identity of sequences is desired, the Needle algorithm in the European Molecular Biology Open Software Suite ("EMBOSS") can be used to determine percent identity.

[0113] Efficacy: As used herein, the term "efficacy" refers to the activity of a drug in terms of the concentration or amount of the drug that produces a defined effect, such as an antibody (or its antigen-binding fragment) with inhibitory activity. For example, an antibody that is capable of producing a certain effect at a specified dose is more potent than another antibody that requires twice the amount (dose) required to produce the same effect. Efficacy can be measured using any suitable functional assay, such as functional ELISA and cell-based assays, in which the degree of myostatin activation, such as activation triggered by a protease (e.g., mTLL2), can be measured in the presence or absence of a test substance (e.g., an inhibitory antibody).

[0114] Prevent / preventing: As used herein, the terms "preventing" and "prevent" 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.

[0115] Pro / latent myostatin: As used herein, the term "promyostatin / latent myostatin" refers to promyostatin, latent myostatin, or both (i.e., the prototype 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) comprising two promyostatin polypeptides. During biosynthesis, the N-terminal signal peptide is cleaved. Promyostatin homodimers are proteolytic substrates for intracellular furin, which is cleaved between the prodomain and the growth factor domain. The furin-cleaved homodimeric complex remains associated ("latent myostatin") until activation, which releases the growth factor from the latent complex. The human sequence of various promyostatin polypeptides is provided as SEQ ID NO: 52.

[0116] The terms "pro-myostatin," "promyostatin," or "promyostatin," also known as "proGDF8," refer to an inactive precursor of mature myostatin comprising a disulfide-linked homodimer, each molecule of which comprises an amino-terminal prodomain covalently bound to a carboxy-terminal mature myostatin domain. In one embodiment, "promyostatin" is not cleaved by proprotein convertases or proteases from the BMP / tolloid family. Exemplary promyostatin sequences, variants thereof, and methods of producing promyostatin are well known in the art and described in more detail herein. In the context of polypeptide sequences, the terms "human proGDF8" or "human promyostatin" refer to the amino acid sequence set forth in SEQ ID NO: 52, which reflects a single polypeptide chain.

[0117] As used herein, the term "latent myostatin or latent-myostatin" refers to an inactive precursor of mature myostatin that comprises a disulfide-linked homodimer, each molecule of which comprises an amino-terminal prodomain non-covalently bound to a carboxyl-terminal mature myostatin domain. In one embodiment, "latent myostatin" is produced from promyostatin that has been cleaved by a proprotein convertase but not by a protease from the BMP / tolloid family. In another embodiment, "latent myostatin" can be produced by combining the prodomain with the carboxyl-terminal mature myostatin domain in vitro and allowing it to fold correctly. See, e.g., Sengle et al. J. Biol. Chem., 286(7):5087-5099, 2011. Exemplary latent myostatin sequences, variants thereof, and methods of producing latent myostatin proteins are well known in the art and are described in more detail herein.

[0118] Pre-GDF8 (human):

[0119]

[0120] Pro-GDF8 (rat):

[0121]

[0122] Pre-GDF8 (mouse):

[0123] MSPINMLYFNGKEQIIYGKIPAMVVDRCGCS (SEQ ID NO: 54).

[0124] Pre-GDF8 (Cynomolgus Monkey):

[0125]

[0126] Exemplary pre-GDF8 sequences in humans, rats, mice, and cynomolgus monkeys are provided above. In these pre-GDF8 sequences, the proprotein convertase cleavage site is indicated in bold, while the tolloid protease site is underlined. In some embodiments, the proprotein convertase cleavage site comprises amino acid residues 240 to 243 of SEQ ID NOs: 52-55. In some embodiments, the tolloid protease site comprises amino acid residues 74-75 of SEQ ID NOs: 52-55. It should be understood that the exemplary pre-GDF8 sequences provided herein are not intended to be limiting, and additional pre-GDF8 sequences from other species (including any isoforms thereof) are within the scope of this disclosure.

[0127] As previously described, the prodomain of the myostatin polypeptide comprises several domains (see, e.g., PCT / US2014 / 036933). These include, for example, the Straight Jacket region, the Fastner region, the Arm region, the Fingers region 1, the Fingers region 2, the Latency loop, the α-1 helical region, and the 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 comprises at least one amino acid residue from the "KALDEN" (SEQ ID NO: 118) polypeptide segment within the Arm region of the prodomain. In some embodiments, the amino acid residue within the Arm region of the prodomain that contacts the antibody upon antigen binding is a residue that is not conserved between myostatin and GDF11. In some embodiments, such residues are K, E, and / or N (shown in bold above) of the polypeptide segment. In some embodiments, the epitope includes at least one amino acid residue from the "FVQILRLIKPMKDGTRYTGIRSLK" (SEQ ID NO: 57) polypeptide segment within the Arm region of the prodomain. In some embodiments, such residues are F, Q, L, Y, R, S, and / or K of the polypeptide segment (shown in bold above). See Dagbay et al. (J. Biol. Chem. (2020) 295(16): 5404-5418), the contents of which are hereby incorporated in their entirety.

[0128] Serum clearance: As used herein, the term "serum clearance" or "clearance" refers to the relative pharmacokinetic / pharmacodynamic profile of the serum concentration (e.g., circulating levels) of an analyte (e.g., a target protein or protein complex) over time. When the analyte being measured accumulates in the serum, it is said to be slowly cleared. In contrast, when the analyte being measured is rapidly removed ("cleared") from the serum, it is said to be rapidly cleared. In vivo serum clearance can occur via a variety of mechanisms, including, for example, targeted degradation, Fc-mediated internalization, and the like. For example, serum clearance of circulating myostatin can be measured by an assay for measuring antibody binding to free myostatin in serum (circulating myostatin not bound by the antibody). Such an assay can include immobilizing a biotinylated capture antibody known to bind to pro- and latent myostatin, followed by addition of a sample containing myostatin to test the antibody's ability to bind to free myostatin, and adding a detection antibody with a detectable marker. The capture antibody known to bind myostatin can be a biotinylated antibody capable of binding to latent myostatin, such as biotinylated Ab2 or a biotinylated antibody of the disclosure (e.g., biotinylated Abl09, Abl33, or Abl41), and the detection antibody can be a ruthenium-labeled antibody known to bind to both latent myostatin and mature myostatin.

[0129] Slow-twitch muscle: As used herein, the term "slow-twitch" muscle, "slow-twitch type 1" muscle or "type I" muscle refers to muscles rich in type I muscle fibers and frequently used, more posturally, and helps achieve long-endurance feats, such as long-distance running. As used herein, the term "fast-twitch" muscle, "fast-twitch type 2" muscle or "type II" muscle refers to muscles that provide higher energy output and intensity and are used in powerful bursts of movement (such as sprinting), but such muscles fatigue faster and cannot be used repeatedly. Fast-twitch muscles are divided into two types of fiber types: moderate fast-twitch fibers (type IIA) and fast-twitch fibers (type IIB or IIx). Moderate fast-twitch fibers are thicker than slow-twitch fibers, contract faster and wear out faster. When the body approaches maximum force, the most powerful and least endurable fast-twitch fibers are activated. Although most muscles tend to contain a mixture of multiple fiber types, different muscles contain different ratios of fiber types. During development or in response to certain events (e.g., exercise, disease, injury, etc.), fiber types within a muscle or muscle group can undergo fiber type switching, resulting in phenotypic changes in muscle physiology.

[0130] Solution Equilibrium Titration (SET): SET is an assay by which the binding between two molecules in solution at equilibrium (such as an antigen and an antibody that binds to the antigen) can be measured. For example, Meso-Scale Discovery ("MSD")-based SET or MSD-SET is a format for determining the dissociation constant of protein-protein interactions at equilibrium with particularly high affinity, such as picomolar affinity binding of an antibody to its antigen (see, e.g., Ducata et al. (2015) J Biomolecular Screening 20(10):1256-1267). SET-based assays are particularly useful for determining KD values ​​for antibodies with subnanomolar (e.g., picomolar) affinities.

[0131] Specific / Specificity: The terms "specific" or "specificity" as used in the context of an interaction between members of a specific binding pair (e.g., a ligand and a binding site, an antibody and an antigen, biotin and avidin) refer to the selective reactivity of the interaction. In the context of antibodies, the phrase "specifically binds to" and similar phrases refer to the ability of the antibody (or an antigenically reactive fragment thereof) to bind (i.e., "specifically binds") to a predetermined target antigen (or fragment thereof), as opposed to other entities. Specific binding is understood to be preferential binding to an antigen, epitope, receptor ligand, or binding partner, for example, at least 100-fold, 200-fold, 500-fold, or 1,000-fold relative to a control nonspecific antigen, epitope, receptor ligand, or binding partner. As used herein, "specific binding" may also refer to a specific binding affinity based on binding kinetics (e.g., K on , K off , and K D For example, if the ligand has 10 -3 sec -1 or smaller, 10 -4 sec -1 or smaller, 10 -5 sec -1 or smaller, or 10 -6 sec -1 or smaller K off and / or 10 -8 M or smaller, 10 -9 M or smaller, 10 -10 M or smaller, or 10 -11 M or smaller, or 10 -12 A KD of M or less, e.g., as determined by a suitable in vitro binding assay (such as BLI (e.g. ), surface plasmon resonance (SPR) (e.g. Biacore TM) and ELISA), it can be understood as specifically binding to its target. It should be 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 more than one ligand but can still be considered specific based on binding preference compared to a nonspecific antigen and / or having certain binding kinetic parameters. Methods for selecting appropriate nonspecific controls are within the capabilities of those skilled in the art. Binding assays are typically performed under physiological conditions.

[0132] Stoichiometry: As used herein, the term "stoichiometry" or "binding stoichiometry" refers to the configuration (e.g., the total mass of the complex comprising the components in a certain ratio) of an antibody (or antigen-binding fragment) and its antigen whereby they interact under predetermined conditions. The binding stoichiometry between an antibody ("Ab") and an antigen ("Ag") can be determined using, for example, intact immunoglobulins such as monoclonal antibodies ("mAb") or fragments such as Fab (monovalent) and F(ab')2 (divalent). In the context of the present disclosure, the antigen is the promyostatin / latent myostatin complex, a homodimer containing two binding sites for each antigen (one on each monomer). For example, a mAb (such as Ab2) can bind to Ag in a 1:1 Ab:Ag configuration, resulting in the first arm of the mAb interacting with the first binding site on Ag, and the second arm of the mAb interacting with the second binding site on Ag (Dagbay et al. (J. Biol. Chem. [Molecular Biochemistry] (2020) 295(16): 5404-5418)). In contrast, on average, two antibody molecules can simultaneously interact with one antigen molecule in a 2:1 Ab:Ag configuration. Similarly, on average, one antibody molecule can simultaneously interact with two antigen molecules in a 1:2 Ab:Ag configuration. In some embodiments, in the context of protein complex formation (e.g., protein-protein interaction) such as an immune complex (e.g., an antibody-antigen complex), the concept of stoichiometry considers the ratio of the components that form the complex to the total mass of the complex. For example, when mAb and Ag are mixed in solution at a total protein concentration of about 3.5 to 8.0 mg / mL, mAb and antigen (promyostatin / latent myostatin complex) can form an immune complex comprising 1 mAb molecule and 1 antigen molecule; 1 mAb molecule and 2 antigen molecules; 2 mAb molecules and 2 antigen molecules, or a mixture thereof. The stoichiometry can be measured by analytical SEC-MALS. For example, mAb and Ag can be present in a 1: 1, 2: 1, or 3: 1 ratio (e.g., as a mAb: Ag mixture) or vice versa, wherein the total protein concentration is between about 3.5 mg / mL (e.g., about 15 μM mAb and Ag each) and 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 duration (such as 1 to 48 hours, preferably about 24 hours).

[0133] Subject: As used herein, the term "subject" is a subject to whom one or more of the therapies described herein is administered. In a clinical setting, the terms "subject" and "patient" are used interchangeably. In some embodiments, the subject is a mammalian subject, such as a companion animal (e.g., dog, cat, etc.), farm animal (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.

[0134] Surface Plasmon Resonance (SPR): Surface plasmon resonance is an optical phenomenon that enables real-time detection of unlabeled interactors. SPR-based biosensors, such as those available from Biacore TM Biosensors based on the present invention can be used to measure biomolecular interactions, including protein-protein interactions, such as antigen-antibody binding. This technology is widely known in the art and is applicable to the determination of parameters such as binding affinity, kinetic rate constants and thermodynamics.

[0135] 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 fat cells, such as white fat, brown fat, and beige fat, and includes fat storage in different parts of the body, such as essential fat, subcutaneous fat, and visceral fat. Total fat mass can be measured or estimated by any method known in the art, including by measuring skin folds with calipers, measuring the circumference of certain body parts, dual-energy X-ray absorptiometry (DXA), static pressure weighing, air displacement plethysmography, bioelectrical impedance analysis, bioimpedance spectroscopy, electrical impedance myography, three-dimensional body scanners, multi-chamber models, or magnetic resonance imaging. The term "increase in fat mass" or "decrease in fat mass" refers to the change in measured fat mass compared to a baseline measurement. For example, a subject with a metabolic disorder may exhibit a loss of fat mass or a loss of visceral fat mass after treatment for 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 adipose tissue composed primarily of fat located deep within the abdominal organs, for example, in the abdominal region of a subject's body and around a subject's major organs, such as the liver, kidneys, pancreas, intestines, and heart.

[0136] Treat or prevent: The terms "treating," "treat," and "treatment" are used interchangeably herein. The term "treating" of a condition or disease of a subject refers to the act of providing a therapeutic regimen that is intended to cure, heal, alleviate, relieve, alter, remedy, delay the progression of, alleviate, improve, or affect the medical condition or at least one symptom of the condition, including slowing or delaying its progression. Thus, the term treatment does not necessarily require a complete cure of the disease or condition. In one embodiment, treating a subject alleviates the symptoms of the disease or condition by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0137] Weight loss: Weight loss refers to a decrease in body weight, regardless of the loss of one or more specific tissues. For example, weight loss alone does not distinguish between a loss of fat mass and a loss of muscle mass. An overall decrease in total body weight does not necessarily reflect an improvement in body composition.

[0138] Weight management: As used herein, the term "weight management" encompasses measures taken to lose weight, maintain weight, and reduce adipose tissue, increase lean body mass, or otherwise improve or maintain body composition. Clinically meaningful successful weight management may or may not be accompanied by overall weight loss. Thus, weight management may include a diet (e.g., a calorie-restricted diet, such as reducing calorie intake or reducing calorie absorption), an exercise regimen, and / or drug therapy (e.g., treatment comprising a myostatin inhibitor) to reduce the amount of total body weight, reduce the amount of total fat mass, reduce the amount of visceral fat mass, increase metabolic rate, increase the amount of lean body mass, and / or increase the ratio of muscle to fat, or otherwise improve body composition in a subject.

[0139] Weight-related conditions: As used herein, the term "weight-related condition" or "weight-related problem" refers to one or more medical conditions associated with excess fat mass (i.e., other than being overweight or obese), where the subject's excess 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.

[0140] General structural features of novel antibodies and their antigen-binding fragments

[0141] The present disclosure provides a novel class of antibodies capable of inhibiting myostatin activation. Such antibodies bind to the promyostatin / latent myostatin complex, but do not bind to free mature myostatin that is not associated with 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 one or more residues of KALDEN (SEQ ID NO: 118) (amino acid residues 205-210 of human promyostatin). This is an epitope previously identified to confer inhibitory activity to protease-induced myostatin activation, such as in a previously identified myostatin inhibitor (Ab2). 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).

[0142] When the VH and VL sequences are combined, while retaining the general binding region, in some embodiments, the novel antibodies and antigen-binding fragments disclosed herein share no more than 70% sequence identity with Ab2. Notably, 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 in Ab2.

[0143] In some embodiments, the antibodies and fragments disclosed herein share no more than 70% 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 sequence identity (preferably no more than 20%) with the L-CDR1 of Ab2. In some embodiments, the L-CDR2 of the antibody shares less than 30% sequence identity with the L-CDR2 of Ab2. In some embodiments, the L-CDR3 of the antibody shares no more than 20% sequence identity (preferably no more than 10%) with the L-CDR3 of Ab2. Preferred antibodies or fragments according to the present disclosure are fully human antibodies / fragments. In various embodiments, the preferred antibodies and fragments disclosed herein exhibit at least the characteristics of category 1 in Table 1 below. In some embodiments, preferred antibodies and fragments exhibit characteristics in category 1 and at least the characteristics of another category in Table 1 below. In some embodiments, preferred antibodies and fragments exhibit characteristics in category 1 and at least the characteristics of the other two categories in Table 1 below. In some embodiments, preferred antibodies and fragments exhibit properties in Category 1 and exhibit properties of at least three additional categories in Table 1 below. In some embodiments, preferred antibodies and fragments exhibit properties in Category 1 and exhibit properties of at least four additional categories in Table 1 below. In some embodiments, preferred antibodies and fragments exhibit properties in Category 1 and exhibit properties of at least five additional categories in Table 1 below. In some embodiments, preferred antibodies and fragments exhibit properties of all categories in Table 1 below.

[0144] Table 1. Antibody characteristics and characterization.

[0145]

[0146]

[0147]

[0148] Characterization of novel antibodies and antigen-binding fragments

[0149] A. Binding Selectivity

[0150] When determining binding selectivity, any suitable in vitro binding assay technique, such as BLI (e.g. ), SPR (such as Biacore TM) or ELISA, can be used to measure antibody-antigen interactions. Typically, recombinantly expressed and purified proteins are used as antigens for binding assays (see, e.g., PCT / US2014 / 036933). As demonstrated herein, when the growth factor is not associated with the prodomain, the novel antibodies and antigen-binding fragments disclosed herein selectively target promyostatin and / or latent myostatin dimer complex, but do not bind to free mature myostatin. Binding to proGDF11, proactivin A, proactivin B, mature GDF11, mature activin A, or mature activin B is not detectable. In some embodiments, the antibody or antigen-binding fragment selectively binds promyostatin and latent myostatin, but does not bind to mature GDF11, as measured by ELISA.

[0151] In preferred embodiments, selective binding to pro-myostatin / latent myostatin over mature myostatin (such as provided by any of Ab 101-141) can preemptively prevent activation, whereas antibodies that bind to mature myostatin only exert their effects after the activation event and / or may exhibit more off-target binding.

[0152] In some embodiments, preferred antibodies exhibit species cross-reactivity with human, cynomolgus monkey, rat and / or mouse promyostatin / latent myostatin with similar binding characteristics. Most preferably, such antibodies exhibit species cross-reactivity with human, cynomolgus monkey and mouse promyostatin / latent myostatin with similar binding characteristics.

[0153] B. Binding region, epitope

[0154] In certain embodiments, the present disclosure encompasses antibodies or antigen-binding fragments thereof that bind to a region of the prodomain of the promyostatin / 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, as numbered according to SEQ ID NO:52) and / or one or more amino acid residues of KALDEN (SEQ ID NO:118) (amino acid positions 205-210 of human promyostatin, as numbered according to SEQ ID NO:52).

[0155] In certain embodiments, the binding to the above-mentioned one or more residues is determined by assessing the cross-blocking that occurs with an antibody (e.g., Ab2) known to be bound to the epitope. A cross-blocking antibody pair indicates that there is a substantially overlapping binding region between the two antibodies. In certain embodiments, epitope binning can be performed to determine whether the two antibodies cross-block each other. In certain embodiments, premix binning can be used to determine epitope binning, wherein the first antibody or Fab is fixed on a biosensor and the binding between the first antibody or Fab is determined using a premixed complex comprising a second antibody or Fab that is bound to the antigen. In certain embodiments, epitope binning can be determined using sandwich binning, wherein the first antibody or Fab is fixed on a biosensor, and the progressive binding of the antigen to the second antibody or Fab is subsequently measured. In certain embodiments, the cross-blocking antibody of the present disclosure is bound to the above-mentioned one or more amino acid residues.

[0156] C. Binding affinity

[0157] In some embodiments, the SPR (e.g., Biacore TM ), by measuring the equilibrium dissociation constant by solution equilibrium titration (SET), or by using BLI-based assays (e.g. ) assay, the anti-promyostatin / latent myostatin antibodies or antigen-binding fragments thereof suitable for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments thereof that bind to promyostatin / latent myostatin with high affinity. In some embodiments, the KD is determined by a BLI-based assay (e.g., ) to determine binding affinity. In some embodiments, binding affinity is determined by SET. In some embodiments, SET can be used to measure circulating myostatin levels (e.g., see Example 3). Preferably, the binding affinity is determined by SPR (e.g., Biacore TM ) Determine binding affinity.

[0158] In some embodiments, preferably as determined by an SPR-based in vitro binding assay (such as Biacore TM ), the antibodies or antigen-binding fragments provided herein have a value of less than 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 The equilibrium dissociation constant (K D ) binds to pro-myostatin / latent myostatin. In some embodiments, the antibody or antigen-binding fragment binds to pro-myostatin / latent myostatin at nanomolar or subnanomolar concentrations. DBinding to promyostatin / latent myostatin. For example, an anti-promyostatin / latent myostatin antibody or antigen-binding fragment thereof can bind to promyostatin / latent myostatin with an affinity between 5 pM and 500 nM (e.g., between 50 pM and 100 nM, e.g., between 500 pM and 50 nM, e.g., between 50 pM and 5 nM, e.g., between 0.5 nM and 2 nM). In some embodiments, the disclosure encompasses antibodies or antigen-binding fragments that compete or cross-compete with any of the antibodies described herein for binding to promyostatin / 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 preferred embodiments, as determined by an in vitro binding assay based on SPR (such as Biacore TM ), the antibody binds pro-myostatin or latent myostatin with a KD of less than 1.0 nM, as measured by PCR.

[0159] In some embodiments, preferably as determined by an SPR-based in vitro binding assay (such as Biacore TM ), the antibodies or antigen-binding fragments thereof provided herein are measured at 10 -11 M to 10 -8 K in the M range D Binds to human promyostatin / latent myostatin. In some embodiments, the antibody or antigen-binding fragment thereof is a protein with a K of less than 5 nM. D In some embodiments, the antibody or antigen-binding fragment thereof binds to pro-myostatin / latent myostatin with a K of less than 1 nM. D Binds to promyostatin / latent myostatin. In some embodiments, the antibody or antigen-binding fragment thereof binds to promyostatin / latent myostatin with a KD of less than 0.5 nM. In some embodiments, the antibody or antigen-binding fragment thereof binds to promyostatin / latent myostatin with a KD of less than 0.1 nM. In some embodiments, the antibody or antigen-binding fragment thereof binds to promyostatin / latent myostatin with a KD of at least 10-fold as compared to Ab2 disclosed in PCT / US2015 / 059468. D Binds to promyostatin / latent myostatin. In some embodiments, affinity or binding kinetics are determined by a BLI-based in vitro binding assay. or Biacore TMWhen measuring such binding profiles, the assay is performed according to the manufacturer's instructions unless otherwise stated. In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment provided herein that binds to promyostatin / latent myostatin with a KD of less than 1 nM, e.g., selected from Abl01, Abl02, Abl03, Abl04, Abl05, Abl06, Abl07, Abl08, Abl09, Abl21, Abl23, Abl25, Abl27, Abl28, Abl33, Abl34, Abl35, Abl36, Abl37, Abl38, Abl39, Abl40, and Abl41. In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment provided herein that binds to promyostatin / latent myostatin with a KD of less than 0.7 nM (e.g., less than 0.6 nM) (e.g., selected from Abl02, Abl05, Abl09, Abl30, Abl31, Abl33, Abl38, Abl39, and Abl40). In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment provided herein that binds to promyostatin / latent myostatin with a KD of less than 0.5 nM, e.g., selected from Abl02, Abl05, Abl09, Abl30, Abl31, Abl33, Abl38, Abl39, and Abl40. In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment provided herein that binds to promyostatin / latent myostatin with a KD of less than 0.2 nM, e.g., Abl09, Abl33, Abl38, Abl39, or Abl40. In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment provided herein that binds to promyostatin / latent myostatin with a KD of less than 0.1 nM, e.g., Abl33.

[0160] In some embodiments, the binding of proteins to the target gene is determined by an in vitro SPR-based binding assay (such as Biacore TM ), the novel antibodies or antigen-binding fragments thereof encompassed by the present disclosure bind to recombinant human promyostatin / latent myostatin with a bivalent KD of less than 1 nM (i.e., <1 nM) as measured according to the manufacturer's instructions (e.g., using the exemplary protocol set forth in Example 1). In some embodiments, the novel anti-promyostatin / latent myostatin antibodies or antigen-binding fragments thereof bind to recombinant human promyostatin / latent myostatin with a KD of less than or equal to 1 nM (e.g., less than 0.1 nM).

[0161] In some embodiments, the biolayer interferometry (BLI)-based assays (such as ), surface plasmon resonance (SPR)-based assays (such as Biacore TM ), MesoScale Discovery (MSD) immunoassays (such as MSD-solution equilibrium titration or MSD-SET). In some embodiments, KD is determined by a BLI-based assay (e.g., by ) to determine KD. In a preferred embodiment, the KD is determined by an SPR-based assay (e.g., Biacore TM ) to determine KD.

[0162] D. Inhibitory efficacy

[0163] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are capable of binding to promyostatin / latent myostatin and thereby inhibiting promyostatin / latent myostatin from being proteolytically activated into mature myostatin. In some cases, the antibodies or antigen-binding fragments thereof described herein can inhibit the proteolytic activation of promyostatin / latent myostatin by at least 20%, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some cases, the antibodies described herein can inhibit the proteolytic cleavage of promyostatin by a proprotein convertase (e.g., furin) by at least 20%, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some cases, the antibodies described herein, or antigen-binding fragments thereof, can inhibit the proteolytic cleavage of promyostatin or latent myostatin by a tolloid protease (e.g., mTLL2) by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some embodiments, antibodies (e.g., Ab109 and Ab130) can inhibit the proteolytic cleavage of promyostatin / latent myostatin by a tolloid protease (e.g., mTLL2) with an IC50 of less than 0.4 nM.

[0164] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are capable of binding to pro-myostatin / latent myostatin and inhibiting myostatin activity. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can inhibit myostatin signaling by at least 20%, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some embodiments, inhibition of myostatin signaling can be measured by conventional methods, for example, using the myostatin activation assay 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.

[0165] It should be understood that any suitable method can be used to measure and / or quantify, for example, the extent of proteolytic cleavage of myostatin by proprotein convertase and / or tolloid protease. In some embodiments, enzyme-linked immunosorbent assay (ELISA) is used to measure and / or quantify the extent of proteolytic cleavage of myostatin. For example, ELISA can be used to measure the level of released growth factors (e.g., mature myostatin). As another example, antibodies or antigen-binding fragments thereof that specifically bind to promyostatin, latent myostatin, and / or mature myostatin can be used in ELISA to measure the level of a specific form of myostatin (e.g., promyostatin / latent myostatin / mature myostatin), or to quantify the extent of proteolytic cleavage of myostatin. In some embodiments, immunoprecipitation is used, 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 to measure and / or quantify the extent of proteolytic cleavage of myostatin.

[0166] According to the novel antibodies and antigen-binding fragments thereof disclosed herein (e.g., any one of Abl01-Ab141) highly potently inhibit the activation step of myostatin in 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 Abl02, Abl05, Abl09, Abl23, Abl12, Abl30, Abl31, Abl32, Abl33, Abl34, Abl35, Abl36, Abl37, Abl38, Abl39, Abl40, or Abl41, 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, in order to measure the inhibitory efficacy of myostatin activation inhibitors (such as those disclosed herein), in some embodiments, an ELISA-based in vitro efficacy assay ("functional ELISA") can be used. In certain embodiments, a test substance (such as a test antibody) can be pre-cultivated with recombinant human latent myostatin to form an immune complex. Subsequently, a Tolloid protease (preferably mTLL-2) can be added to the immune mixture to trigger the release of mature myostatin by proteolytic cleavage. If the test antibody can block the activation induced by mTLL-2, the latent myostatin complex does not release mature myostatin. On the other hand, if the test antibody does not inhibit myostatin activation, the mTLL-2 process causes myostatin to be released from the latent myostatin complex. After the Tolloid / mTLL-2 treatment step, the amount of free (released) mature myostatin can be measured by ELISA in the presence of the test antibody. The ELISA assay can include a plate coated with a myostatin capture reagent. In certain embodiments, the myostatin capture reagent is an antibody or fusion construct in conjunction with mature myostatin. In certain embodiments, the fusion construct is an ActRII-Fc fusion protein, which is a ligand trap. Free mature myostatin present in the assay mixture is captured on the ELISA plate, and the amount of bound mature myostatin can be measured by any suitable method, such as a biotin-streptavidin-based detection reagent. Functional ELISA experiments can be performed at room temperature (e.g., 20° C.-25° C.).

[0167] In some embodiments, highly potent antibodies and antigen-binding fragments thereof include Abl02, Abl09, Abl30, Abl32, Abl33, and Abl41.

[0168] E. Binding Stoichiometry

[0169] In some embodiments, anti-promyostatin / latent myostatin antibodies or antigen-binding fragments thereof suitable for practicing various embodiments of the present disclosure comprise an antibody capable of binding to promyostatin / latent myostatin at an antibody:promyostatin / latent myostatin stoichiometry of about 1:2. The antibody:promyostatin / latent myostatin stoichiometry can be determined using any method known in the art, including SEC-MALS (size exclusion chromatography coupled with multi-angle light scattering).

[0170] In some embodiments, the monoclonal antibodies of the present disclosure bind to human promyostatin / latent myostatin with a 1:2 antibody:antigen stoichiometry. In some embodiments, the monoclonal antibodies of the present disclosure bind to human promyostatin / latent myostatin in both a 1:2 antibody:antigen configuration and a daisy-chain format. In some embodiments, the monoclonal antibodies of the present disclosure bind to human promyostatin / latent myostatin in a daisy-chain format. In some embodiments, the Fab fragments of the monoclonal antibodies of the present disclosure bind to human promyostatin / latent myostatin with a 2:1 Fab:antigen stoichiometry. In some embodiments, the antibody binds to human promyostatin / latent myostatin with a 1:2 mAb:Ag binding stoichiometry as measured by analytical SEC-MALS, wherein the mAb and Ag are present in a 1:1, 2:1, or 3:1 stoichiometry (e.g., as a mAb:Ag mixture), wherein the total protein concentration is between about 3.5 mg / mL (e.g., about 15 μM each of mAb and Ag) and about 8 mg / mL (e.g., about 45 μM mAb and about 15 μM Ag), and immune complexes are allowed to form at room temperature at neutral pH for 24 hours. In some embodiments, the mAb:Ag mixture further comprises oligomeric complexes, including 2:1 mAb:Ag complexes and / or 2:2 mAb:Ag complexes. In some embodiments, the mAb:Ag mixture does not comprise detectable levels of polydaisy-chains as measured by analytical SEC-MALS.

[0171] In some embodiments, the antibodies or antigen-binding fragments disclosed herein are capable of reducing the total serum myostatin level in a subject compared to background levels in which the total serum myostatin comprises antibody-antigen immune complexes. Although myostatin is believed to act locally, as opposed to through a circulating pool, it is conceivable that high levels of circulating immune complexes (therapeutic antibodies bound to latent myostatin) may reach tissues where the bound latent myostatin may dissociate from the inhibitory antibody at some point, leading to inadvertent activation of the tissue. In such situations, antibodies that enable rapid serum clearance of myostatin may reduce the risk of inadvertent myostatin activation.

[0172] In some embodiments, as measured by analytical SEC-MALS, the antibody binds to human promyostatin / latent myostatin with a 1:2 mAb:Ag binding stoichiometry, wherein mAb and Ag are present in a 1:1, 2:1, or 3:1 stoichiometry, wherein the total protein concentration is in the range of about 3.5 mg / mL to about 8 mg / mL, and the complex is allowed to form at room temperature for, for example, 24 hours. In some embodiments, the antibodies or antigen-binding fragments disclosed herein form larger immune complexes (e.g., 1:2 mAb:Ag or greater) with promyostatin / latent myostatin. In some embodiments, these larger complexes may comprise oligomers greater than 1 mAb:antigen, but smaller than a daisy chain (e.g., oligomers of 500 kDa or less). Such larger immune-oligomer complexes may be advantageous, for example, for promoting faster clearance and / or better target binding in tissues with higher local concentrations of myostatin. In some embodiments, larger immune complexes (e.g., multiple daisy chains) comprise complexes greater than 500 kDa in size and, for example, may promote faster serum clearance. Without being bound by a particular theory, it is contemplated that larger immune complex formation (eg, oligomers or daisy chains) may facilitate clearance through, for example, enhanced FcRn interaction.

[0173] F. pH dependence

[0174] In some embodiments, anti-promyostatin / latent myostatin antibodies or antigen-binding fragments thereof suitable for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments thereof that exhibit pH-sensitive binding to promyostatin / latent myostatin, resulting in the antibody or antigen-binding fragment thereof binding to promyostatin / 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 antibody or antigen-binding fragment exhibits pH-sensitive affinity, resulting in an off-rate (i.e., off-rate or Kd) at acidic pH that is at least 10 times greater than the off-rate at neutral pH. In one embodiment, a BLI-based assay (such as (e.g. Octet )) measures the pH sensitivity binding characteristics of an antibody or its antigen binding fragment. In some embodiments, the dissociation rate (K) of an antibody at two or more pH levels can be compared. off pH sensitivity is measured by dissociation rate. In some embodiments, the antibody or fragment that binds to promyostatin / latent myostatin dissociates from the antigen faster at acidic pH (e.g., pH 5.5) than at neutral pH (e.g., pH 7.4).

[0175] In some embodiments, pH dependence can be expressed as a ratio of a first off-rate at a first pH level to a second off-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 off-rate divided by the second off-rate.

[0176] In some embodiments, such as those described in Example 1, the off-rate is measured by known in vitro binding techniques, such as BLI-based assays (e.g. ).

[0177] In some embodiments, the antibodies or antigen-binding fragments thereof according to the present disclosure are pH-dependent binders, characterized in that the ratio of the off-rate at acidic pH levels to the off-rate at neutral pH levels, as determined by the off-rate at pH 5.5 divided by the off-rate at pH 7.4, as measured by a BLI-based binding assay, is 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.

[0178] In some embodiments, antibodies with high pH sensitivity (e.g., faster dissociation under acidic conditions compared to neutral conditions) can contribute to more robust recycling ability in vivo compared to antibodies with lower pH sensitivity (which can contribute to an increased serum half-life of the antibody in vivo).

[0179] Accordingly, the present disclosure includes antibodies that selectively bind to human promyostatin / latent myostatin, which antibodies bind 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 one or more amino acid residues of KALDEN (SEQ ID NO: 118) (amino acid residues 205-210 of human promyostatin) and thereby inhibit myostatin activation as measured by a functional ELISA (which measures inhibition of myostatin activation by mTLL2) with an IC50 of less than 1 nM, wherein the antibody has a K for dissociation from promyostatin / latent myostatin at acidic pH of off The rate is at least 10 times greater than at neutral pH, wherein optionally the acidic pH is 5.5 and the neutral pH is 7.4.

[0180] G. Developability

[0181] In terms of antibody engineering design, the antibodies or antigen-binding fragments disclosed herein can be fully human antibodies of the IgG1 subtype or the IgG4 subtype. For the latter, in some embodiments, the antibody comprises an Adair mutation (S228P). Thus, a hinge stabilization skeleton is obtained to reduce the binding to Fc gamma receptors, with the aim of minimizing effector functions. In some embodiments, the variable region used is located on a preferred framework using only germline amino acids, with the aim of reducing the possibility of undesired immunogenicity.

[0182] In terms of expression profiles, in some embodiments, candidate antibodies are selected based, at least in part, on the ability to express highly transiently (e.g., 100-200 mg / L), for example, at a research scale in shake flasks using suitable mammalian cells, such as 293 Expi cells. Additionally, preferred antibodies may exhibit high monomer content following protein A purification. In preferred embodiments, protein A purified antibody samples exhibit >85% monomer based on small-scale, transient expression.

[0183] The developability profile can be assessed based on well-known parameters. In some embodiments, the antibodies do not show measurable multireactivity as measured by baculovirus particle ELISA or multispecific reagent (PSR). In some embodiments, the aggregation properties of the antibodies are tested by affinity capture self-interaction nanoparticle spectroscopy (AC-SINS). In some embodiments, the antibodies do not show measurable self-association as measured by affinity capture self-interaction nanoparticle spectroscopy (AC-SINS) (Δλmax (nM) <5). Typically, gold nanoparticles are coated with polyclonal antibodies specific for human monoclonal antibodies and the monoclonal antibodies are captured by the conjugate. The multivalency of the monoclonal antibody conjugate expands the attractive self-interaction, that is, the aggregation between the adsorbed antibodies. The interparticle spacing is thereby shortened and detected based on the color change of the gold colloid solution, which can be quantified based on the change in the wavelength of maximum absorbance (plasmon wavelength). The 530nm plasmon wavelength can be used as a reporting value for unaggregated gold nanoparticles. Antibodies that tend to self-aggregate shift the plasmon wavelength toward the red end of the spectrum. In some embodiments, a shift in the plasmon wavelength of greater than 5 nm (i.e., >5 nm) can be used as a cutoff value to indicate a self-interacting antibody. In some embodiments, an antibody that does not show measurable self-association (e.g., aggregation) as measured by AC-SINS is selected from AblOl, AblO2, AblO3, Abl34, AblO4, AblO5, AblO6, AblO7, AblO8, AblO9, Abl33, Abl35, and Abl41.

[0184] In some embodiments, ELISA can be used to detect non-specific binding to baculovirus particles (BV-ELISA) to assess the multispecificity of novel antibodies. In some embodiments, an arbitrary cutoff value of 1000 RFU can be set based on the mean + 5x standard deviation of control IgG and no antibody. Antibodies that do not show measurable multireactivity using baculovirus particle ELISA include but are not limited to: Abl01, Abl02, Abl03, Abl34, Abl04, Abl05, Abl06, Abl07, Abl08, Abl09, Abl33, Abl35, and Abl41.

[0185] In some embodiments, the antibodies exhibit relatively low hydrophobic interactions as measured by retention time in a hydrophobic chromatography column, indicating a low potential for self-interaction. In some embodiments, minimal aggregation is observed in a 4-week accelerated degradation / stability study.

[0186] With respect to in vivo configuration, in some embodiments, pharmacokinetic studies in non-human primates (e.g., cynomolgus monkeys) predict the half-life in humans. Preferably, such studies predict a half-life in humans of approximately 28 days. In some embodiments, an assay suitable for determining the pharmacokinetics of myostatin-inhibiting antibodies may be used. The assay may be performed by immobilizing promyostatin on a surface (e.g., 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, to enhance specificity, particularly for human clinical use, a mouse anti-human IgG4 Fc fragment conjugated to a suitable detection agent (e.g., ruthenium red) may be used.

[0187] In some embodiments, the novel antibodies and antigen-binding fragments thereof according to the present disclosure are modified to reduce susceptibility to deamination and oxidation. In some embodiments, such modifications include one or more modifications at or around a region of the protein containing amino acid residues NG. In some embodiments, the novel antibodies and antigen-binding fragments thereof according to the present disclosure are modified to reduce susceptibility to isomerization. In some embodiments, such modifications include one or more modifications at or around a region of the protein containing amino acid residues DG.

[0188] H. Reduce or prevent serum accumulation

[0189] It has been previously observed that certain myostatin selective activation inhibitors (such as apilomab) can cause elevated levels of circulating latent myostatin (e.g., latent myostatin-antibody immune complexes) in subjects treated with the antibody. See, for example, PCT / US2016 / 052014, the contents of which are hereby incorporated in their entirety. The accumulation of serum latent myostatin relative to baseline has been used as a pharmacodynamic biomarker for apilomab. However, certain antibodies disclosed herein (e.g., Abl02, Abl30, Abl09, Abl32, Abl33, Abl41) unexpectedly exhibit enhanced serum clearance 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., Abl09, Abl32), or reduced accumulation of circulating latent myostatin in serum samples from subjects administered the antibodies (e.g., Abl02, Abl30, Abl33, Abl41), compared to the enhanced accumulation seen with apilitutinib. Without wishing to be bound by theory, it is contemplated that the enhanced serum clearance of latent myostatin associated with certain antibodies disclosed herein (e.g., Abl02, Abl30, Abl09, Abl32, Abl33, Abl41) may be due, in part, to the larger pH differences exhibited by certain antibodies disclosed herein (e.g., Abl02, Abl30, Abl09, Abl32, Abl33, Abl41). It is also contemplated that differences in binding stoichiometry may contribute to enhanced serum clearance of latent myostatin. In some embodiments, the higher order stoichiometry of certain antibodies disclosed herein may contribute to enhanced serum clearance or reduced serum accumulation thereof. For example, it is contemplated that, in some embodiments, the single 1:2 antibody:antigen stoichiometric peak observed when measuring the stoichiometry of Abl09 and Abl32, compared to apilitutinib bound at a 1:1 antibody:antigen stoichiometry, may contribute to enhanced serum clearance of latent myostatin, thereby preventing accumulation of circulating latent myostatin above baseline levels. In some embodiments, the 1:2 and 2:1 antibody:antigen dual stoichiometry of Abl02, Abl30, Abl33, and Abl41 can contribute to enhanced serum clearance of latent myostatin compared to apilituzumab, thereby preventing accumulation of circulating latent myostatin levels in a serum sample of a subject; accumulation of circulating latent myostatin levels did not occur with the use of the antibodies compared to apilituzumab.

[0190] Although higher order stoichiometry (e.g., 1:2 and / or 2:1) of antibody: promyostatin was observed with certain antibodies of the disclosure (e.g., Abl02, Abl30, Abl09, Abl32, Abl33, Abl41), negative stain electron microscopy at lower protein concentrations (e.g., 0.01-0.015 mg / ml) demonstrated that certain antibodies (e.g., Ab2, Abl02, Abl30, Abl09, Abl32, Abl33, Abl41) can also form a 1:1 stoichiometry. In some embodiments, certain of these antibodies (Abl02, Abl30, Abl09, Abl32, Abl33, Abl41) can form higher order stoichiometry and a 1:1 stoichiometry (the antibodies are capable of both). Without wishing to be bound by theory, it is expected that in the circulation, circulating levels of myostatin:antibody complexes in subjects administered Ab109 may favor the formation of a 1:1 stoichiometry; and in other situations where target myostatin concentrations are high, for example, target tissues such as muscle and other depots, and on cell surfaces where the antibody is being cleared and local concentrations of immune complexes are forming, 1:2 and 2:1 stoichiometries are favored and promote target clearance and / or prevent target accumulation.

[0191] In some embodiments, the antibodies disclosed herein are capable of reducing the serum concentration of total myostatin or latent myostatin. In some embodiments, the antibody may comprise Abl02, Abl30, or an antigen-binding fragment thereof (e.g., to achieve higher clearance and lower accumulation than Ab2). In some embodiments, the antibody may comprise Abl33, Abl41, or an antigen-binding fragment thereof (e.g., to achieve higher clearance and lower accumulation than Ab2). In some embodiments, the antibody may comprise Abl09, Abl32, or an antigen-binding fragment thereof (e.g., to achieve even higher clearance and lower accumulation than Abl02, Abl30, Abl33, or Abl41). Without wishing to be bound by theory, it is contemplated that faster serum clearance may be associated with larger immune complexes (e.g., multiple daisy chains) formed in vivo and that larger immune complex formation (e.g., oligomers) may promote clearance, for example, by enhancing FcRn interactions. In some embodiments, this enhanced clearance of total or latent myostatin associated with the antibodies disclosed herein is achieved without engineering (e.g., introducing mutations) the Fc region (see, e.g., Muramatsu et al., Sci Rep. 2021; 11:2160), thereby minimizing the risk of undesirable immunogenicity.

[0192] In some embodiments, anti-promyostatin / latent myostatin antibodies or antigen-binding fragments thereof suitable for implementing 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 reduces circulating total latent myostatin in the subject by at least 10% (e.g., at least 20%, 30%, 40%, 50%, 75%, 80%, 90% or more) compared to baseline.

[0193] In some embodiments, the anti-promyostatin / latent myostatin antibodies or antigen-binding fragments thereof suitable for implementing the various embodiments of the present disclosure include antibodies or antigen-binding fragments that increase myostatin clearance in serum. In some embodiments, administration of the antibodies or antigen-binding fragments provided herein to a subject can increase myostatin clearance in 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, administration of the anti-promyostatin / latent myostatin antibodies or antigen-binding fragments provided herein can result in higher clearance of myostatin in the subject's serum (e.g., at least 10%, 20%, 30%, 40%, 50%, 75%, 80%, 90% or more clearance) compared to administration of a similar dose of another anti-promyostatin / latent myostatin antibody or antigen-binding fragment known in the art (e.g., compared to Ab2 provided in PCT / US2015 / 059468).

[0194] 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 selective binding to promyostatin / latent myostatin, pH-dependent binding, and binding region (i.e., epitope), can surprisingly provide unique serum clearance profiles in vivo (e.g., faster clearance of immune complexes compared to Ab2), such antibodies include, for example, Abl09 and Abl30.

[0195] Target engagement assays of antibodies, i.e., pharmacodynamic assays, can be performed by measuring the binding of the antibody 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, the sample is treated with acid to convert all forms of myostatin to mature growth factor; 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.

[0196] In another embodiment, a pharmacodynamic assay can be performed by measuring the binding of the antibody to serum free latent myostatin (i.e., circulating latent myostatin that is not bound by the antibody). Such an assay can be performed by immobilizing streptavidin, binding a biotinylated antibody known to bind to latent myostatin, adding latent myostatin, and testing the antibody's ability to bind to free latent myostatin by 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. Following administration of a single dose (e.g., 2 to 20 mg / kg) of the novel myostatin inhibitors disclosed herein to mice, a rapid decrease in serum free latent myostatin was observed within one day, which remained at undetectable or nearly undetectable levels for at least 42 days, indicating that target engagement was durable and the antibodies had inhibitory activity.

[0197] I. In vivo efficacy

[0198] In some embodiments, anti-myostatin / latent myostatin antibodies or antigen-binding fragments thereof suitable for implementing 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) prevent muscle atrophy; 2) maintain or increase muscle mass; and / or 3) maintain overall body weight. In some embodiments, one or more effects can be tested in vivo using a dexamethasone-induced atrophy injury model (e.g., as described in Example 2 of the present disclosure). In some embodiments, administration of one of the novel anti-myostatin / latent myostatin antibodies or antigen-binding fragments thereof disclosed herein can produce one or more of the following effects: 1) induce overall weight loss;

[0199] 2) attenuate weight gain; 3) maintain or increase lean muscle mass; 4) reduce fat mass; and / or 5) alter the ratio of muscle to fat.

[0200] In some embodiments, administration of the novel anti-promyostatin / latent myostatin antibodies or antigen-binding fragments thereof disclosed herein in combination with standard-of-care treatments for diabetes and / or obesity (e.g., a GLP-1 pathway activator, such as semaglutide, tilportide, AMG-133 (a GLP-1 receptor agonist / GIP-1 receptor antagonist being developed by Amgen), or danglitazone (an oral GLP-1 receptor agonist being developed by Pfizer)) can result in greater efficacy than administration of the standard-of-care treatment alone. For example, administration of the combination therapy results in increased weight loss or greater attenuation of weight gain, increased lean muscle mass or attenuated loss of lean muscle mass, and / or decreased fat mass compared to administration of the standard-of-care treatment alone.

[0201] Non-limiting examples of novel antibodies and antigen-binding fragments thereof

[0202] In some embodiments, anti-promyostatin / latent myostatin antibodies or antigen-binding fragments thereof suitable for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments that exhibit one or more surprising unique properties (e.g., compared to antibodies of the prior art, such as Ab2 provided in PCT / US2015 / 059468). In some embodiments, the properties include one or more (e.g., all) of the following: 1) as measured by an SPR-based in vitro binding assay (e.g., Biacore TM), binds to promyostatin / latent myostatin with a divalent KD (e.g., F(ab')2 or mAb) of less than 1 nM as measured by ; 2) the binding to promyostatin / latent myostatin is pH sensitive, e.g., resulting in a higher binding affinity for promyostatin / latent myostatin at physiological pH (e.g., pH 7.0-7.5, e.g., pH 7.4) than at acidic pH (e.g., pH 4.0-6.5, e.g., pH 7.4). 5.5) has an on-rate that is at least 9-fold, e.g., at least 10-fold greater than that under the conditions described herein; 3) is capable of binding to promyostatin / latent myostatin with a 1:2 mAb:promyostatin / latent myostatin stoichiometry; 4) is capable of inhibiting protease-induced myostatin activation in vitro with an IC50 of less than 1 nM as measured by a functional ELISA as described herein; 5) does not cause serum accumulation of total or latent myostatin levels or reduces circulating total or latent myostatin levels (e.g., enhances serum clearance of myostatin); and / or 6) is capable of inhibiting protease-induced myostatin activation in vitro with an IC50 of less than 1 nM as measured by an SPR-based in vitro binding assay (e.g., Biacore TM ), capable of monovalently binding to latent myostatin with a KD of less than 50 nM as measured by IgG4. In some embodiments, the antibody or antigen-binding fragment further comprises an IgG4 constant domain.

[0203] In some embodiments, anti-myostatin antibodies or antigen-binding fragments thereof suitable for implementing various embodiments of the present disclosure include antibodies or antigen-binding fragments that exhibit one of the six unique characteristics described above. In some embodiments, the antibodies or antigen-binding fragments thereof exhibit two of the above unique characteristics. In some embodiments, the antibodies or antigen-binding fragments thereof exhibit three of the above unique characteristics. In some embodiments, the antibodies or antigen-binding fragments thereof exhibit four of the above unique characteristics. In some embodiments, the antibodies or antigen-binding fragments thereof exhibit five of the above unique characteristics. In some embodiments, the antibodies or antigen-binding fragments thereof exhibit all six of the above unique characteristics. Any subgroup combination of the six characteristics is contemplated herein.

[0204] The inventors have surprisingly found that a subset of antibodies derived from or bound to the same epitope as Ab2 provided in PCT / US2015 / 059468 has all six of the above-mentioned characteristics. These antibodies, described in further detail below, are particularly useful for implementing various embodiments of the present disclosure. In certain embodiments, the antibody or antigen-binding fragment thereof comprises six CDR groups and / or variable domain groups of Ab102, Ab109, Ab130, Ab132, Ab133, or Ab141 (e.g., as defined by their respective heavy and light chain sequences) or any one of those antibodies.

[0205] In some embodiments, anti-promyostatin / latent myostatin antibodies or antigen-binding fragments thereof suitable for implementing various embodiments of the present disclosure include antibodies or antigen-binding fragments that specifically bind to promyostatin / latent myostatin. In some embodiments, such antibodies and antigen-binding fragments bind to an epitope within the prodomain, wherein numbering is according to SEQ ID NO: 52, the epitope comprising one or more (e.g., all) of amino acid residues F147, Q149, L151, Y183, S168, Q149, L151, Y163, S168, K170, K205, and L207 (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, wherein, numbered according to SEQ ID NO: 52, the epitope comprises one or more (e.g., all) of amino acid residues F147, Q149, L151, Y186, S168, K170, K205, and / or L207.

[0206] In some embodiments, the off-rate of the antibody or antigen-binding fragment is at least 10 times greater 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., Abl09, Abl30, Abl32, Abl33, or Abl41.

[0207] In some embodiments, the antibody or antigen-binding fragment has a 1:2 antibody:promyostatin / latentmyostatin binding stoichiometry. In some embodiments, the antibody is an antibody provided herein, e.g., Abl05, Abl09, Abl30, Abl33, or Abl41.

[0208] In some embodiments, the binding of proteins to the target gene is determined by an in vitro SPR-based binding assay (such as Biacore TM ), the antibody or antigen-binding fragment binds to promyostatin / latent myostatin with a binding Kd of less than or equal to 0.1 nM, as measured by . In some embodiments, the antibody is an antibody provided herein, e.g., AblOl, AblO2, AblO4, AblO5, AblO7, AblO9, Abl33, or Abl41.

[0209] In some embodiments, anti-promyostatin / latent myostatin antibodies or antigen-binding fragments thereof suitable for implementing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising a constant domain of the IgG1 subtype or IgG4 subtype. In some embodiments, the antibody comprising an IgG1 or IgG4 constant domain further comprises 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.

[0210] In any of the embodiments disclosed herein, the antibody or antigen-binding fragment may comprise HCDR1 of SEQ ID NO: 201; HCDR2 of SEQ ID NO: 202, wherein X1 is T or A; HCDR3 of SEQ ID NO: 203; LCDR1 of SEQ ID NO: 204; LCDR2 of SEQ ID NO: 205; and LCDR3 of SEQ ID NO: 206, wherein X1 is M or Q and X2 is P or G, numbered according to the Kabat numbering system.

[0211] In any of the embodiments disclosed herein, the antibody or antigen-binding fragment may comprise HCDR1 of SEQ ID NO: 293; HCDR2 of SEQ ID NO: 279; HCDR3 of SEQ ID NO: 296; LCDR1 of SEQ ID NO: 281; LCDR2 of EVS; and LCDR3 of SEQ ID NO: 297, wherein X1 is P or G and is numbered according to the Clothia numbering system.

[0212] In any of the embodiments disclosed herein, the antibody or antigen-binding fragment may comprise HCDR1 of SEQ ID NO: 293; HCDR2 of SEQ ID NO: 294, wherein X1 is T or A; HCDR3 of SEQ ID NO: 257; LCDR1 of SEQ ID NO: 258; LCDR2 of EVS; and LCDR3 of SEQ ID NO: 292, wherein X1 is M or Q and X2 is P or G, numbered according to the IMGT numbering system.

[0213] In some embodiments, antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising the following 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 SFTGSGGX1YYPDSVKG (SEQ ID NO: 202), wherein 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), wherein X1 is M or Q, and X2 is P or G, wherein the CDR sequences are numbered according to the Kabat numbering system.

[0214] In some embodiments, antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising the following 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 X1QATQFPRP (SEQ ID NO: 210), wherein X1 is M or Q, wherein the CDR sequences are numbered according to Kabat.

[0215] In some embodiments, antibodies or antigen-binding fragments useful in practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising six complementary determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein 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); CDRL1 comprises the sequence RX1SQSX2LHSX3X4HNFLH (SEQ ID NO: 212), wherein 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); NO: 213), wherein X1 is A or V; X2 is R or L; X3 is V or A, and CDRL3 comprises the sequence QQX1TQYPPT (SEQ ID NO: 214), wherein X1 is Q or Y, wherein the CDR sequences are numbered according to Kabat.

[0216] In some embodiments, antibodies or antigen-binding fragments useful in practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising the following six complementary determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises the sequence SYGMS (SEQ ID NO: 201); CDRH2 comprises the sequence SX1TGSGGX2X3YX4X5X6X7X8X9 (SEQ ID NO: 512), wherein X1 is I or F; X2 is E, T, or A; 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, and CDRH3 comprises the sequence DLLX1RFLEWSHYYGMDV (SEQ ID NO: 513), wherein X1 is V or I; CDRL1 comprises the sequence RSSQSLLHSSGHNFLH (SEQ ID NO: 514). NO: 204); CDRL2 comprises the sequence EX1SNRX2X3 (SEQ ID NO: 514), wherein X1 is T or V; X2 is A or V; X3 is P or S; and CDRL3 comprises 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; X5 is P or T, wherein these CDR sequences are numbered according to Kabat.

[0217] In some embodiments, 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 SX1TGSGGX2TYYPDSVKG (SEQ ID NO: 275), wherein X1 is F or I, and X2 is E or A; CDRH3 comprises the sequence DLLX1RFLEWSHYYGMDV (SEQ ID NO: 272), wherein X1 is I or V; CDRL1 comprises the sequence RSSQSLLHSSGHNFLH (SEQ ID NO: 204); CDRL2 comprises the sequence ETSNRX1X2 (SEQ ID NO: 276), wherein X1 is V or A and X2 is P or S; and CDRL3 comprises the sequence X1QQX2TQX3PX4X5 (SEQ ID NO: NO: 277), wherein 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, wherein the CDR sequences are numbered according to the Kabat numbering system.

[0218] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises the sequence GFTFX1SY (SEQ ID NO: 278), wherein X is S or T; CDRH2 comprises the sequence TGSGG (SEQ ID NO: 279); CDRH3 comprises the sequence LLX1RFLEWSHYYGMD (SEQ ID NO: 280), wherein X1 is I or V; CDRL1 comprises the sequence SQSLLHSSGHNF (SEQ ID NO: 281); CDRL2 comprises the sequence EX1S, wherein X1 is T or V; and CDRL3 comprises the sequence XXXXX6, wherein 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; wherein the CDR sequences are numbered according to the Chothia numbering system.

[0219] In some embodiments, the antibody or antigen-binding fragment comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises the sequence GFTFTSSYG (SEQ ID NO: 284); CDRH2 comprises the sequence X1TGSGGX2T (SEQ ID NO: 285), wherein X1 is F or I and X2 is E, T, or A; CDRH3 comprises the sequence ARDLLVRFLEWSHYYGMDV (SEQ ID NO: 286); CDRL1 comprises the sequence QSLLHSSGHNF (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), wherein 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, wherein the CDR sequences are numbered according to the IMGT numbering system.

[0220] In some embodiments, an anti-promyostatin / latentmyostatin antibody, or antigen-binding portion thereof, suitable for practicing various embodiments of the present disclosure comprises the following six CDRs: a CDRH1 comprising GFTFSSYG (SEQ ID NO: 3); a CDRH2 comprising FTGSGGX1 (SEQ ID NO: 291), wherein X1 is selected from the group consisting of T and A; a CDRH3 comprising ARDLLIRFLEWSHYYGMDV (SEQ ID NO: 257); a CDRL1 comprising QSLLHSSGHNF (SEQ ID NO: 258); a CDRL2 comprising EVSNRVS (SEQ ID NO: 289); and a CDRL3 comprising X1QQTQYPX2T (SEQ ID NO: 292), wherein X1 is selected from the group consisting of M and Q, and X2 is selected from the group consisting of 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), wherein X1 is P or G.

[0221] In some embodiments, antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising the following six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or a combination thereof, wherein 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, as defined according to the Kabat numbering system. In some embodiments, preferred antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising the following six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or a combination thereof, wherein CDRH1 comprises SEQ ID NO: 201, CDRH2 comprises any one of SEQ ID NO: 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 NO: 223, 225, or 227, as defined according to the Kabat numbering system.

[0222] In some embodiments, antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising the following six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or a combination thereof, wherein CDRH1 comprises SEQ ID NO: 228, CDRH2 comprises SEQ ID NO: 229, CDRH3 comprises SEQ ID NO: 230, CDRL1 comprises SEQ ID NO: 231, CDRL2 comprises ETS, and CDRL3 comprises SEQ ID NO: 233, as defined according to the Chothia numbering system. In some embodiments, preferred antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising the following six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or a combination thereof, wherein CDRH1 comprises SEQ ID NO: 234, CDRH2 comprises SEQ ID NO: 235, CDRH3 comprises SEQ ID NO: 236, CDRL1 comprises SEQ ID NO: 237, CDRL2 comprises EVS, and CDRL3 comprises any one of SEQ ID NO: 239 or 240, as defined by the Chothia numbering system.

[0223] In some embodiments, antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising the following six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or a combination thereof, wherein 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 ETS, and CDRL3 comprises SEQ ID NO: 255 or 264, as defined by the IMGT numbering system. In some embodiments, preferred antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising the following six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or a combination thereof, wherein CDRH1 comprises SEQ ID NO: 250, CDRH2 comprises SEQ ID NO: 256, CDRH3 comprises SEQ ID NO: 257, CDRL1 comprises SEQ ID NO: 258, CDRL2 comprises EVS, and CDRL3 comprises any one of SEQ ID NOs: 260, 261, or 263, as defined by the IMGT numbering system.

[0224] In some embodiments, antibodies or antigen-binding fragments used to implement various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises any one of SEQ ID NOs: 400, 402, 409, 420 and the light chain variable domain comprises any one of SEQ ID NOs: 410, 412, 419, 421, 422.

[0225] In some embodiments, antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments 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, preferred antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising a heavy chain variable domain comprising any one of SEQ ID NO: 402, 409, or 420 and a light chain variable domain comprising any one of SEQ ID NO: 412, 419, or 421. In some embodiments, preferred antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments 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, preferred antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments 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, preferred antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments 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, preferred antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure include antibodies or antigen-binding fragments comprising a heavy chain variable domain comprising SEQ ID NO: 420 and a light chain variable domain comprising SEQ ID NO: 422.

[0226] In some embodiments, the preferred antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure are Abl02, Abl09, Abl30, Abl32, Abl33, or Abl41. In some embodiments, the preferred antibodies or antigen-binding fragments for practicing various embodiments of the present disclosure are Abl09, Abl33, or Abl41.

[0227] In some embodiments, the anti-promyostatin / latent myostatin antibody, or antigen-binding portion thereof, comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of any one of the antibodies shown in Tables 2a-f. The present disclosure also encompasses any nucleic acid sequence encoding a molecule comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3 as provided by any one of the antibodies shown in Tables 2a-f.

[0228] In some embodiments, the consensus CDR sequences provided in Table 2a are based on antibodies Abl09, Abl32, and Abl33. In some embodiments, the consensus CDR sequences provided in Table 2a are based on antibodies Abl02 and Abl30. In some embodiments, the consensus CDR sequences provided in Table 2a are based on all antibodies shown in Table 2d.

[0229] In some embodiments, the consensus CDR sequences provided in Table 2b and Table 2c are based on antibodies Ab 102, 109, 130, 132, and 133. In some embodiments, the consensus CDR sequences provided in Table 2b and Table 2c are based on antibodies Abl09, Abl33, and Abl41.

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244] In some embodiments, anti-promyostatin / latentmyostatin antibodies, or antigen-binding portions thereof, suitable for practicing various embodiments of the present disclosure comprise: 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, as defined according to the Kabat numbering system.

[0245] In some embodiments, anti-promyostatin / latent myostatin antibodies, or antigen-binding portions thereof, suitable for practicing various embodiments of the present disclosure comprise: 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, as defined according to the Chothia numbering system.

[0246] In some embodiments, anti-promyostatin / latentmyostatin antibodies, or antigen-binding portions thereof, suitable for practicing various embodiments of the present disclosure comprise: 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 according to the Kabat numbering system.

[0247] In some embodiments, anti-promyostatin / latent myostatin antibodies, or antigen-binding portions thereof, suitable for practicing various embodiments of the present disclosure comprise: 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, as defined according to the Chothia numbering system.

[0248] In some embodiments, anti-promyostatin / latent myostatin antibodies, or antigen-binding portions thereof, suitable for practicing various embodiments of the present disclosure comprise: 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 sequences set forth in SEQ ID NOs: 260, 262, 290, as defined by the IMGT numbering system.

[0249] In some embodiments, anti-promyostatin / latent myostatin antibodies, or antigen-binding portions thereof, suitable for practicing various embodiments of the present disclosure comprise: 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, as defined according to the Kabat or IMGT numbering systems.

[0250] In some embodiments, anti-promyostatin / latent myostatin antibodies, or antigen-binding portions thereof, suitable for practicing various embodiments of the present disclosure comprise: a CDRH1 comprising the sequence set forth in SEQ ID NO: 648; a CDRH2 comprising the sequence set forth in SEQ ID NO: 654; a CDRH3 comprising the sequence set forth in SEQ ID NO: 655; a CDRL1 comprising the sequence set forth in SEQ ID NO: 656; a CDRL2 comprising the amino acid sequence AAS; and a CDRL3 comprising the sequence set forth in SEQ ID NO: 657, as defined according to the Chothia numbering system.

[0251] In some embodiments, the present disclosure encompasses anti-promyostatin / latent myostatin antibodies, or antigen-binding portions thereof, having one or more CDR sequences containing up to 5, 4, 3, 2, or 1 variations (e.g., 1 or 2 or 3 substitutions, insertions, and / or deletions) in amino acid residues compared to the corresponding CDR region in any one of the SEQ ID NOs shown in Tables 2a-f. In some embodiments, the present disclosure encompasses anti-promyostatin / latent myostatin antibodies, or antigen-binding portions thereof, comprising one or more CDR sequences (e.g., a set of all six CDRs) corresponding to the set of CDRs illustrated in any one of the SEQ ID NOs shown in Tables 2a-f, e.g., the set of SEQ ID NOs identified in the Tables for the specific antibodies in the Tables.

[0252] Table 3. Variable domain sequences of exemplary antibodies.

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261] Table 4. Complete chain sequences of exemplary antibodies.

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284] In some examples, the disclosure encompasses anti-promyostatin / latentmyostatin antibodies, or antigen-binding portions thereof, comprising a heavy chain variable domain, a light chain variable domain, or a pair of heavy and light chain variable domains from Table 3 above.

[0285] In various embodiments, the anti-promyostatin / latentmyostatin 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.

[0286] In various embodiments, the anti-promyostatin / latentmyostatin 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.

[0287] In various embodiments, the anti-promyostatin / latentmyostatin 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.

[0288] In various embodiments, the anti-promyostatin / latentmyostatin 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.

[0289] In various embodiments, the anti-promyostatin / latent myostatin antibody or antigen-binding fragment thereof is Abl02 or Abl30. In various embodiments, the anti-promyostatin / latent myostatin antibody or antigen-binding fragment thereof is Abl09, Abl32, or Abl33.

[0290] In one embodiment, the anti-promyostatin / latentmyostatin 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.

[0291] In another embodiment, the anti-promyostatin / latentmyostatin 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.

[0292] In another embodiment, the anti-promyostatin / latentmyostatin 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.

[0293] In another embodiment, the anti-promyostatin / latentmyostatin 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.

[0294] In yet another embodiment, the anti-promyostatin / latentmyostatin 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.

[0295] In some embodiments, the anti-promyostatin / latent myostatin antibodies or antigen-binding fragments thereof of the present disclosure include any antibodies or antigen-binding fragments 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, the anti-promyostatin / latent myostatin antibodies or antigen-binding fragments thereof of the present disclosure include any antibodies 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.

[0296] In some embodiments, the present disclosure encompasses anti-myostatin / 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 of the sequences described herein. In some embodiments, the anti-myostatin / latent myostatin antibodies or antigen-binding fragments thereof comprise 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, the anti-myostatin / latent myostatin antibodies or antigen-binding fragments thereof comprise 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, the at least 90% identical heavy chain variable domain does not comprise any variation within any of the CDR sequences provided herein. In some embodiments, the light chain variable domains that are at least 90% identical do not comprise any variation within any of the CDR sequences provided herein. For example, in some embodiments, the sequence variation in the heavy or light chain variable domains (e.g., 90%, 95%, 98%, or 99%) occurs outside of the CDR sequences.

[0297] In some embodiments, the anti-promyostatin / latent myostatin antibodies or antigen-binding fragments thereof encompassed by the present disclosure comprise a variable domain sequence (i.e., the sum of the combined heavy chain variable domain and light chain variable domain) that is less than 70% identical to the variable domain sequence of Ab2 provided in PCT / US2015 / 059468. In some embodiments, the anti-promyostatin / latent myostatin antibodies encompassed by the present disclosure comprise 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 comprise a heavy chain variable domain that is also 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, the anti-promyostatin / latentmyostatin 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.

[0298] In some embodiments, the 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 antibodies comprise an IgG1 constant domain subtype or an IgG4 subtype. For the latter, in some embodiments, the antibodies comprise an Adair mutation (S228P).

[0299] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein include IgG constant domains or constant domain mutations that confer desired properties. For example, to avoid potential complications due to Fab-arm exchange (known to occur in the case of native IgG4 mAbs), the antibodies or antigen-binding fragments thereof provided herein may include IgG1 constant domains or stabilizing "Adair" mutations, such as in the original IgG4 (Angal S. et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody," Mol Immunol [Molecular Immunology] 30, 105-108; 1993), wherein serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline, resulting in an IgG1-like (CPPCP (SEQ ID NO: 58)) hinge sequence. Therefore, any antibody can include a stable 'Adair' mutation or the amino acid sequence CPPCP (SEQ ID NO: 58).

[0300] In some embodiments, the anti-myostatin / latent myostatin antibodies or antigen-binding portions thereof of the present disclosure may comprise an antibody constant region or portion thereof. For example, the VL domain may be attached to a light chain constant domain, such as Cκ or Cλ, at its C-terminus. Similarly, the 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 include a suitable constant region (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, pp. 91-3242, National Institutes of Health Publications, Bethesda, MD (1991)). Thus, antibodies within the scope of the present disclosure may include VH and VL domains, or antigen-binding portions thereof, in combination with any suitable constant region.

[0301] In certain embodiments, the VH and / or VL domains can be reverted to germline sequences, for example, by mutating the framework regions (FRs) of these domains using conventional molecular biology techniques to match those produced by germline cells. For example, the VH and / or VL domains can be restored to the germline sequences of IgHV3-30 (SEQ ID NO: 36) and / or IgLV1-44 (SEQ ID NO: 37), respectively. It should be understood that any VH and / or VL domain can be restored to any suitable germline sequence. In other embodiments, the FR sequence remains different from the consensus germline sequence.

[0302] In some embodiments, the anti-pro-myostatin / latent-myostatin antibody or antigen-binding fragment may or may not include the framework regions of the antibodies shown in SEQ ID NOs: 400- 421. In some embodiments, the anti-pro-latent-myostatin antibody is a murine antibody and includes murine framework region sequences.

[0303] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein specifically bind to pro-myostatin / latent myostatin. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein bind at or near the tolloid cleavage site of pro-myostatin / latent myostatin, or at or near the tolloid docking site of pro-myostatin / latent myostatin. In some embodiments, an antibody binds near the tolloid cleavage site or near the tolloid docking site if the antibody binds within 15 or fewer amino acid residues of the tolloid cleavage site or tolloid docking site. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein bind within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the tolloid cleavage site or tolloid docking site. In some embodiments, the antibody binds at or near the tolloid cleavage site of GDF8. For example, the antibody can bind to the amino acid sequence shown in SEQ ID NO: 62PKAPPLRELIDQYDVQRDDSSDG SLEDDDYHAT (SEQ ID NO: 62). In other embodiments, the antibodies or antigen-binding fragments thereof provided herein can bind at or near the proprotein convertase cleavage site of promyostatin / latent myostatin, or at or near the proprotein convertase docking site of promyostatin / latent myostatin. In some embodiments, if the antibody or antigen-binding fragment thereof binds within 15 or fewer amino acid residues of the proprotein convertase cleavage site or the proprotein convertase docking site, it binds near the proprotein convertase cleavage site or the proprotein convertase docking site. In some embodiments, any of the antibodies or antigen-binding fragments thereof provided herein bind within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the proprotein convertase cleavage site or the proprotein convertase docking site. In some embodiments, the antibody binds at or near the proprotein convertase cleavage site of GDF8. For example, the antibody can bind to the amino acid sequence set forth in SEQ ID NO: 63 (GLNPFLEVKVTDTPKRSRRDFGLD CDEHSTESRC).

[0304] In some embodiments, the epitope bound by the antibodies or antigen-binding fragments thereof disclosed herein includes at least one amino acid residue in KALDENH (SEQ ID NO: 118) and / or FVQILRLIKP MKDGTRYTGIRSLK (SEQ ID NO: 57).

[0305] In some embodiments, based on the numbering of the human proGDF8 sequence set forth in SEQ ID NO: 52, the epitope bound by the antibodies or antigen-binding fragments thereof disclosed herein includes one or more amino acid residues in F147, Q149, L151, Y163, R167, S168, K170, K205, L207, E209, and N210, which correspond to F170, Q172, L174, Y186, R190, S191, K193, K228, L230, E232, and N233, respectively, based on the numbering of Dagbay et al., J. Biol. Chem. (2020), 295(16): 5404-5418. In some embodiments, such antibodies or antigen-binding fragments bind to an epitope within the prodomain of human myostatin, wherein numbering is according to SEQ ID NO: 52 disclosed herein, and the epitope comprises one or more (e.g., all) of amino acid residues F147, Q149, L151, Y186, S168, Q149, L151, Y163, S168, K170, K205, and / or L207. In some embodiments, such antibodies and antigen-binding fragments bind to an epitope within the prodomain of human myostatin, wherein numbering is according to SEQ ID NO: 52, and the epitope comprises one or more (e.g., all) of amino acid residues F147, Q149, L151, Y186, K170, K205, and / or L207. 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 above-recited amino acid residues. In some embodiments, the antibody is Abl02 or Abl30. In some embodiments, the antibody is Abl09, Abl32, or Abl33. In some embodiments, the antibody is Abl09, Abl33, or Abl41.

[0306] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein specifically bind to pro-myostatin / latent myostatin as compared to other forms of myostatin and / or other members of the TGFβ growth factor family. Members of the TGFβ growth factor family include, but are not limited to, AMH, ARTN, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDNF, INHA, INHBA, INHBB, INHBC, INHBE, LEFTY1, LEFTY2, NODAL, NRTN, PSPN, TGFβ1, TGFβ2, and TGFβ3 proteins. In some embodiments, the antibodies or antigen-binding fragments thereof bind to promyostatin / latent myostatin with an affinity that is at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1,000-fold greater than that of other members of the TGFβ family of growth factors. In some embodiments, the antibodies or antigen-binding fragments thereof bind to promyostatin / latent myostatin with an affinity that is at least 1,000-fold greater than that of other members of the TGFβ family of growth factors. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to promyostatin / latent myostatin with an affinity that is at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1,000-fold greater than that of one or more forms of GDF11 or mature myostatin. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to promyostatin / latent myostatin with an affinity that is at least 1,000 times higher than that bound 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 inhibitory activity against promyostatin / latent myostatin proteolytic cleavage (e.g., by proprotein convertase or tolloid protease) than other members of the TGFβ family (such as pro / latent GDF11) that is at least 2 times, 5 times, 10 times, 50 times, 100 times, 200 times, 500 times, 1,000 times higher. In another embodiment, the antibodies or antigen-binding fragments thereof disclosed herein do not bind to GDF11. Without wishing to be 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 members of the TGFβ family (e.g., compared to antibodies that cross-react with both myostatin and GDF11).An example of such potential toxicity is related to impaired bone strength associated with GDF11 inhibition, as recently reported in Suh et al., Proceedings of the National Academy of Sciences, March 2020, 117(9): 4910-4920, the contents of which are hereby incorporated in their entirety.

[0307] Clearing antibodies

[0308] Certain embodiments of the present disclosure relate to removal antibodies. As used herein, "removal antibody" or its antigen-binding fragment refers to an antibody or its antigen-binding fragment that binds to a cell surface neonatal Fc receptor (FcRn) with pH-sensitive antigen binding and at least a threshold level at neutral or physiological pH. In certain embodiments, removal antibodies or their antigen-binding portion thereof bind to neonatal Fc receptor FcRn at neutral pH. For example, removal antibodies can bind to FcRn within a pH range of 7.0 to 7.6. In certain embodiments, removal antibodies or their antigen-binding portion thereof can bind to an antigen at an antigen binding site and bind to cell FcRn via the Fc portion of an antibody. In certain embodiments, removal antibodies or their antigen-binding portion thereof can then be internalized, releasing antigens in acidic endosomes, and their endosomes can be degraded. In certain embodiments, removal antibodies or their antigen-binding portion thereof that are no longer bound to antigens can then be released (e.g., by exocytosis) back into serum by cells.

[0309] In some embodiments, FcRn in the vascular endothelium (e.g., of a subject) extends the half-life of the clearance antibody, or its antigen-binding portion thereof. In some embodiments, the vascular endothelial cells internalize the clearance antibody, or its antigen-binding portion thereof, which in some embodiments binds to an antigen, such as myostatin (e.g., promyostatin, latent myostatin, or spare myostatin). In some embodiments, the clearance antibody, or its antigen-binding portion thereof, is recycled back into the bloodstream. In some embodiments, the clearance antibody, or its 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 the clearance antibody refers to an antibody, or its antigen-binding portion thereof, that generates the clearance antibody, or its antigen-binding portion thereof (e.g., before engineering the Fc portion of a conventional antibody to bind to FcRn with higher affinity at pH 7). In some embodiments, the half-life of the cleared antibody, or antigen-binding portion thereof, in the serum of a subject is at least 1%, 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 100%, 150%, 200%, or 250% longer compared to a conventional counterpart.

[0310] In some embodiments, the Fc portion of the clearance antibody binds to FcRn. In some embodiments, the Fc portion of the clearance antibody binds to FcRn at 10 -3 M to 10 -8 K in the M range D In some embodiments, the clearing antibody binds to FcRn at pH 7.4 with a K in the range D Binding to FcRn: 10 -3 M to 10 -7 M, 10 -3 M to 10 -6 M, 10 -3 M to 10 -5 M, 10 -3 M to 10 -4 M, 10 -4 M to 10 -8 M, 10 -4 M to 10 -7 M, 10 -4 M to 10 -6 M, 10 -4 M to 10 -5 M, 10 -5 M to 10 -8 M, 10 -5 M to 10 -7 M, 10 -5 M to 10 -6 M, 10 -6 M to 10 -8 M, 10 -6 M to 10 -7 M, or 10 -7 M to 10 -8 M. In some embodiments, FcRn binds to the CH2-CH3 hinge region of a clearance antibody. In some embodiments, FcRn binds to the same region as Protein A or Protein G. In some embodiments, FcRn and FcγR bind to different binding sites. In some embodiments, amino acid residue AA in the Fc region of a clearance antibody is required for binding to FcRn. In some embodiments, amino acid residue AA in the Fc region of a clearance antibody influences binding to FcRn.

[0311] In some embodiments, any of the antibodies or antigen-binding fragments provided herein are engineered to bind to FcRn with greater affinity. In some embodiments, any of the antibodies or antigen-binding fragments provided herein are engineered to bind to FcRn with greater affinity at pH 7.4. In some embodiments, the affinity of the antibody or antigen-binding fragment thereof for FcRn is increased to extend its pharmacokinetic (PK) properties compared to its conventional counterparts. For example, in some embodiments, the clearance antibody causes fewer adverse reactions due to its efficacy at lower doses. In some embodiments, the clearance antibody or its antigen-binding portion is administered less frequently. In some embodiments, the clearance antibody or its antigen-binding portion increases the transcytosis of certain tissue types. In some embodiments, the clearance antibody or its antigen-binding portion enhances the efficiency of transplacental delivery. In some embodiments, the production cost of the clearance antibody or its antigen-binding portion is low.

[0312] In some embodiments, any of the antibodies or antigen-binding fragments provided herein are engineered to bind to FcRn with lower affinity. In some embodiments, any of the antibodies or antigen-binding fragments provided herein are engineered to bind to FcRn with lower affinity at pH 7.4. In some embodiments, the affinity of the removal antibody or its antigen-binding portion thereof to FcRn is reduced compared to their conventional counterparts to reduce their pharmacokinetic (PK) properties. For example, in some embodiments, the removal antibody or its antigen-binding portion thereof is cleared more quickly for imaging and / or radioimmunotherapy. In some embodiments, the removal antibody or its antigen-binding portion thereof promotes the removal of endogenous pathogenic antibodies as a treatment for autoimmune diseases. In some embodiments, the removal antibody or its antigen-binding portion thereof reduces the risk of adverse pregnancy outcomes, which may be caused by the transplacental transport of material fetal-specific antibodies.

[0313] In some embodiments, the depleting 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 depleting antibody, or antigen-binding portion thereof, has reduced affinity for the antigen at acidic pH (e.g., a pH ranging from 5.5 to 6.5) compared to physiological pH (e.g., pH 7.4).

[0314] It should be understood that any of the antibodies or antigen-binding fragments provided herein can be engineered to dissociate from the antigen based on changes in pH (e.g., pH-sensitive antibodies). In some embodiments, the clearance antibodies or antigen-binding portions thereof provided herein are engineered to bind to the antigen in a pH-dependent manner. In some embodiments, the clearance antibodies or antigen-binding portions thereof provided herein are engineered to bind to FcRn in a pH-dependent manner. In some embodiments, the clearance antibodies or antigen-binding portions thereof provided herein are internalized by endocytosis. In some embodiments, the clearance antibodies or antigen-binding portions thereof provided herein are internalized by FcRn binding. In some embodiments, the endocytosed clearance antibodies or antigen-binding portions thereof release the antigen in the endosome. In some embodiments, the clearance antibodies or antigen-binding portions thereof are recycled back to the cell surface. In some embodiments, the clearance antibodies remain attached to the cells. In some embodiments, the endocytosed clearance antibodies or antigen-binding portions thereof are recycled back to the plasma. It should be understood that the Fc portion of any of the antibodies or antigen-binding fragments thereof provided herein can be engineered to have different FcRn binding activities. In some embodiments, the FcRn binding activity affects the time it takes for the antigen to be cleared by the clearance antibody. In some embodiments, the clearance antibodies can be long-acting or fast-acting clearance antibodies.

[0315] In some embodiments, converting a conventional therapeutic antibody or antigen-binding portion thereof to a clearing 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 clearing 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 clearing 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.

[0316] In some embodiments, an appropriate dose of a clearance antibody or antigen-binding portion thereof can be selected empirically for use in therapy. In some embodiments, a high dose of a clearance antibody or antigen-binding portion thereof can saturate FcRn, thereby generating an antibody that stabilizes the antigen in the serum without internalization. In some embodiments, the clearance antibody or antigen-binding portion thereof is administered once a day, once a week, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every eight weeks, once every 10 weeks, once every 12 weeks, once every 16 weeks, once every 20 weeks, or once every 24 weeks.

[0317] In some embodiments, any of the antibodies or antigen-binding fragments provided herein may be modified or engineered to be a clearance antibody. In some embodiments, any of the antibodies or antigen-binding fragments provided herein may be converted into a clearance antibody using any suitable method. For example, methods suitable for making clearance 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 [Public Library of Science Comprehensive] 8 (5): e63236; and Igawa et al., "pH-dependent antigen-binding antibodies as a novel therapeutic modality," Biochimica et Biophysica Acta [Biological and Biophysical Journal] 1844 (2014) 1943-1950; the contents of each of these documents are hereby incorporated by reference. However, it should be understood that the methods for preparing clearance antibodies or antigen-binding portions thereof as provided herein are not intended to be limiting. Thus, additional methods for preparing cleared antibodies, or antigen-binding portions thereof, are within the scope of the present disclosure.

[0318] Some aspects of the present disclosure are based on the recognition that the affinity (e.g., measured as K) of any of the anti-promyostatin / latent myostatin antibodies or antigen-binding fragments thereof provided herein is D In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have a K for binding to promyostatin / latent myostatin at a relatively low pH (e.g., a pH in the range of 4.0-6.5, e.g., pH 5.5) compared to a relatively high pH (e.g., a pH in the range of 7.0-7.6, e.g., pH 7.4). D In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to promyostatin / latent myostatin with a K of 1.5 Å when the pH is between 4.0 and 6.5 (e.g., pH 5.5). D Range is 10 -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to promyostatin / latent myostatin at a pH between 7.0 and 7.6 (e.g., pH 7.4). D Range is 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to promyostatin / latent myostatin at a pH between 4.0 and 6.5 (e.g., pH 5.5). D At least 2 times, at least 10 times, at least 20 times, at least 25 times, at least 50 times, at least 75 times, at least 100 times, at least 200 times, at least 250 times, at least 500 times, at least 1000 times, at least 5000 times, or at least 10000 times greater than a pH between 7.0 and 7.6 (e.g., pH 7.4).

[0319] Antibodies and antigen-binding fragments that compete with novel anti-promyostatin / latent myostatin antibodies or antigen-binding fragments thereof for antigen binding

[0320] Certain embodiments of the present disclosure relate to antibodies and antigen-binding fragments thereof that compete or cross-compete with any of the antibodies or antigen-binding fragments thereof provided herein for binding to an antigen. Preferably, the antigen is human latent myostatin.

[0321] In some embodiments, the 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 antibodies or antigen-binding fragments cross-compete with Ab2 or Apitrezumab for binding to human promyostatin / latent myostatin. The antibodies or antigen-binding fragments may cross-compete with Ab2 for binding to human promyostatin / latent myostatin as described herein. In some embodiments, such antibodies or antigen-binding fragments bind to an epitope within the prodomain of human myostatin, wherein numbering is according to SEQ ID NO: 52 disclosed herein, the epitope comprises one or more (e.g., all) of amino acid residues F147, Q149, L151, Y186, S168, Q149, L151, Y163, S168, K170, K205, and / or L207. In some embodiments, such antibodies and antigen-binding fragments bind to an epitope within the prodomain of human myostatin, wherein numbering is according to SEQ ID NO: 52, the epitope comprises one or more (e.g., all) of amino acid residues F147, Q149, L151, Y186, K170, K205, and / or L207. In some embodiments, such antibodies or antigen-binding fragments comprise a HCDR3 paratope that contains no more than two amino acid differences compared to SEQ ID NO: 220. In some embodiments, such antibodies or antigen-binding fragments comprise a HCDR3 sequence comprising a leucine at amino acid position 3 and a tryptophan at amino acid position 9, as numbered according to SEQ ID NO: 220. In some embodiments, such antibodies or antigen-binding fragments comprise a 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, as numbered according to SEQ ID NO: 220.

[0322] In another embodiment, the antibody, or antigen-binding portion thereof, competes or cross-competes for binding to any antigen provided herein (e.g., promyostatin / latent myostatin), wherein the equilibrium dissociation constant between the antibody and the protein is K D Less than 10 -8In other embodiments, the antibodies or antigen-binding portions thereof compete or cross-compete for binding to any antigen provided herein, wherein K D The range is 10 -11 M to 10 -8 In a preferred embodiment, the binding of TM ) measured, the antibody has a divalent K of less than 1 nM D .

[0323] Certain embodiments of the present disclosure relate to antibodies and antigen-binding fragments thereof that compete with any of the antibodies or antigen-binding fragments thereof provided herein for binding to promyostatin / latent myostatin. In some embodiments, the antibody or antigen-binding portion thereof binds to promyostatin / latent myostatin at the same epitope as any of the antibodies or antigen-binding fragments thereof provided herein. In another embodiment, the antibody or antigen-binding portion thereof competes for binding to promyostatin / latent myostatin, wherein the equilibrium dissociation constant K between the antibody or antigen-binding portion thereof and promyostatin / latent myostatin is D Less than 10 -6 In other embodiments, an antibody or antigen thereof that competes with any of the antibodies or antigen-binding portions thereof provided herein binds to promyostatin / latent myostatin, wherein K D The range is 10 -11 M to 10 -8 M.

[0324] Any antibody or antigen-binding fragment thereof provided herein can be characterized using any suitable method. For example, one method is to identify the epitope that antigen binds, or "epitope mapping". There are many methods suitable for mapping and characterizing the position of epitopes on proteins, including crystal structures of parsed antibody-antigen complexes, competitive assays, gene fragment expression assays, and assays based on synthetic peptides, such as those described in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999. In another example, epitope mapping can be used to determine the sequence bound to the antibody or its antigen-binding portion. The epitope can be a linear epitope (i.e., contained in a single stretch of amino acids) or a conformational epitope formed by the three-dimensional interaction of amino acids, which may not necessarily be contained in a single stretch (primary structure linear sequence). Peptides of different lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinant) and used for binding assays with antibodies. In another example, the epi-position combined with antibody or its antigen-binding portion thereof can be determined in system screening by using overlapping peptides derived from target antigen sequence and determining the combination of antibody or its antigen-binding portion thereof. According to gene fragment expression assay, the open reading frame encoding target antigen is randomly or by specific gene structure fragmentation, and the reactivity of expressed antigen fragment and antibody to be tested is determined. For example, gene fragment can be produced by PCR, and then in the presence of radioactive amino acids, in vitro transcription and translation into protein are made. Then the combination of antibody or its antigen-binding portion thereof and radiolabeled antigen fragment is determined by immunoprecipitation and gel electrophoresis. Some epi-positions can also be identified by using a large library (phage library) of random peptide sequences displayed on phage particle surfaces. Alternatively, the combination of the overlapping peptide fragment library determined in simple binding assay and test antibody or its antigen-binding portion thereof can be tested. In another example, mutagenesis, domain exchange experiments and alanine scanning mutagenesis of antigen-binding domains can be carried out to identify epi-positions in combination with required, sufficient and / or necessary residues. For example, domain swap experiments can be performed using mutants of the target antigen in which various fragments of the promyostatin / latent myostatin polypeptide have been 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 an antibody, or antigen-binding portion thereof, to the mutant promyostatin / latent myostatin, the importance of a particular antigenic fragment to the antibody, or antigen-binding portion thereof, can be assessed.

[0325] Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen ("reference antibody") to determine whether an antibody, or its antigen-binding portion thereof, ("test antibody") binds to the same epitope as the other antibody, or its antigen-binding portion thereof. Competition assays are well known to those skilled in the art. When 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 each other for binding to the antigen. It is well understood in the art that such antibodies bind to the same or overlapping epitopes within the antigen.

[0326] Any suitable method as described herein, such as epitope mapping methods, can be used to determine whether an anti-myostatin / latent myostatin antibody or its antigen-binding portion binds to one or more specific residues / segments in a promyostatin / latent myostatin as described herein. In addition, the interaction of an antibody or its antigen-binding portion with one or more defined residues in a promyostatin / latent myostatin can be determined by conventional techniques. For example, a crystal structure can be determined, and the distances between residues in a promyostatin / latent myostatin and one or more residues in an antibody or its antigen-binding portion can be determined accordingly. Based on such distances, it can be determined whether a specific residue in a promyostatin / latent myostatin interacts with one or more residues in an antibody or its antigen-binding portion. In addition, suitable methods (such as competition assays and targeted mutagenesis assays) can be used to determine the preferential binding of a candidate anti-myostatin / latent myostatin antibody or its antigen-binding portion to a promyostatin / latent myostatin compared to another target (such as a mutant promyostatin / latent myostatin).

[0327] In one embodiment, the disclosure encompasses a method for identifying an antibody or antigen-binding fragment thereof that competitively binds to one or more antibodies provided herein, wherein the method comprises screening for antibodies or antigen-binding fragments that compete or cross-compete with one or more antibodies provided herein (e.g., any of Abl01-141, e.g., any of Abl02, Abl09, Abl30, Abl32, or Abl33) and determining the antibody or antigen-binding fragment that exhibits one or more (e.g., all) of the following properties: binding to a protein with a KD 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 TM) binds to promyostatin / latent myostatin; binding to promyostatin / latent myostatin has high pH sensitivity (e.g., greater than 10x, as determined by comparing off-rates at pH 5.5 / 7.4), and / or a 2:1 Fab:promyostatin homodimer binding stoichiometry. In some embodiments, the disclosure encompasses antibodies identified and / or prepared according to the disclosed methods.

[0328] Modification

[0329] In some embodiments, the antibodies and antigen-binding fragments of the present disclosure may be modified, for example, by attachment 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 detecting and isolating pro-myostatin / latent myostatin. Detectable substances can be coupled or conjugated directly or indirectly through an intermediate (e.g., a linker) to the polypeptides of the present disclosure 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, dichlorotriazinamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include 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-emitters or other radioisotopes, such as, for example, iodine (131I, 125I, 123I, 121I ), carbon (14C), sulfur (35S), tritium (3H), indium (115mIn, 113mIn, 112In, 111In), and technetium (99Tc, 99mTc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (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 or indirectly through an intermediate (e.g., a linker) to an anti-promyostatin / latent myostatin antibody, or an antigen-binding portion thereof, of the present disclosure using suitable techniques. The anti-promyostatin / latent myostatin antibody, or an antigen-binding portion thereof, conjugated to a detectable substance can be used in diagnostic assays as described herein.

[0330] As shown herein, the present disclosure includes novel antibodies and antigen-binding fragments thereof that can selectively inhibit the activation of myostatin. However, unlike the inhibitors previously described in PCT / US2016 / 052014, at least some of the novel antibodies disclosed herein (e.g., Abl09, Abl05, Abl30, and Abl33) bind to latent myostatin with sufficiently high monovalent affinity, resulting in one arm of the antibody (e.g., Fab) being able to interact with the antigen. Without wishing to be bound by theory, it is contemplated that such high monovalent binding affinity can provide flexibility for designing myostatin inhibitors that incorporate six CDRs, variable domains (VH and / or VL), or corresponding Fab domains into engineered constructs, such as bispecific antibodies and other forms containing the antigen-binding portion of an antibody. The present disclosure encompasses such recombinant engineered constructs.

[0331] Pharmaceutical composition

[0332] 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-promyostatin / 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. "Pharmaceutically acceptable" means that the carrier is compatible with the active ingredient of the composition (and preferably capable of stabilizing the active ingredient) and is not harmful to the subject to be treated. Examples of pharmaceutically acceptable excipients (carriers, including buffers) will be apparent to those skilled in the art and have been previously described. See, for example, Remington: The Science and Practice of Pharmacy, 20th ed. (2000) Lippincott Williams and Wilkins, ed. KE Hoover. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; and chloride), benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as TWEEN®. TM 、PLURONICS TM or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0333] In one example, the pharmaceutical compositions described herein contain more than one myostatin inhibitor that recognizes different epitopes / residues of the target antigen, such as more than one anti-promyostatin / latent myostatin antibody or antigen-binding portion thereof.

[0334] In some examples, the pharmaceutical compositions described herein comprise emulsion-based or lipid-based formulations, such as liposomes containing a myostatin inhibitor (e.g., an anti-promyostatin / 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); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be prepared by reverse phase evaporation with a mixture containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine.

[0335] Liposomes are produced by extruding liposomes through filters of defined pore size to produce liposomes with the desired diameter.

[0336] Anti-promyostatin / latent myostatin antibodies or antigen-binding portions thereof can also be entrapped in microcapsules, such as microcapsules prepared by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Exemplary techniques have been described previously, see, for example, Remington, The Science and Practice of Pharmacy, 20th ed. Mack Publishing (2000).

[0337] In other embodiments, the pharmaceutical compositions described herein can be formulated into sustained-release forms. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies or their antigen-binding portions thereof, which are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Patent 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 (e.g., LUPRONDEPOT™ (injectable microspheres consisting of lactic acid-glycolic acid copolymers and leuprorelin acetate)), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0338] Pharmaceutical compositions for in vivo administration must be sterile. This has been accomplished, for example, by filtration through a sterile filtration membrane. Therapeutic antibody compositions are generally placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle.

[0339] The pharmaceutical compositions described herein may be in unit dosage form such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

[0340] Compositions in pharmaceutically acceptable, preferably sterile, solvents can be nebulized using gases. Nebulized solutions can be inhaled directly from the nebulizing device or the nebulizing device can be attached to a mask, hood, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered, preferably orally or nasally, from devices that deliver the formulation in an appropriate manner.

[0341] Reagent test kit

[0342] The present disclosure also provides a kit for alleviating diseases / conditions associated with myopathy or metabolic disorders (e.g., diabetes, obesity, or metabolic syndrome). Such a kit may include one or more containers comprising any of the anti-promyostatin / latent myostatin antibodies or antigen-binding fragments thereof disclosed herein (e.g., Ab102, Ab109, Ab130, Ab132, Ab133, or Ab141). In some embodiments, such a kit may further comprise one or more additional therapeutic agents, such as one or more GLP-1 receptor agonists. In some embodiments, such a kit may further comprise one or more diagnostic agents.

[0343] 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-promyostatin / 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 the target disease 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 still other embodiments, the instructions include a description of administering the antibody to an individual at risk for the target disease.

[0344] Instructions for treatment using any of the anti-promyostatin / latent myostatin antibodies or antigen-binding fragments thereof disclosed herein (e.g., Ab102, Ab109, Ab130, Ab132, Ab133, or Ab141) typically include information about the dose, dosing schedule, and route of administration for the intended therapy. The container may be a unit dose, bulk package (e.g., multi-dose package), or subunit dose. The instructions provided in the kit of the present disclosure are typically written instructions on a label or package insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0345] The label or package insert indicates that the composition is used to treat, delay the onset of, and / or alleviate a disease or condition associated with a muscle disorder (e.g., myopathy) or associated with a metabolic disorder (e.g., diabetes, obesity, and / or metabolic syndrome). Instructions for practicing any method described herein can be provided.

[0346] The kits disclosed herein are packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, cans, flexible packaging (e.g., sealed Mylar or plastic bags), etc. Also contemplated are packaging for use in combination with specific devices, such as inhalers, nasal administration devices (e.g., nebulizers), or infusion devices such as miniature pumps. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with 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 a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an anti-promyostatin / latent myostatin antibody or an antigen-binding fragment thereof, such as those described herein.

[0347] The kit may optionally provide additional components, such as a buffer and explanatory information. Typically, the kit comprises a container and a label or package insert on or associated with the container. In certain embodiments, the disclosure provides an article comprising the contents of the above-mentioned kit.

[0348] Generation of anti-promyostatin / latent myostatin antibodies or antigen-binding fragments thereof

[0349] A variety of methods can be used to obtain the antibodies or antigen-binding fragments thereof disclosed herein. For example, recombinant DNA methods can be used to produce antibodies and antigen-binding fragments thereof. Monoclonal antibodies and antigen-binding fragments thereof can also be produced by generating hybridomas according to known methods (see, for example, Kohler and Milstein (1975) Nature, 256: 495-499). Standard methods such as enzyme-linked immunosorbent assay (ELISA) and BLI or SPR (e.g., or Biacore TM ) analysis and screening of hybridomas formed in this manner to identify one or more hybridomas that produce antibodies or antigen-binding portions thereof that specifically bind to a specified antigen. Any form of a specific antigen can be used as an immunogen, such as a recombinant antigen, a naturally occurring form, any variant or fragment thereof, and an antigenic peptide thereof (e.g., any epitope described herein as a linear epitope or as a conformational epitope within a scaffold). An exemplary method for preparing antibodies and antigen-binding portions thereof includes screening a protein expression library, such as a phage or ribosome display library, that expresses an antibody or fragment thereof (e.g., scFv). Phage display is described, for example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985) Science, 228: 1315-1317; Clackson et al. (1991) Nature, 352: 624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597; WO 92 / 18619; WO 91 / 17271; WO 92 / 20791; WO 92 / 15679; WO 93 / 01288; WO 92 / 01047; WO 92 / 09690; and WO 90 / 02809.

[0350] In addition to using display libraries, a given antigen (eg, promyostatin) can be used to immunize a non-human animal, such as a rodent, eg, a mouse, hamster, or rat. In one embodiment, the non-human animal is a mouse.

[0351] In another embodiment, monoclonal antibodies are obtained from non-human animals and then modified, for example, chimerized, using appropriate recombinant DNA techniques. Various methods for preparing chimeric antibodies have been described. See, for example, Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., European Patent Publication EP 171496; European Patent Publication No. 0173494, British Patent GB 2177096B.

[0352] For additional antibody production techniques, see Antibodies: A Laboratory Manual, Harlow et al., eds., Cold Spring Harbor Laboratory, 1988. The present disclosure is not necessarily limited to any particular source, production method or other specific characteristics of the antibodies.

[0353] Some aspects of the present disclosure relate to host cells transformed with polynucleotides or vectors. 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 can be maintained outside the chromosome. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial cell, an insect cell, a fungal cell, a plant cell, an animal cell, or a human cell. In some embodiments, the fungal cell is, for example, a fungal cell of the genus Saccharomyces, especially a fungal cell of the species Saccharomyces cerevisiae. The term "prokaryotic" 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, for example, Escherichia coli, Salmonella typhimurium, Serratia marcescens, and Bacillus subtilis. The term "eukaryotic" includes yeast, higher plants, insects, and vertebrate cells, such as mammalian cells, such as NSO and CHO cells. Depending on the host used in the recombinant production procedure, the antibody or immunoglobulin chain encoded by the polynucleotide may 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., NSO and CHO cells) can be transformed with a polynucleotide or vector encoding any one of the antibodies disclosed herein (e.g., Abl01-Ab141) or its antigen-binding fragment, or with a portion thereof (e.g., a first cell can be transformed with a polynucleotide or vector encoding the heavy chain of any one of Abl01-Ab141 or its antigen-binding fragment and a second cell can be transformed with a polynucleotide or vector encoding the light chain of any one of Abl01-Ab141 or its antigen-binding fragment).

[0354] In some embodiments, once the vector has been incorporated into an appropriate host, the host can be maintained under conditions suitable for high-level expression of the nucleotide sequence, and, if desired, immunoglobulin light chains, heavy chains, light chain / heavy chain dimers, or complete antibodies, antigen-binding fragments, or other immunoglobulin forms can then be collected and purified; see, Beychok, Cells of Immunoglobulin Synthesis, Academic Press, New York (1979). Thus, the polynucleotide or vector is introduced into the cell, which then produces the antibody or antigen-binding fragment. For example, a host cell (e.g., NSO or CHO cell) transformed with a polynucleotide or vector encoding any of the antibodies disclosed herein (e.g., Abl01-Ab141) or an antigen-binding fragment thereof, or a portion thereof, can express the antibody or antigen-binding fragment (or the heavy or light chain of the antibody). In addition, transgenic animals (preferably mammals) comprising the above-described host cells can be used for large-scale production of antibodies or antibody fragments. Large-scale production typically refers to bioreactors (e.g., cell culture) of 250 liters or larger, such as 250 L, 500 L, 1000 L, 1500 L, 2000 L, 3000 L, 4000 L, 5000 L, 6000 L or larger.

[0355] The transformed host cells can be grown in a fermentation tank and cultured using any suitable technology to achieve optimal cell growth. After expression, complete antibodies, dimers thereof, single light chains and heavy chains, other immunoglobulin forms or Fabs can be purified according to the standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, etc.; See, Scopes, "Protein Purification", Springer Verlag, New York (1982). Antibodies or Fabs can then be isolated from growth medium, cell lysates or cell membrane fractions. Any conventional means, such as preparative chromatographic separation and immunological separation, such as involving the use of a monoclonal or polyclonal antibody for example, an antibody constant region, can be separated and purified for example through microbial expression of antibodies or Fabs. Any of Ab101-Ab141 produced by host cells or its Fabs can be collected, or the heavy and light chains of antibodies or Fabs can be collected from separate cells and then combined to form complete antibodies or their Fabs.

[0356] Aspects of the present disclosure relate to hybridomas, which provide a source of monoclonal antibodies that can be extended indefinitely. As an alternative to obtaining immunoglobulins directly from hybridoma cultures, immortal hybridoma cells can be used as the source of the heavy chain and light chain loci of rearrangement, for subsequent expression and / or genetic manipulation. The antibody genes of rearrangement can be reverse transcribed from appropriate mRNA to produce cDNA. In certain embodiments, the heavy chain constant region can be exchanged or eliminated together with the heavy chain constant region of different isotypes. Variable regions can be connected to encode single-chain Fv regions. Multiple Fv regions can be connected to impart the binding ability of more than one target, or chimeric heavy and light chain combinations can be used. Any suitable method can be used for cloning antibody variable regions and producing recombinant antibodies and antigen-binding portion thereof.

[0357] In some embodiments, appropriate 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 may be advantageous for efficient processing and production. Typical mammalian cells that can be used 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 suitable for host cell growth and expression of the coding sequence. The antibodies or antigen-binding fragments can be recovered by isolating them from the culture. The expression system can be designed to include a signal peptide so that the resulting antibody is secreted into the culture medium; however, intracellular production is also possible.

[0358] The present disclosure also includes polynucleotides encoding at least the immunoglobulin chain variable region of an antibody described herein (e.g., any one of 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 of the hybridomas described above.

[0359] The polynucleotide encoding the antibody or antigen-binding fragment can be, for example, DNA, cDNA, RNA, or a synthetically produced DNA or RNA or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides, alone or in combination. In some embodiments, the polynucleotide is part of a vector. Such a vector may comprise additional genes, such as marker genes that allow selection of the vector in a suitable host cell and under suitable conditions.

[0360] In certain embodiments, polynucleotide is effectively connected with the expression control sequence that allows to express in prokaryotic or eukaryotic cell.The expression of polynucleotide comprises that polynucleotide is transcribed into translatable mRNA.The regulatory element that guarantees to express in eukaryotic cell (preferably mammalian cell) is well known to those skilled in the art.They can comprise the regulatory sequence that promotes transcription initiation and optionally promote the poly A signal that transcription termination and transcript are stable.Other regulatory element can comprise transcription and translation enhancer, and / or natural related or heterologous promoter region.The possible regulatory element that allows to realize expression in prokaryotic host cell comprises for example PL, Lac, Trp or Tac promoter in Escherichia coli, and the regulatory element example that allows to realize expression in eukaryotic host cell is AOX1 or GAL1 promoter in yeast, or CMV promoter, SV40 promoter, RSV promoter (Rous sarcoma virus), CMV enhancer (cytomegalovirus), SV40 enhancer or hemoglobin intron in mammal and other animal cells.

[0361] In some embodiments, the present invention provides the method for the present invention to provide the polypeptide of the present invention.Except the element responsible for transcription initiation, such regulatory element can also include a transcription termination signal, such as the SV40-poly A site or the tk-poly A site in the polynucleotide downstream.In addition, according to the expression system used, the leader sequence that can be directed to the cell compartment or secreted into the substratum can be added to the coding sequence of the polynucleotide and has been previously described.One or more leader sequences are assembled together with translation, initiation and termination sequence in appropriate stages, and preferably, the leader sequence can guide the protein of translation or its part to be secreted into, for example, the extracellular medium.Optionally, the heterologous polynucleotide sequence of encoding fusion protein can be used, this fusion protein includes C or N-terminal recognition peptides that give desired characteristics (for example, the stability of the recombinant product expressed or simplifying purification).

[0362] In some embodiments, the polynucleotide encoding at least the variable domains of the light chain and / or heavy chain can encode the variable domains of two immunoglobulin chains or only one. Similarly, one or more polynucleotides can be under the control of the same promoter or can be controlled for expression separately. In addition, some aspects relate to vectors, particularly plasmids, cosmids, viruses and phages conventionally used in genetic engineering, which vectors contain polynucleotides encoding the variable domains of the immunoglobulin chains of antibodies or antigen-binding fragments; optionally in combination with a polynucleotide encoding the variable domains of another immunoglobulin chain of an antibody.

[0363] In some embodiments, expression control sequences are provided as a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell, but control sequences of prokaryotic hosts may also be used. Expression vectors derived from viruses (such as retroviruses, poxviruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses) can be used to deliver polynucleotides or vectors to a target cell population (e.g., cells are engineered 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 reconstructed into liposomes for delivery to target cells. Vectors containing polynucleotides (e.g., heavy and / or light variable domains of immunoglobulin chain coding sequences and expression control sequences) can be transferred to host cells by suitable methods, which methods depend on the type of cell host.

[0364] Myostatin

[0365] In some embodiments, the human promyostatin / latent myostatin is wild-type human promyostatin / latent myostatin having a human kappa signal peptide and an N-terminal His6 tag (SEQ ID NO: 941) that is cleaved upon expression (promyostatin). Promyostatin undergoes incomplete furin cleavage during expression. In some embodiments, the human promyostatin / latent myostatin has the amino acid sequence of SEQ ID NO: 135 as shown below:

[0366]

[0367] In some embodiments, the sequence encoding the human immunoglobulin kappa signal peptide is MDMRVPAQLLGLLLLWFSGVLG (SEQ ID NO: 136).

[0368] 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.

[0369] In some embodiments, the sequence encoding the human myostatin prodomain is SEQ ID NO: 137 as shown below:

[0370] NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLETAPNISKDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSS KIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPK(SEQ ID NO:137).

[0371] In some embodiments, the RSRR furin cleavage site of human promyostatin (SEQ ID NO:943) is the sequence in bold in SEQ ID NO: 135.

[0372] In some embodiments, the sequence encoding human mature myostatin growth factor is as shown in SEQ ID NO: 138:

[0373] DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS (SEQ ID NO: 138).

[0374] In some embodiments, the DNA sequence encoding human promyostatin is SEQ ID NO: 139 as shown below:

[0375]

[0376] In some embodiments, when the protein is expressed, the signal peptide is cleaved from the rest of the molecule.

[0377] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein bind to an epitope or promyostatin. In some embodiments, the antibodies or antigen-binding fragments are antibodies or antigen-binding fragments disclosed herein that compete with Ab2 for antigen binding. The variable domain and full-length antibody sequences of Ab2 are disclosed in PCT / US2015 / 059468 and PCT / US2016 / 052014.

[0378] The role of myostatin in muscle homeostasis and metabolic regulation

[0379] Skeletal muscle accounts for approximately 40% of body weight and is a dynamic organ that turns over at a rate of 1%-2% per day. Myostatin is believed to play a key role in maintaining muscle homeostasis in both health and disease. Myostatin can induce muscle atrophy through its inhibition of myoblast proliferation, increase ubiquitin-proteasome activity, and downregulate IGF-Akt pathway activity. These well-recognized effects are seen in a variety of atrophy-inducing conditions, including injury, disease (such as cachexia), disuse, and space flight, demonstrating the importance of myostatin signaling mechanisms. Given this important role, extensive research has been devoted to inhibiting the effects of myostatin in vivo. Indeed, inhibition of the myostatin pathway has been shown to promote muscle growth and maintain muscle mass.

[0380] In addition, muscle is known to be the body's main protein reservoir and therefore contributes to amino acid homeostasis and metabolism. Protein in muscle can serve as an energy source (i.e., broken down to produce energy) together with glucose (produced as glycogen and stored primarily in the liver and muscle) and lipids (stored in adipose tissue). Obstacles or imbalances in the utilization or movement of these energy sinks in the body can at least partially contribute to various types of metabolic dysregulation. Therefore, it is considered that myostatin can play a direct role in metabolic regulation (e.g., enhancing "metabolic adaptability") by coordinating the balance between glucose, fat and / or muscle breakdown and synthesis / storage in the body, such as adaptability or flexibility in terms of fuel sources, the ability to change metabolism to meet the needs of an organism or tissue in a state of exercise, or to change metabolism based on available fuel, thereby contributing to the overall metabolic regulation (e.g., energy expenditure) in the body. In fact, although myostatin has been primarily regarded as a key regulatory element of muscle metabolism since its discovery in 1997, the efficacy of myostatin as a metabolic regulatory element has been shown in the latest research results. See, for example, PCT / US2018 / 012686. Accordingly, it is contemplated herein that inhibition of myostatin (e.g., by the antibodies or antigen-binding fragments disclosed herein) may, in certain embodiments, be useful in treating a variety of conditions associated with muscle and / or metabolic dysregulation, as discussed in more detail below.

[0381] Therapeutic uses

[0382] In various embodiments, pharmaceutical compositions comprising the antibodies or antigen-binding fragments disclosed herein (e.g., compositions comprising any one of Ab101-Ab141 or an antigen-binding fragment thereof) are suitable for administration to human patients to treat or prevent diseases and conditions associated with myostatin signaling, such as conditions requiring reduction of myostatin signaling. In some embodiments, the antibodies and antigen-binding fragments disclosed herein can be used to improve the metabolic health of patients. Suitable diseases and conditions include, for example, muscle disorders such as muscular dystrophy and myopathy; metabolic disorders such as obesity and diabetes; bone and connective tissue disorders or conditions such as bone loss, anterior cruciate ligament (ACL) repair, and osteogenesis imperfecta (OI); cardiovascular disease such as heart disease (e.g., heart failure, e.g., patients with type 2 diabetes or obesity); and chronic inflammation and inflammatory diseases such as chronic kidney disease (CKD), idiopathic pulmonary fibrosis (IPF), and fatty liver disease (e.g., NAFLD, NASH). Thus, the present disclosure encompasses therapeutic uses of such antibodies and antigen-binding fragments thereof (e.g., engineered constructs incorporating such fragments) for treating one or more of these diseases and conditions. In some embodiments, the myostatin selective inhibitor is an antibody or antigen-binding fragment thereof comprising all six CDRs of any one of Abl01-Ab141 (e.g., the set of SEQ ID NOs identified for the specific antibodies in Table 2d-Table 2f). In some embodiments, the myostatin selective inhibitor is an antibody or antigen-binding fragment thereof comprising the heavy and light chain variable domains of any one of Abl01-Ab141 (e.g., the SEQ ID NO pairs identified for the specific antibodies in Table 3). In some embodiments, the myostatin selective inhibitor is an antibody or antigen-binding fragment thereof comprising the heavy and light chains of any one of Abl01-Ab141 (e.g., the SEQ ID NO pairs identified for the specific antibodies in Table 4). In some embodiments, the antibody sequences are those of Abl09, Abl33, or Abl41, or antigen-binding fragments thereof. In some embodiments, the antibody is selected from Abl01, Abl02, Abl03, Abl04, Abl05, Abl06, Abl07, Abl08, Abl09, Abl10, Abl11, Abl12, Abl13, Abl14, Abl15, Abl16, Abl17, Abl18, Abl19, Abl20, Abl21, Abl22, Abl23, Abl24, Abl25, Abl26, Abl27, Abl28, Abl29, Abl30, Abl31, Abl32, Abl33, Abl34, Abl35, Abl36, Abl37, Abl38, Abl39, Abl40 and Abl41, or an antigen-binding fragment thereof.In a preferred embodiment, the antibody is selected from Ab 109, Ab 133, and Ab 141. The antibody or antigen-binding fragment can 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 these diseases or conditions.

[0383] The diseases and conditions encompassed by the present disclosure include, but are not limited to, metabolic disorders, such as metabolic syndrome, obesity, type 2 diabetes (T2DM) (e.g., adult-onset diabetes), prediabetes, and the T2DM associated with obesity. T2DM is a chronic and progressive metabolic disease characterized by elevated blood glucose levels, which, if not treated, can damage multiple organs, leading to complications, such as cardiovascular disease, neuropathy, nephropathy (e.g., diabetic nephropathy), and retinopathy (e.g., diabetic retinopathy). Obesity is a risk factor for multiple serious medical conditions, including cardiovascular disease, prediabetes, T2DM, NAFLD, NASH, certain types of cancer, Alzheimer's disease, etc. Therefore, treating obesity can treat or improve these conditions. In some embodiments of the present disclosure, the patient is treated without TGFβ inhibitors (e.g., TGFβ1 inhibitors). In certain embodiments, the patient suffers from metabolic liver disease, wherein optionally, the metabolic liver disease comprises NAFLD, wherein further optionally, the metabolic liver disease has not yet progressed to NASH, wherein optionally, the metabolic liver disease has not yet progressed to fibrosis, or wherein optionally, the metabolic liver disease has not yet progressed to cirrhosis.

[0384] In some embodiments, preferred myostatin inhibitors for therapeutic use are myostatin selective inhibitors, such as any of the myostatin selective antibodies or antigen-binding fragments disclosed herein, for example, antibodies or antigen-binding fragments that selectively bind to promyostatin / latent myostatin, such as any of Ab101-Ab141. Without wishing to be bound by theory, it may be beneficial to use a myostatin selective inhibitor to provide the desired therapeutic efficacy while avoiding undesirable side effects, such as those associated with non-selective myostatin inhibition. In some embodiments, the desired therapeutic efficacy includes at least one (e.g., all) of the following: loss of fat mass over lean body mass; maintenance of fat mass loss; prevention of muscle loss; increase in muscle mass; increased endurance; reduction in fatigue; prevention of bone loss; improved blood sugar levels; and / or improved liver health. In some embodiments, it may be beneficial to avoid GDF11 inhibition, which has been reported to promote metabolic health but is also associated with side effects. See, e.g., 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), the contents of which are hereby incorporated in their entirety.

[0385] In some embodiments, combining a selective myostatin activation inhibitor with a GLP-1 pathway activator, a GIP activator or antagonist, or a glucagon modulator can provide additive or synergistic benefits to the subject's metabolic health, such as preventing muscle loss during weight loss, allowing for greater amounts of fat loss, including visceral fat. Combining a selective myostatin activation inhibitor with a GLP-1 pathway activator (e.g., a GLP-1 receptor agonist) can also increase the durability of the therapeutic effect, such as maintaining muscle mass, which can promote the durability of metabolic benefits.

[0386] Exemplary conditions for which the compositions and methods of the present disclosure are applicable are further described below.

[0387] Metabolic disorders and diseases

[0388] In various embodiments, disclosed herein is a method for treating or preventing a metabolic disease in a subject. Metabolic disease (also referred to as metabolic disease, illness or metabolic condition) is generally associated with glucose, lipid / fat and / or protein / nitrogen metabolism abnormalities or osmotic pressure regulation abnormalities, and has the pathological findings caused by such a patient's condition. Many metabolic disorders disclosed herein share certain features, such as they are associated with: fat-free mass or lean muscle mass loss, excessive fat mass, lower metabolic rate, insulin resistance, lack of ability to regulate blood sugar, weight gain and / or body mass index increase. In some cases, such metabolic disease or illness can be triggered or aggravated by the drug therapy received by the patient.

[0389] The present disclosure is based, at least in part, on the discovery that administering 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 the subject's physiological and functional characteristics.

[0390] Other examples of metabolic diseases 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 diabetes associated with obesity), obesity (e.g., adult-onset obesity, diet-induced obesity, pediatric 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, kidney disease (e.g., chronic kidney disease), syndrome X, fatty liver disease, metabolic bone disease. Spinal cord injury (SCI) (e.g., complete or incomplete / partial SCI), hypometabolic states, dual diabetes, and Cushing's disease (also known as Cushing's syndrome). In some embodiments, metabolic diseases include diseases associated with impaired neural signaling or partial denervation. In some embodiments, metabolic diseases include conditions (e.g., side effects) caused by or associated with certain drugs.

[0391] Additional diseases or conditions associated with metabolic disturbances and / or body composition will be apparent to those skilled in the art and are within the scope of the present disclosure.

[0392] As discussed in more detail herein, metabolic disturbances may occur secondary to, or as a result of, muscle conditions or disorders. Since muscle homeostasis is linked to amino acid / protein metabolism, it is further contemplated that myostatin inhibition may in turn regulate nitrogen metabolism and nitrogen transport in the body. In muscle catabolism, muscle tissue is broken down into its building blocks, amino acids, which can be considered the primary reservoir (and thus source) of nitrogen. Nitrogen is an element in ammonia that is highly toxic to the body and is excreted in the human body as urea. When nitrogen metabolism is dysregulated, possible consequences include imbalances in fluid retention, which can manifest as systemic or localized edema (e.g., congestion or fluid overload).

[0393] Diabetes is a prevalent metabolic disease that refers to a group of metabolic disorders characterized by high blood sugar (glucose) levels, which are caused by the secretion or action of insulin, or both. Insulin is a hormone released by the pancreas in response to elevated blood sugar (glucose) levels. 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.

[0394] In type 2 diabetes (T2DM) (also known as non-insulin-dependent diabetes mellitus, NDDM), the pancreas continues to produce insulin, sometimes even at higher levels than normal. However, the body develops resistance to its effects, resulting in a relative lack of insulin. As the pancreas continues to produce insulin and the body becomes insulin-resistant, the insulin-producing cells in the islets of Langerhans in the pancreas may become depleted, limiting or losing their ability to continue producing insulin. Type 2 diabetes can occur in children and adolescents, but usually begins after the age of 30 and gradually becomes more common with age: approximately 15% of people over the age of 70 suffer from type 2 diabetes. Obesity is a risk factor for type 2 diabetes, and 80% to 90% of people with this disorder are obese.

[0395] In some embodiments, diabetes includes prediabetes. "Prediabetes" refers to one or more early diabetic conditions, including impaired glucose utilization, abnormal or impaired fasting glucose levels, impaired glucose tolerance, impaired insulin sensitivity, and insulin resistance. Prediabetes is a major risk factor for developing type 2 diabetes, cardiovascular disease, and mortality. There has been significant development of therapeutic interventions to prevent the onset or progression of type 2 diabetes by effectively treating prediabetes.

[0396] In some embodiments, diabetes includes dual diabetes, which is a combination of type 1 diabetes and type 2 diabetes with features of insulin resistance.

[0397] Diabetes and prediabetes can be diagnosed by administering a glucose tolerance test, which can be measured by drawing venous blood from a fasting or non-fasting subject. It can also be diagnosed by measuring blood hemoglobin A1C (A1C) levels, which is a glycosylated form of hemoglobin that reflects the average amount of glucose in the blood over the past two to three months. Normal, prediabetic, and diabetic A1C levels are known in the art and can vary with age. In some embodiments, the expected value for a normal fasting blood glucose concentration can be between about 70 mg / dL (3.9 mmol / L) and about 100 mg / dL (5.6 mmol / L). In some embodiments, prediabetes is associated with a hemoglobin A1C level 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 a fasting glucose level greater than about 125 mg / dL or a hemoglobin A1C level greater than about 6.9 mmol / L.

[0398] Clinically, diabetes is usually divided into several basic categories.The main examples of these categories include, autoimmune diabetes, non-insulin dependent diabetes mellitus (type 1 NDDM), insulin-dependent diabetes mellitus (type 2 IDDM), non-autoimmune diabetes, non-insulin dependent diabetes mellitus (type 2 NIDDM) and maturity-onset diabetes of the young (MODY).Another category (commonly referred to as secondary) refers to the diabetes caused by some identifiable medical conditions, and these medical conditions cause or allow diabetic syndrome to occur.The example of secondary category includes the diabetes caused by pancreatic disease, hormone abnormalities, the diabetes induced by drugs or chemicals, the diabetes caused by insulin receptor abnormalities, the diabetes related to genetic syndrome and the diabetes of other causes of disease.(see, for example, Harrison's (1996) the 14th edition, see, for example, McGraw-Hill [McGraw-Hill Press]).

[0399] Obesity is another prevalent metabolic disease that can be treated or prevented by the methods of the present disclosure. "Obesity" refers to a chronic condition defined by excess body fat. Normal body fat content (expressed as a percentage of body weight) for women is between 25%-30% and for men between 18%-23%. Women with more than 30% body fat and men with more than 25% body fat are considered obese. Any clinically relevant definition can be used to define obesity. For example, in adults, body mass index (BMI, kg / m2) is frequently used as a measure of overweight and obesity, where overweight is defined as BMI 25-29.9 kg / m2, obesity is defined as a BMI equal to or greater than 30 kg / m2, and morbid obesity is defined as a BMI greater than 40 kg / m2. Adult obesity can also be defined as central obesity measured by waist circumference, where an increased waist circumference is defined as being equal to or greater than 102 cm for men and equal to or greater than 88 cm for women.

[0400] The experimenter suffering from obesity can show other symptoms, such as increase in fasting plasma glucose, glucose intolerance, heart failure, hypertension, insulin resistance, increase in fasting plasma triglycerides, reduction in fasting high-density lipoprotein (HDL) levels, prediabetes, increase in blood pressure, stroke, heart failure, obstructive sleep apnea, reproductive hormone damage, obstructive sleep apnea, osteoarthritis, gallstones, gastroesophageal reflux or nephropathy. Obesity may also cause various orthopedic problems, skin disorders and swelling of the feet and ankles. The serious complications of obesity include the much higher risk of coronary artery disease and its major risk factors type II diabetes, hyperlipidemia and hypertension. Most of the morbidity related to obesity is relevant to type II diabetes, because poorly controlled diabetes and obesity cause a series of symptoms, which are collectively referred to as syndrome X or metabolic syndrome. In certain embodiments, obesity is sarcopenia obesity. In certain embodiments, the experimenter suffering from obesity is carrying out a caloric restriction program.

[0401] In one aspect, the methods of the present disclosure are suitable for treating all forms of obesity, including diabetes-related obesity, metabolic syndrome-related obesity, single gene disorder-related obesity, antipsychotic drug use-related obesity, glucocorticoid-related obesity, and hypothalamic obesity.

[0402] In another aspect, the method of the present disclosure is suitable for treating or preventing metabolic diseases, such as obesity syndrome.Term " obesity syndrome " refers to any disease or the patient's condition that causes experimenter to be very fat or overweight.As with other metabolic diseases, the people suffering from obesity syndrome are usually relevant with the loss of fat-free or lean muscle mass, excessive fat amount, lower metabolic rate, insulin resistance, lack the ability to regulate blood sugar, weight gain and / or body mass index increase.In certain embodiments, obesity syndrome is selected from the group consisting of: Prader-Willi syndrome (Prader Willi), the obesity syndrome related to genetic disorder and the obesity syndrome related to hypothalamic disorder.

[0403] The methods disclosed herein are also suitable for treating or preventing metabolic diseases, such as metabolic syndrome. As used herein, "metabolic syndrome" refers to the concept of a group of metabolic risk factors that are clustered in a single individual and lead to a high risk of developing diabetes and / or cardiovascular disease. The main features of metabolic syndrome include insulin resistance, hypertension (high blood pressure), dyscholesterolemia, dyslipidemia, dystriglycerideemia, increased risk of coagulation, and excess weight (especially abdominal) 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 (male, equal to or greater than 40 inches (102 cm); female, equal to or greater than 35 inches (88 cm)); (2) high triglycerides (equal to or greater than 150 mg / dL); (3) low high-density lipoprotein cholesterol (HDL) (male, less than 40 mg / dL; female, less than 50 mg / dL); (4) high blood pressure (equal to or greater than 130 / 85 mm Hg); and (5) high fasting glucose (equal to or greater than 100 mg / dL).

[0404] Body composition can be measured by a variety of methods, including dual-energy X-ray absorptiometry (DEXA). Whole-body scans using DEXA provide generally accurate and precise measurements of body composition, including bone mineral content, bone mineral density, lean tissue mass, adipose tissue mass, and the fractional contribution of fat.

[0405] Obesity is a risk factor for cardiovascular disease. Compared to individuals of normal weight, obese individuals experience cardiovascular disease events earlier, live with cardiovascular disease for a greater portion of their lives, and have a shorter life expectancy. 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 cardiovascular mortality (independent of other cardiovascular risk factors). Visceral obesity promotes systemic and vascular inflammation, which underlies the atherosclerotic process. Obesity-induced inflammation increases the likelihood of LDL oxidation, thereby promoting atherogenesis. Insulin resistance is associated with dyslipidemia and metabolic syndrome, which are associated with atherosclerosis. Endothelial dysfunction in obesity (e.g., due to reduced bioavailability of nitric oxide in response to inflammation and oxidative stress) also contributes to the progression of atherosclerosis. Obesity has also been associated with abnormalities in the coronary microvasculature and pericardial coronary arteries. Another aspect of the present disclosure includes methods of treating a subject suffering from a metabolic disease or condition associated with aging. Exemplary diseases and conditions associated with aging include, but are not limited to, sarcopenia (age-related muscle loss), frailty, and androgen deficiency.

[0406] Therefore, the method of the present disclosure is suitable for treating or preventing metabolic diseases, such as cardiovascular diseases, for example, the cardiovascular diseases associated with metabolic syndrome.Term " cardiovascular disease " refers to any disease of the heart or blood vessels.Cardiovascular or heart disease include but are not limited to such as angina pectoris, arrhythmia, coronary artery disease (CAD), coronary heart disease, cardiomyopathy (including dilated cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy and diabetic cardiomyopathy), heart attack (myocardial infarction), heart failure (such as acute heart failure (AHF), chronic heart failure (CHF) or normal ejection fraction heart failure (HfpEF)), hypertrophic cardiomyopathy, mitral valve regurgitation, mitral valve prolapse, pulmonary valve stenosis etc.Vascular disease includes but is not limited to such as peripheral vascular disease, arterial disease, carotid artery disease, deep vein thrombosis, venous disease and atherosclerosis.In certain embodiments, the subject with heart failure is resistant to diuretic therapy.In another embodiment, the subject with heart failure responds poorly to diuretic therapy. It is contemplated 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 (e.g., GLP-1 receptor agonists). In preferred embodiments, the myostatin inhibitor is a myostatin selective inhibitor. In particularly preferred embodiments, the myostatin inhibitor is selected from the novel antibodies disclosed herein, e.g., any one of Abl01-Ab141. In a most preferred embodiment, the myostatin selective inhibitor is selected from Abl09, Abl33, and Abl41.

[0407] For example, pulmonary edema and renal congestion are frequently observed in patients with heart failure associated with decreased cardiac output. Pulmonary congestion is actually the most frequent cause of hospitalization in this clinical setting and is associated with a poor prognosis. Similarly, in pathological conditions involving osmotic pressure regulation disorders, affected individuals may be particularly sensitive to salt intake, which may cause or exacerbate fluid overload. Therefore, for subjects with fluid retention or volume overload (such as subjects with osmotic pressure regulation disorders and subjects with heart failure (e.g., chronic heart failure)), current guidelines indicate that diuretic therapy should be used to attempt to eliminate congestion (see, for example, Regolisti et al., Nephrology in Point of Care 2016; 2(1): e73-e87). However, in many cases, diuretic therapy is ineffective or the subject is refractory to diuretic treatment. Myostatin inhibition according to the present disclosure may provide clinical benefits to such patients. In particular, the methods of the present disclosure are suitable for enhancing the response of subjects who are refractory to diuretic therapy, or who have not responded well to diuretic therapy. For example, administration of a myostatin inhibitor can reduce diuretic dosage requirements and / or improve control of symptoms (such as CHF symptoms); improve cardiac function; and / or prevent pathological cardiac remodeling or other chronic deterioration of cardiac function. Myostatin inhibition achieved using the inhibitors described herein also reduces the risk of CHF exacerbations, such as acute pulmonary edema attacks.

[0408] For subjects at higher risk of developing acute pulmonary edema (such as those receiving IV fluids, blood transfusions, or fluid transfers), the myostatin inhibitors disclosed herein can be administered prophylactically. For example, a myostatin inhibitor can be administered prophylactically to a subject with congestive heart failure who requires a blood transfusion during a blood transfusion to prevent the onset of acute pulmonary edema during the transfusion. For subjects with CHF and / or other volume overload states, who develop hyponatremia due to volume overload themselves or due to the use of diuretics to treat volume overload, the myostatin inhibitors disclosed herein can be administered to treat hyponatremia and / or to increase the dose of diuretics when the dose of diuretics is limited by hyponatremia as a side effect. However, in general, the myostatin inhibitors disclosed herein can be used to treat hyponatremia regardless of the underlying etiology.

[0409] For other volume overload states that require high doses of diuretics (e.g., renal failure or liver disease), the myostatin inhibitors disclosed herein reduce the necessary diuretic doses; improve control of symptoms such as peripheral edema or internal congestion (including pleural effusions, ascites, hepatic congestion, or intraocular volume overload that can cause retinal detachment); and / or reduce the risk of pulmonary edema.

[0410] Metabolic disorders and diseases treated according to the methods provided herein also include metabolic conditions that affect the liver. Non-alcoholic fatty liver disease (NAFLD) is a liver disease spectrum associated with metabolic and cardiovascular disorders and is strongly associated with metabolic syndrome (Godoy-Matos (2020) Diabetes Metab Synd [Diabetes and Metabolic Syndrome] 12:50). Metabolic conditions that affect the liver include non-alcoholic steatohepatitis (NASH), NAFLD, hereditary hemochromatosis, α-1 antitrypsin deficiency, and Wilson's disease (Wilson disease). When excessive tissue deposition causes fibrotic scars, fibrosis (thickening of connective tissue) occurs in both NASH and NAFLD in the late stages. Fibroblast growth factor 21 (FGF21) and FGF19 have been observed to reduce hepatic steatosis. Accordingly, in some embodiments, the subject receives 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 for treatment of liver disease: Hydronidone, BIO89-100, Efruxifermin, Pegbelfermin, Aldafermin, MK-3655, PRI-724, Selonsertib, CC-90001, Epeleuton, Elafibranor, Saroglitazar, Lanifibranor, Pemafibrate, ZSP0678, Obeticholic acid, Acid), Cilofexor, Nidufexor, TERN-101, Vonafexor, EDP-305, Tropifexor, JKB-121, JKB-122, Semaglutide, Telportide, Cotudotepide, HM-15211, Resmetirom, VK2809, Cenicriviroc, Belapectin, GB1211, Azemiglitazone potassium Potassium), deuterium-stabilized 1-pioglitazone, Aramchol, PF-05221304, Firsocostat, ZSP1601, Epeleuton, PXL-770, ALS-L1023, Namodenoson, TVB-2640, LPCN 1144, HepaStem, BMS-986263, Foralumab, Elobixibat, Apararenone, PF-06835919, ARO-HSD, and CB4211.

[0411] The compositions and methods of the present disclosure are also suitable for treating or preventing metabolic diseases associated with a hypometabolic state. The term "hypometabolic state" refers to a state of reduced metabolism or metabolic activity, in which the body cannot produce enough energy. Patients in a hypometabolic state are typically associated with a lower metabolic rate, loss of fat-free or lean muscle mass, excessive increase in fat mass, insulin resistance, lack of ability to regulate blood sugar, weight gain, and / or an increase in body mass index. In some embodiments, the hypometabolic state is selected from the group consisting of: a state associated with prolonged immobilization, a state associated with bed rest, a state associated with cast immobilization, a state associated with stroke, a state associated with resection, and a postoperative state. In some embodiments, the hypometabolic state is a postoperative state, such as paraspinal muscle atrophy after lumbar surgery. In one embodiment, paraspinal muscle atrophy is nerve damage-dependent muscle atrophy. In one embodiment, the surgery is spinal surgery. In one embodiment, the spinal surgery is a lumbar spine surgery or lumbar spine 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, or the like.

[0412] In some embodiments, the myostatin inhibitors of the present invention can be used to attenuate spinal cord injury (SCI)-induced sublesional muscle mass loss and overall body weight loss, while also reducing the mass of unwanted adipose tissue (such as white and visceral adipose tissue). Subjects treated with myostatin inhibitor therapy can also demonstrate significant improvements in their motor function, muscle strength, and motor coordination and balance skills.

[0413] In another aspect, the methods of the present disclosure are suitable for treating or preventing metabolic diseases such as Cushing's disease, which is also known as Cushing's syndrome or hypercortisolism. The term "Cushing's disease" refers to a series of signs and symptoms caused by long-term 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 suitable as an alternative or additional treatment option to current standard of care treatments for patients with Cushing's disease.

[0414] Accordingly, the present disclosure provides methods for treating or preventing metabolic diseases in human subjects. These methods include selecting a human subject with a metabolic disease and administering an effective amount of a myostatin inhibitor (e.g., Ab102, Ab109, Ab130, Ab132, or Ab133) to the human subject to treat or prevent the metabolic disease in the human subject. The myostatin inhibitor is preferably a myostatin selective inhibitor. The myostatin selective inhibitor is more preferably an antibody or antigen-binding fragment thereof that specifically binds to promyostatin / latent myostatin but does not bind to GDF11, such as Ab102, Ab109, Ab130, Ab132, or Ab133. Antibodies that specifically recognize promyostatin / latent myostatin but do not recognize GDF11 are beneficial and avoid adverse toxicity caused by off-target binding of antibodies to GDF11 in the subject. In one embodiment, the subject is a pediatric subject. In one embodiment, the subject is a subject aged 2-19 years (inclusive). In one embodiment, the subject is a subject 12 years of age or older (eg, 12-17 years of age, inclusive).

[0415] In one embodiment, the monoclonal antibody specifically binds to and inhibits the activation step of myostatin / GDF8. In some embodiments, such antibodies bind to pro-myostatin 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 its fragment binds to a bound form (e.g., intramuscularly) of inactive myostatin (e.g., pro-myostatin), which has the ability to act locally on tissue-associated myostatin within the disease niche. In some embodiments, the antibody or its fragment 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 that may have endocrine or systemic effects. In any of such embodiments, preferred myostatin inhibitors for practicing the methods of the present disclosure are those that are selective for myostatin but do not antagonize other members of the growth factor / cytokine TGFβ superfamily (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) may produce harmful or unwanted side effects or adverse events. In any of such embodiments, preferred myostatin inhibitors for practicing the methods of the present disclosure comprise Abl02, Abl09, Abl30, Abl32, or Abl33.

[0416] weight management

[0417] Currently available obesity therapies, such as GLP-1 receptor agonists, focus almost entirely on weight loss. In contrast, the present disclosure considers the quality of weight management, rather than simply weight loss, to achieve improved metabolic health. To this end, it is contemplated that incorporating a myostatin inhibitor into a weight management regimen may achieve one or more of the following: preferential loss of fat mass over lean body mass; maintenance of fat mass loss; prevention of muscle loss; increase in muscle mass; increased endurance; reduced fatigue; prevention of bone loss; improved blood glucose levels; and / or improved 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 combination with another therapy aimed at addressing metabolic dysregulation.

[0418] The present disclosure further provides methods for promoting robust weight loss (e.g., loss of fat mass (i.e., body fat weight) without concomitant loss of lean muscle mass) in healthy subjects (e.g., fitness enthusiasts) or subjects with metabolic diseases such as obesity (e.g., diet-induced obesity), metabolic syndrome, and / or type 2 diabetes (T2DM). Compared to dieting alone (e.g., calorie-restricted dieting, low-carbohydrate diet, ketogenic diet, vegan diet, etc.) (wherein both fat stores and muscle mass lose weight during the diet), 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) results in a loss of fat stores while avoiding muscle loss. In some embodiments, administration of a myostatin inhibitor results in a loss of about 5%, about 10%, about 15%, or about 20% or more of the subject's fat mass during treatment.

[0419] By middle age, body fat mass increases with age in both men and women. Specifically, subjects are at higher risk for cardiovascular disease, metabolic syndrome, hypertension, diabetes, or dyslipidemia associated with abdominal fat than subjects without abdominal obesity. Fat mass, or abdominal 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 greater in men and 88 cm or greater in women.

[0420] 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., Abl02, Abl09, Abl30, Abl32, Abl33, or Abl41) can result in more robust weight loss compared to dieting or administering other standard therapies alone due to: maintaining a higher metabolic rate; improved cardiometabolic benefits (such as lipid profile, glucose metabolism, cardiovascular risk, etc.); and higher fat (e.g., total fat or visceral fat and / or other harmful fat levels) reduction. In addition, administration of a myostatin inhibitor therapy can prevent or reduce muscle atrophy and / or bone loss that can occur with a diet (e.g., a calorie-restricted diet, a low-carbohydrate diet, a ketogenic diet, etc.). Overall, administration of a myostatin inhibitor disclosed herein (e.g., an anti-myostatin antibody or antigen-binding fragment thereof disclosed herein, e.g., Abl02, Abl09, Abl30, Abl32, Abl33, or Abl41) can increase the ratio of muscle to fat in a subject.

[0421] In subjects with metabolic diseases (e.g., obesity, metabolic syndrome, and / or diabetes, such as T2DM), administration of a myostatin inhibitor disclosed herein (e.g., an anti-myostatin antibody or antigen-binding fragment thereof disclosed herein, such as Abl02, Abl09, Abl30, Abl32, Abl33, or Abl41) can prevent or alleviate a decrease in the subject's metabolic rate and prevent or reduce lean muscle loss. Myostatin inhibitor therapy can be combined with another standard therapy (e.g., a diet, such as a calorie-restricted diet). In some embodiments, a moderate calorie-restricted diet is recommended because it provides better patient compliance and better long-term results because the subject does not have to adhere to a strict, aggressive diet, such as an aggressive calorie-restricted diet.

[0422] Such treatments are particularly useful for subjects with limited physical activity, such as those with orthopedic injuries, spinal cord injuries, musculoskeletal diseases, pulmonary disorders, cardiovascular disorders, neurological disorders, severe obesity, and the like. In such subjects, administration of a myostatin inhibitor therapy can prevent muscle atrophy and / or bone loss, which are more pronounced in these subjects due to their limited physical activity. In some embodiments, such therapy enables subjects with limited physical activity to adopt a more robust diet, such as a calorically restricted diet, because the subjects are no longer limited by muscle loss or bone loss due to the administration of the myostatin inhibitor.

[0423] In some embodiments, the subject is following a diet regimen but not an exercise regimen. In some embodiments, the subject is following an exercise regimen but not a diet regimen. In some embodiments, the subject is following a diet regimen and an exercise regimen. Examples of diet regimens include, but are not limited to, caloric restriction (e.g., reducing caloric intake or reducing caloric absorption), a diet with improved nutrients (e.g., high protein, low fat, low carbohydrate, ketone, original diet, etc.), or changing the time of food intake (e.g., intermittent fasting, increasing feeding frequency, etc.), or changing the combination of time, portion size, and nutrients (e.g., more frequent small meals, containing high protein, low fat, and / or other nutrient restrictions).

[0424] Although the availability of GLP-1 receptor agonists has had a huge impact on obesity therapy in recent years, rapid weight loss is associated with decreased bone density. Applicants have previously shown that selective inhibition of myostatin can strengthen bones (PCT / US2018 / 012686, the contents of which are hereby incorporated in their entirety). This, coupled with the observation that GDF11 can exert a beneficial effect on bones, suggests that myostatin selective methods may provide advantages over non-selective methods in preventing bone loss during obesity treatment. For example, Suh et al. reported that GDF11 promotes osteogenesis and follistatin increases muscle mass, but weakens bones (Proc Natl Acad Sci US A. [Proceedings of the National Academy of Sciences of the United States] March 3, 2020; 117 (9): 4910-4920; doi: 10.1073 / pnas.1916034117). Therefore, it is contemplated that inhibition of GDF11 may be detrimental to bone health, and that selectivity of myostatin inhibition (eg, relative to activin A or GDF11 inhibition) may be beneficial in preventing bone loss.

[0425] Accordingly, the present disclosure encompasses methods for preventing bone loss using myostatin selective inhibitors, 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 does not inhibit GDF11 or activin A. In some embodiments, myostatin selective inhibitors include, but are not limited to, neutralizing antibodies that bind to mature myostatin and thereby prevent or interfere with receptor binding, and activating antibodies that bind to promyostatin / latent myostatin and inhibit myostatin activation. In some embodiments, the myostatin selective inhibitor is any of the antibodies or antigen-binding fragments disclosed herein. In preferred embodiments, the myostatin inhibitor is an inhibitor that selectively targets promyostatin / latent myostatin, such as Abl09, Abl33, or Abl41. In a preferred embodiment, the myostatin inhibitor is Abl09.

[0426] Diseases associated with impaired nerve signaling

[0427] The antibodies and antigen-binding fragments disclosed herein may be useful in intervening in conditions involving a lack of communication between muscles and their innervating neurons. The spinal cord houses the primary nerves that control motor function. Thus, the present disclosure provides methods for treating or preventing diseases associated with impaired neural signaling between neurons in a subject (e.g., a human subject) and target tissue expressing myostatin. The condition may be, for example, an injury-based condition (e.g., spinal cord injury) or a genetic condition (e.g., caused by a genetic mutation, such as SMA).

[0428] In some embodiments, the method comprises administering 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) to a subject suffering from a disease associated with impaired neural signaling between neurons and target tissues, thereby treating or preventing the subject's disease associated with impaired neural signaling. Preferably, the antibody or antigen-binding fragment thereof specifically binds to promyostatin / 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 implementing these embodiments include Abl02, Abl09, Abl30, Abl32, Abl33, and Abl41.

[0429] As used herein, the term "disease in which neural signaling is impaired" refers to any disease or condition caused by or associated with an interruption in signal transduction or communication between neurons and their target tissues (e.g., muscle tissue, brain tissue, liver tissue, vascular tissue, or adipose tissue). In some embodiments, neural signaling is impaired due to damage to the neuronal structure, where the neurons are unable to transmit signals to their targets. In other embodiments, the neuronal structure remains intact, but there is functional damage or deficiency, such as a blockage at the neuromuscular junction, which affects the ability of the neurons to transmit signals.

[0430] In some embodiments, "disease in which neural signal conduction is impaired" refers to a disease or condition associated with neurectomy (e.g., partial loss or interference of nerve distribution to its target or neuronal afferents to its target (such as muscle)). In some embodiments, injury induces neurectomy. In some embodiments, neurectomy is associated with a disease (such as a genetic disease). In the case of a genetic disease, in some embodiments, the patient can be diagnosed with a genetic disease by genetic screening. In some embodiments, such genetic screening can be performed on a fetus, a newborn, or a pediatric subject. Non-limiting examples of diseases in which neural signal conduction is impaired include, for example, vocal cord paralysis / 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).

[0431] Spinal cord injury

[0432] The method of the present disclosure is suitable for treating or preventing the condition in which nerve signal conduction is impaired due to nerve damage. In certain embodiments, such a condition ...

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to promyostatin / 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 SFTGSGGX1YYPDSVKG (SEQ ID NO: 202), wherein 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), wherein X1 is M or Q, and X2 is P or G, wherein the CDR sequences are numbered according to the Kabat numbering system.

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 the sequence of any one of SEQ ID NOs: 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 the sequence of any one of SEQ ID NOs: 223, 225, 227, or 298, as defined according to the Kabat numbering system.

3. The antibody or antigen-binding fragment thereof of claim 1 or claim 2, comprising 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 according to the Kabat numbering system.

4. The antibody or antigen-binding fragment thereof of claim 1 or claim 2, comprising 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 according to the Kabat numbering system.

5. The antibody or antigen-binding fragment thereof of claim 1 or claim 2, comprising 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 according to the Kabat numbering system.

6. The antibody or antigen-binding fragment thereof of claim 1 or claim 2, comprising 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, as defined according to the Kabat numbering system.

7. 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. The antibody or antigen-binding fragment of claim 7, comprising a variable domain sequence pair 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. 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. The antibody or antigen-binding fragment thereof of claim 9, comprising a heavy chain and light chain sequence pair 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. An antibody or antigen-binding fragment thereof that specifically binds to promyostatin / 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 X1QATQFPRP (SEQ ID NO: 210), wherein X1 is M or Q, and wherein the CDR sequences are numbered according to Kabat.

12. The antibody or antigen-binding fragment thereof of claim 11, wherein 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, as defined according to the Kabat numbering system.

13. The antibody or antigen-binding fragment thereof of claim 11 or claim 12, wherein 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, as defined according to the Kabat numbering system.

14. The antibody or antigen-binding fragment thereof of claim 11 or claim 12, wherein 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, as defined according to the Kabat numbering system.

15. The antibody or antigen-binding fragment thereof of claim 11 or claim 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. The antibody or antigen-binding fragment of claim 7, comprising a variable domain sequence pair comprising SEQ ID NOs: 400 and 410, or SEQ ID NOs: 407 and 417.

17. The antibody or antigen-binding fragment thereof of claim 11 or claim 12, comprising a heavy chain sequence that is at least 70% identical to any one of SEQ ID NO: 501 or 505, and / or a light chain sequence that is at least 70% identical to any one of SEQ ID NO: 502 or 506.

18. The antibody or antigen-binding fragment thereof of claim 9, comprising a heavy chain and a light chain sequence pair 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 promyostatin / 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 GFTFSSYG (SEQ ID NO: 3); CDRH2 comprises the sequence FTGSGGX1 (SEQ ID NO: 291), wherein X1 is selected from the group consisting of 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); and CDRL3 comprises the sequence X1QQTQYPX2T (SEQ ID NO: 292), wherein X1 is M or Q and X2 is selected from P and G, wherein the CDR sequences are numbered according to IMGT.

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

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

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

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

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

25. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment has an equilibrium dissociation constant, KD, of less than 5 nM, wherein the antibody or antigen-binding fragment optionally 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. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment is capable of inhibiting mTLL-2-induced myostatin activation with an IC50 of less than 1 nM as measured by functional ELISA.

27. The antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the antibody or antigen-binding fragment has a 2:1 Fab:promyostatin binding stoichiometry, e.g., when the antibody and antigen are each mixed at 15 μM and allowed to form an immune complex at neutral pH, and wherein the binding stoichiometry is measured by analytical size exclusion chromatography (SEC).

28. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the pH-sensitive binding of the antibody or antigen-binding fragment is at least 9-fold, e.g., at least 10-fold, higher than that of 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 the preceding claims, wherein the antibody or antigen-binding fragment is capable of reducing serum total myostatin levels compared to background levels.

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

31. An antibody or antigen-binding fragment thereof that specifically binds to promyostatin / 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 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); and CDRL3 comprises the amino acid sequence MQQTQYPPT (SEQ ID NO: 223), wherein the CDR sequences are numbered according to Kabat.

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 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.

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 at least 95% identical thereto; and / or a light chain sequence comprising the amino acid sequence of SEQ ID NO: 504, or a sequence at least 95% identical thereto.

34. The antibody or antigen-binding fragment of any one of claims 31-33, 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 promyostatin / latent myostatin or to a region of the prodomain of human promyostatin / latent myostatin located 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, as numbered according to SEQ ID NO:52) and / or one or more amino acid residues of the sequence KALDEN (SEQ ID NO:118) (amino acid positions 205-210 of human promyostatin, as numbered according to SEQ ID NO:52), wherein the antibody or antigen-binding fragment is not Ab2; Wherein such as by BLI based in vitro binding assay (eg ), the antibody binds to the antigen in a pH-dependent manner; and / or wherein the antibody 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., comprising detecting the presence of mature myostatin in an assay mixture comprising an antibody-antigen immune complex and mTLL-2.

36. The antibody or antigen-binding fragment thereof of claim 35, wherein i) the L-CDR1 of the antibody shares no more than 20% sequence identity with the L-CDR1 of Ab2; ii) the L-CDR2 of the antibody shares no more than 30% sequence identity with the L-CDR2 of Ab2; and / or iii) the L-CDR3 of the antibody shares no more than 10% sequence identity with the L-CDR3 of Ab2, or wherein 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. The antibody or antigen-binding fragment thereof of claim 35 or claim 36, wherein the antibody dissociates from the bound antigen at a higher rate under acidic conditions than under neutral conditions.

38. The antibody or antigen-binding fragment thereof of any one of claims 35-37, wherein the antibody is detected by an in vitro binding assay based on SPR (e.g., Bicore TM ), the antibody or antigen-binding fragment binds pro / latent myostatin with a divalent 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 39. The antibody or antigen-binding fragment of any one of claims 35-38, wherein the antibody is capable of binding to the antigen with a 1:2 antibody:antigen stoichiometry when the antibody and antigen are mixed at 15 μM each and allowed to form an immune complex at neutral pH, and wherein the binding stoichiometry is measured by analytical size exclusion chromatography (SEC).

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

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

42. An antibody or antigen-binding fragment thereof that binds to the prodomain of human promyostatin / latent myostatin, wherein the antibody or antigen-binding fragment: (a) As determined by an in vitro SPR-based binding assay (e.g., Biacore TM ), binds promyostatin / 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 (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 a functional ELISA, e.g., wherein the measuring comprises: detecting the presence of mature myostatin in an assay mixture comprising an antibody-antigen immune complex and mTLL-2; (c) As determined by BLI-based in vitro binding assays (e.g. ) dissociates from the bound antigen at a higher rate under acidic conditions than under neutral conditions; and (d) is capable of binding to the antigen with a 1:2 antibody:antigen stoichiometry, for example, when the antibody and antigen are each mixed at 15 μM and allowed to form an immune complex at neutral pH, and wherein the binding stoichiometry is measured by analytical size exclusion chromatography (SEC).

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

44. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of the preceding claims and a pharmaceutically acceptable excipient, wherein: The composition optionally further comprises a second agent, such as a GLP-1 pathway activator (eg, a GLP-1R agonist or a GLP-1 analog) and / or a biguanide (eg, metformin).

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 treating or preventing one or more of the following conditions in a human subject: muscle conditions (e.g., muscle wasting or myopathy), metabolic disorders (e.g., obesity or diabetes), bone conditions (e.g., bone loss), cardiovascular disease (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 disease (e.g., fatty liver disease, NAFLD, or NASH); wherein the treatment comprises administering the antibody, antigen-binding fragment or pharmaceutical composition in an amount effective to treat or prevent the one or more conditions.

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

47. The antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to claim 46, wherein the obesity is pediatric obesity, optionally wherein the antibody or antigen-binding fragment comprises Abl09 or an antigen-binding fragment thereof.

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

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

51. The antibody or antigen-binding fragment thereof, or pharmaceutical composition for use according to claim 45, wherein the one or more conditions comprise a liver disease, wherein optionally, the liver disease comprises fatty liver disease, NAFLD or NASH, wherein optionally, the subject has not been treated with a TGFβ inhibitor (e.g., a TGFβ1 inhibitor).

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

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

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

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, wherein optionally, the subject is undergoing a dietary and / or exercise regimen.

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, tilportide or retaglutide.

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

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

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 the rate of fat accumulation in the subject by at least 10% compared to fat accumulation in the subject before receiving the treatment.

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 the subject's fat mass by at least 5% (e.g., at least 10%, 15%, 20%, 25% or more) compared to a baseline value.

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 the amount of visceral fat in the individual by at least 5% compared to a baseline value.

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 the amount of subcutaneous fat in the individual by at least 5% compared to a baseline value.

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 body mass in the subject.

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

66. 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 useful for treating a 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 the antigen-binding fragment or the pharmaceutical composition and the second agent in an amount effective to treat the metabolic disorder, wherein optionally, the metabolic disorder is obesity, diabetes or both, Further optionally wherein the effective amount of the second agent is lower than the recommended monotherapy dose of the second agent.

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

68. The antibody, antigen-binding fragment, or pharmaceutical composition for use according to claim 66 or claim 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-Nyne syndrome, maple syrup urine disease, Monks 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 disease, or Zellweger syndrome.

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

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

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

72. A myostatin inhibitor for use in treating obesity or improving body composition in a subject, wherein the treatment comprises administering the myostatin inhibitor to the subject in combination with a biguanide, and wherein the subject is not receiving GLP-1 receptor agonist therapy at the time of the administration, and wherein the subject is an overweight or obese subject but is not a diabetic subject, wherein optionally, the biguanide is metformin.

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

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

75. The myostatin inhibitor, myostatin selective inhibitor, biguanide (e.g., metformin) or combination for use according to any one of claims 69-74, wherein the subject: i) Low tolerance to GLP-1 receptor agonists; ii) be a female of childbearing potential; iii) diagnosed with cancer or at risk of developing cancer; and / or iv) At risk of suicidal ideation, self-harm, or depression.

76. A myostatin inhibitor or a myostatin selective inhibitor, a biguanide (e.g., metformin) or a combination for use according to any one of claims 69 to 75, wherein 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 not to activin A or GDF11, wherein optionally, the neutralizing antibody is trogeluzumab or a variant thereof, wherein optionally the myostatin inhibitor does not cause a decrease in bone mineral density as measured by dual-energy x-ray absorptiometry compared to baseline.

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

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

79. Metformin and a non-selective myostatin inhibitor for use according to claim 74, wherein body composition is measured as the ratio of fat mass to lean body mass before and after said administration.

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

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

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

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

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

85. The method of any one of claims 81-84, wherein administration of the myostatin selective inhibitor prevents fat mass regain by greater than 20% compared to subjects following discontinuation of the GLP-1 receptor agonist but without administration of the myostatin selective inhibitor, wherein the myostatin selective inhibitor prevents fat mass regain for up to 6 months from the time of discontinuation of the GLP-1 receptor agonist.

86. The method of any one of claims 81-85, wherein administration of the myostatin selective inhibitor prevents a greater than 20% decrease in the ratio of lean body mass to fat mass compared to a subject after discontinuation of the GLP-1 receptor agonist but without administration of the myostatin selective inhibitor, wherein the myostatin selective inhibitor prevents fat mass rebound for up to 6 months from the time of discontinuation of the GLP-1 receptor agonist.

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

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

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

90. The method of claim 89, wherein the administration reduces fat mass regain compared to a subject following discontinuation of the GLP-1 receptor agonist but without administration of a myostatin selective inhibitor, wherein the myostatin selective inhibitor prevents fat mass regain for up to 6 months from the time of discontinuation of the GLP-1 receptor agonist.

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

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

93. The method of any one of claims 89-92, 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 89-93, wherein the myostatin selective inhibitor comprises the antibody or antigen-binding fragment of any one of claims 1-43.

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

96. The method of claim 95, wherein administration of the myostatin selective inhibitor reduces hepatic fat mass by at least 10% (e.g., 10%, 20%, 25% or more) relative to the subject's hepatic fat mass prior to administration of the myostatin selective inhibitor.

97. A method of improving bone strength and / or preventing bone loss in a subject (e.g., an obese subject), the method 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 bone fracture by at least 10% (e.g., 10%, 20%, 25% or more) compared to a subject not administered the myostatin selective inhibitor.

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

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

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

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 administration of the GLP-1 receptor agonist alone.

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

104. The method of any one of claims 100-103, wherein the subject is receiving or has received metformin.

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

106. A myostatin selective inhibitor for use in treating obesity in a subject or improving body composition in a subject, wherein the treatment comprises administering the myostatin selective inhibitor in combination with a GLP-1 receptor agonist and a biguanide to the subject, wherein the myostatin selective inhibitor, the GLP-1 receptor agonist, and the 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 tilportide.

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

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

110. A myostatin inhibitor for use in treating chronic inflammation in a subject, wherein the treatment comprises administering to the subject an amount of a myostatin inhibitor effective to treat the chronic inflammation, wherein optionally, the chronic inflammation is inflammation associated with a muscle disorder (e.g., inflammation associated with 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. A myostatin inhibitor for use according to claim 110, wherein the myostatin inhibitor is used in combination with an additional therapy, wherein optionally 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. The myostatin inhibitor for use according to claim 110 or claim 111, wherein the myostatin inhibitor is a myostatin selective inhibitor, wherein optionally, the myostatin selective inhibitor is the antibody or antigen-binding fragment of any one of claims 1-40, wherein further optionally, the myostatin selective inhibitor is Abl09, Abl33, Abl41, apilituzumab, trogelutumab, GYM329 or any variant thereof.

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

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