A novel class of MUSK agonists and uses thereof

Abstract

Disclosed herein is a novel class of anti-MuSK agonist antibodies capable of binding a unique epitope of MuSK. The present disclosure further includes the use of such antibodies for the treatment of muscle-related conditions, such as neuromuscular disorders.

Description

[0001] SR64-WO-PCT / 127036-05220

[0002] A NOVEL CLASS OF MuSK AGONISTS AND USES THEREOF

[0003] RELATED APPLICATIONS

[0004] [1] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 744,152 filed January 10, 2025, and U.S. Provisional Application No. 63 / 827,328 filed June 20, 2025, the contents of each of which are expressly incorporated herein by reference in their entireties.

[0005] SEQUENCE LISTING

[0006] [2] The instant application contains a Sequence Listing which has been submitted electronically in Sequence Listing XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 9, 2026 is named 127036-05220.xml and is 194,765 bytes in size.

[0007] FIELD OF THE INVENTION

[0008] [3] This invention generally relates to novel anti-MuSK antibodies capable of activating human MuSK.

[0009] BACKGROUND

[0010] [4] Muscle-specific receptor tyrosine kinase (MuSK) is a receptor tyrosine kinase and transmembrane receptor with a distinct structure. MuSK is highly expressed in skeletal muscle where it plays an important role in the formation and stabilization of neuromuscular synapses / junctions (NMJs). Formation of an NMJ is a complex signaling process that results in the clustering of acetylcholine receptors (AChRs) on the post-synaptic side of the NMJ. Deficiency in the MuSK pathway can result in NMJ dysfunction and neuromuscular disease, through impaired neuromuscular transmission and motor function, causing muscle weakness and / or atrophy.

[0011] [5] The biological process that regulates synaptogenesis at the NMJ is well understood and involves the key players, agrin, its receptor Lrp4, and MuSK (“the agrin-Lrp4-MuSK axis”). Proteinprotein interactions amongst these proteins orchestrate the downstream AChR clustering at the postsynaptic density on muscle, enabling efficient neurotransmission to occur at the NMJ. Defects in any components within this axis can lead to impaired signaling, resulting in motor dysfunction.

[0012] Because MuSK phosphorylation (i.e., activated MuSK) triggers AChR clustering, it is rationalized that targeting this point of the signaling axis could circumvent defects in the upstream events involving agrin and / or Lrp4, making MuSK an advantageous therapeutic target.

[0013] [6] To date, at least two agonistic antibodies have been described, which specifically target MuSK to induce its activation. In a 2018 publication, Cantor et al described an antibody referred to as “#13” which was shown to bind the Fz-like domain and induce phosphorylation of MuSK independent of Lrp4, and showed that antibody #13 (mAb#13) bound the MuSK Fz-like domain alone.. The authors demonstrated that mice treated with the #13 antibody improved innervation of the NMJ and slowed down muscle denervation in a murine model of ALS (Cantor et al, “Preserving Neuromuscular Synapses in ALS by Stimulating MuSK with a Therapeutic Agonist Antibody,” Elife 7:e34375 (2018)). Whilst mAb#13 showed agonistic potency towards murine MuSK, it does not cross-react with human MuSK, significantly limiting its utility as a therapy. More recently, WO 2021 / 212053 also disclosed a

[0014] 1

[0015] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0016] monoclonal antibody (“3B2G2” “3B2g2” or “3B2”) capable of binding the Fz-like domain of human MuSK and stimulating MuSK phosphorylation. The antibodies of WO 2021 / 212053 were described as binding an epitope within the Fz-like domain of human MuSK and binding this region was considered important to achieve optimized binding effects. This antibody was shown to enhance NMJ synapses and rescue lethality in Dok7 mutant mice. A humanized equivalent is being developed as ARGX-119.

[0017] SUMMARY

[0018] [7] The present disclosure provides a novel class of anti-MuSK antibodies capable of activating MuSK with superior potency and physicochemical properties, as compared to prior art. These antibodies bind the extracellular domain (ECD) of MuSK and are capable of inducing phosphorylation of MuSK. Notably, unlike the MuSK agonist antibodies described in prior art that can bind to the Fz-like domain alone, the antibodies disclosed herein require the presence of extra amino acid residues immediately C-terminus to the Fz-like domain for the antibody-antigen binding to occur. More specifically, the inventors recognized that inclusion of the 7 amino acid sequence immediately C-terminus to the Fz-like domain can provide a superior antigen to generate anti-MuSK antibodies with potent agonistic activity. This 7 amino acid-segment C-terminal to the Fz-like domain is herein referred to as the “Truncated Tail.” Without wishing to be bound by a particular theory, it is contemplated that the presence of the Truncated Tail C-terminal to the Fz-like domain mediates high-affinity binding of these antibodies to MuSK, possibly contributing to or stabilizing the epitope either directly or indirectly. Thus, the identification of this distinct epitope of MuSK represents a novel class of MuSK agonist antibodies. Thus, the invention includes the identification of a novel binding region of the MuSK protein that confers agonistic activity of MuSK.

[0019] [8] This discovery also provides improved screening methods for additional MuSK agonist antibodies, which employs the use of a novel antigen that includes a stretch of amino acids outside (C-term to) the Fz-like domain. In preferred embodiments, the stretch of amino acids outside of the Fz-like domain is or comprises the 7 amino acid residues immediately C-terminus to the Fz-like domain (i.e. , “Truncated Tail”). Preferably, additional MuSK agonist antibodies to be identified using the antigen that contains at least a portion of the tail region cross-block (e.g., cross-compete) with the novel antibodies disclosed herein (Ab1, Ab2, Ab3, Ab4, Ab5, Ab6). More preferably, such additional antibodies show species-cross-reactivity to both human and murine MuSK.

[0020] [9] Moreover, data disclosed herein show that the novel anti-MuSK antibodies of the present disclosure possess superior thermostability, as compared to a reference anti-hMuSK antibody of prior art. Greater protein stability may allow improved manufacturability and / or shelf-life of pharmaceutical products.

[0021]

[0010] In some embodiments, the antibodies of the novel class bind (e.g., directly make contact with) one or more amino acid residues of the 7 amino acids.

[0022]

[0011] The novel class of MuSK agonist antibodies described herein show superior properties conferred by the unique epitope, which can be attributable to the Truncated Tail region C-terminal to the Fz-like domain within the ECD of the MuSK protein. By contrast, the Truncated Tail C-terminal to the Fz-like domain is not required in the previously described anti-MuSK antibodies, such as mab#13

[0023] 2

[0024] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0025] and 3B2G2. Advantageously, the antibodies in this novel class show slower off rate (the rate at which an antibody dissociates from its bound antigen) as compared to the previously described MuSK agonists, which may contribute to more durable effects in vivo. Thus, anti-MuSK antibodies disclosed herein represent a novel, unexpected and superior class of MuSK agonists with unique binding properties.

[0026]

[0012] Accordingly, the invention includes an antibody that binds an extracellular domain fragment of human MuSK, wherein the ECD fragment includes the Truncated Tail C-terminal to the Frizzled-like domain. These antibodies require the presence of the Truncated Tail C-terminal to the Fz-like domain and do not bind the MuSK ECD when the C-terminal “tail” segments are absent. In some embodiments, the antibody binds an epitope that comprises one or more amino acid residues of the Truncated Tail of MuSK. In some embodiments, an antibody binds an epitope that comprises one or more amino acid residues of the Truncated Tail and one or more amino acid residues of the Frizzled-like domain. In some embodiments, the antibody binds an epitope that comprises one or more amino acid residues of the Frizzled-like domain wherein the epitope is a conformational epitope that requires the Truncated Tail.

[0027]

[0013] The present disclosure encompasses novel antibodies and antigen-binding fragments thereof that selectively bind to and activate MuSK, as well as therapeutic uses thereof. The binding of such antibodies and antigen-fragments thereof to MuSK potently activates it, leading toAChR clustering and improved formation and maintenance of the neuromuscular junction (NMJ). The antibodies of the present disclosure (such as Ab1 , Ab2, Ab3, Ab4, Ab5 and Ab6) and antigen-binding fragments thereof represent a new class of highly potent anti-MuSK agonist antibodies, distinct from previous antibodies (such as mAb13 and 3B2G2), as demonstrated by their differential binding profiles to human MuSK fragments and their unexpected effects in vitro and in vivo. Unlike previously described MuSK agonists {e.g., anti-MuSK antibodies mAb13 and 3B2G2), however, the antibodies and antigenbinding fragments thereof disclosed herein are capable of binding to fragments of human MuSK consisting of the Fz-like domain of human MuSK when the Truncated tail C-terminal to the Fz-like domain is present, but they do not bind to fragments of MuSK consisting of the Fz-like domain alone, i.e., where the Truncated tail C-terminal to the Fz-like domain is absent. Such antibodies and antigenbinding fragments thereof all bind to MuSK in a common epitope bin that is distinct from mAb13 and 3B2G2. They do not cross-block with mAb13 or 3B2G2, which bound in their own, distinct epitope bin (although mAb13 and 3B2G2 cross-block each other). Moreover, the antibodies and antigen-binding fragments thereof not only induced AChR clustering in vitro at similar potencies as compared to previously disclosed MuSK agonists (and without interference with agrin activation of MuSK), they also surprisingly showed in vivo effects on isolated muscle mass similar to or greater than 3B2G2, in particular leading to comparatively greater increases in muscle force in a mouse model for congenital myasthenic syndrome (with a maximum muscle force at 150 Hz stimulation of at least 30 mN or at least 50% of wild type control treated with HuNeg). In addition, Ab3 and Ab4 are further differentiated from the others by greater binding affinities, and by greater induction of AChR clustering in C2C12 myotubes compared to Ab1 and Ab2.

[0028] 3

[0029] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0030]

[0014] Antibodies Ab1 , Ab2, Ab3 Ab4, Ab5 and Ab6 (and antigen-binding fragments thereof) therefore define an important new class of anti-MuSK agonists with distinct binding features, potent activation of MuSK as measured by induction of AChR clustering, and superior kinetics and / or in vivo efficacy from previous anti-MuSK antibodies such as mAb13 and 3B2G2.

[0031]

[0015] Accordingly, in one aspect, the invention provides an antibody or antigen-binding fragment thereof that binds and activates human muscle-specific kinase (MuSK), wherein the antibody or antigen-binding fragment is capable of binding to a MuSK fragment consisting of SEQ ID NO: 7 but is not capable of binding to a MuSK fragment consisting of SEQ ID NO: 8.

[0032]

[0016] Without being bound by theory, the novel class of antibodies (e.g., Ab1 , Ab2, Ab3, Ab4, Ab5 and Ab6) (and antigen-binding fragments thereof) may bind to an epitope that includes one or more residues on human MuSK within PHLDYNK (SEQ ID NO: 9), because they require the presence of PHLDYNK to bind to human MuSK. Accordingly, in some embodiments, the antibodies of the present disclosure bind an epitope on human MuSK comprising one or more residues within SEQ ID NO: 9. Additionally or alternatively, the antibodies or antigen-binding fragments may bind to a conformational epitope that is formed only in the presence of the Truncated Tail region of MuSK. The confirmational epitope may or may not include one or more amino acid residues within the Truncated Tail. In any of the embodiments, however, the antibodies or antigen-binding fragments require the Truncated tail for binding to MuSK.

[0033]

[0017] Thus, in some embodiments, the antibody or antigen-binding fragment binds to an epitope that comprises one or more amino acid residues of SEQ ID NO: 9 (e.g. two or more, three or more, or four or more amino acid residues of SEQ ID NO: 9). In other embodiments, the antibody or antigenbinding fragment binds to an epitope that does not comprise any amino acids of SEQ ID NO: 9. In any of these embodiments, the antibody may bind to an epitope comprising one or more amino acids of SEQ ID NO: 8.

[0034]

[0018] In some embodiments, the antibody or antigen-binding fragment has a bivalent (e.g. mAb) binding affinity (ko) for human MuSK of < 12 nM, < 11 nM, more preferably < 10 nM, as measured by BioLayer Interferometry (BLI)-based in vitro binding assay. In some embodiments, the antibody or antigen-binding fragment also has a bivalent binding affinity (ko) for mouse MuSK of < 30 nM, as measured by BLI-based in vitro binding assay.

[0035]

[0019] In some embodiments, the antibody or antigen-binding fragment has a monovalent (e.g. Fab) binding affinity for human MuSK of < 2.5 nM, as measured by Surface Plasmon Resonance (SPR)-based in vitro binding assay. In some embodiments, the antibody or antigen-binding fragment has a monovalent binding affinity for mouse MuSK of <25nM, < 20nM, more preferably < 15 nM, as measured by SPR-based in vitro binding assay. The antibody or antigen-binding fragment may alternatively or additionally have a bivalent binding affinity for mouse MuSK of < 2 nM, <1 nM, more preferably < 0.5 nM, as measured by SPR-based in vitro binding assay.

[0036]

[0020] Binding affinity for human or mouse MuSK may be assessed by binding to the human or mouse MuSK extracellular domain, respectively.

[0037] 4

[0038] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0039]

[0021] In some embodiments, the antibody or antigen-binding fragment has an ECso of < 2.5 nM (e.g. < 2 nM, < 1 nM), as measured by a C2C12 myotube AChR clustering assay. In some embodiments, the antibody has an ECso of < 0.5 nM (e.g. < 0.2 nM), as measured by a C2C12 myotube AChR clustering assay.

[0040]

[0022] In some embodiments, the antibody or antigen-binding fragment elicits maximum muscle force at least 25 mN (e.g. at least 30 mN). In some embodiments, the antibody or antigen-binding fragment increases maximum muscle force by at least 20 mN as compared to an isotype matched control IgG. In some embodiments, the antibody or antigen-binding fragment increases maximum muscle force by at least 20 mN as compared to an isotype matched control IgG. The maximum muscle force may be measured at 150 Hz stimulation in an Agrn nmf380 mouse model. In some embodiments, the antibody or antigen-binding fragment increases maximum muscle force at 150 Hz stimulation in an Agrn nmf380 mouse model by at least two-fold or at least three-fold compared to an isotype matched control IgG.

[0041]

[0023] In some embodiments, the antibody or antigen-binding fragment restores maximum muscle force to greater than 30% (e.g. at least 50%, at least 60%) of the maximum muscle force of a wildtype control. In some embodiments, the antibody or antigen-binding fragment restores at least 50% (e.g. at least 60%) of maximum muscle force. The maximum muscle force may be measured at 150 Hz stimulation in an Agrn nmf380 mouse model. The control may be a wild-type control mouse treated with isotype matched control IgG.

[0042]

[0024] In some embodiments, the antibody or antigen-binding fragment a) increases mass of an isolated muscle in an Agrn nmf380 mouse model greater than a control, wherein the isolated muscle is a gastrocnemius or a tibialis anterior muscle, and the control is an antibody that is capable of binding to SEQ ID NO: 8; and / or b) increases mass of an isolated muscle in an Agrn nmf380 mouse model to greater than 50% of a control, wherein the isolated muscle is a gastrocnemius or a tibialis anterior muscle, and the control is a wild-type mouse (e.g. a wild-type control mouse treated with isotype matched control IgG). The antibody that is capable of binding to SEQ ID NO: 8 may be 3B2G2 as described herein.

[0043]

[0025] In some embodiments, the antibody or antigen-binding fragment a) produces a maximum muscle force of at least 25 mN (e.g. at least 30 mN) at 150 Hz stimulation in an Agrn nmf380 mouse model; and / or b) increases a maximum muscle force at 150 Hz stimulation in an Agrn nmf380 mouse model to at least 50% (e.g. at least 60%) of a control, wherein the control is a wild-type mouse (e.g. a wild-type control mouse treated with isotype matched control IgG).

[0044]

[0026] In some embodiments, the antibody or antigen-binding fragment produces an improvement in muscle force of at least 35%, at least 40% or at least 42% in an Agrn nmf380 mouse model compared to a control, wherein the control is a wild-type mouse (e.g. a wild-type control mouse treated with isotype matched control IgG).

[0045] 5

[0046] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0047]

[0027] In some embodiments, the antibody, or antigen-binding fragment increases a maximum muscle force at 150 Hz stimulation in an Agrn nmf380 mouse model to at least 55% of a control, wherein the control is a wild-type mouse (e.g. a wild-type control mouse treated with isotype matched control IgG). In some embodiments, the antibody, or antigen-binding fragment increases a maximum muscle force to at least 60% of a control. In some embodiments, the antibody, or antigen-binding fragment increases a maximum muscle force to at least 65% of a control. The control may be a wild-type mouse {e.g. a wild-type control mouse treated with isotype matched control IgG).

[0048]

[0028] In some embodiments, the antibody, or antigen-binding fragment, produces an improvement in a maximum muscle force at 150 Hz stimulation in an Agrn nmf380 mouse model that is at least 1.7-fold greater than a control, wherein the control is an Agrn nmf380 mouse model treated with isotype matched control IgG. In some embodiments, the antibody, or antigen-binding fragment, produces an improvement in a maximum muscle force that is at least 2-fold greater than a control. In some embodiments, the antibody, or antigen-binding fragment, produces an improvement in a maximum muscle force that is at least 2.2-fold greater than a control. The control may be a an Agrn nmf380 mouse model treated with isotype matched control IgG.

[0049]

[0029] In some embodiments, the muscle force or maximum muscle force is exerted by the plantarflexor muscle group. Thus, the muscle force or maximum muscle force may be measured in the plantarflexor muscle group.

[0050]

[0030] In some embodiments, the antibody or antigen-binding fragment does not comprise a L-CDR1 comprising amino acid residues Q and S as the first two residues.

[0051]

[0031] The antibody or antigen-binding fragment may not compete with Agrin for binding to MuSK. The antibody or antigen-binding fragment may not interfere with exogenous activation of MuSK by Agrin. The antibody or antigen-binding fragment is preferably cross-reactive for human and mouse MuSK. The antibody or antigen-binding fragment may induce MuSK phosphorylation e.g. at amino acid residue Y554, Y577, Y600, Y751 , S752, Y755 and / or Y756 of SEQ ID NO: 1 ). The antibody or antigen-binding fragment activates MuSK and induces acetylcholine receptor (AChR) clustering. The antibody or antigen-binding fragment may activate MuSK and induce acetylcholine receptor (AChR) clustering. For instance, the antibody or antigen-binding fragment may induce AChR clustering in myotubes in vitro {e.g. with an EC50 of less than 10 nM, 8 nM, 4 nM, 1 nM, 0.2 nM, 0.1 nM, as measured by a C2C12 myotube AChR clustering assay).

[0052]

[0032] In some embodiments, the antibody or antigen-binding fragment binds to human MuSK with high affinity. Binding affinity may be determined by an in vitro binding assay, for e.g., a Bio-Layer Interferometry (BLI)-based assay {e.g., Octet®), a Surface Plasmon Resonance (SPR)-based assay {e.g., BIACORE®) or solution equilibrium titration-based assays {e.g., MSD-SET) (preferably by an SPR-based assay).

[0053]

[0033] In some embodiments, the antibody or antigen-binding fragment has all six complementarity determining regions (CDRs), and / or the heavy chain variable region (HCVR) and light chain variable

[0054] 6

[0055] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0056] region (LCVR) pair of any of the antibodies described herein, and particularly an antibody selected from Ab1 , Ab2, Ab3, Ab4, Ab5 or Ab6.

[0057]

[0034] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) comprising a heavy chain (HC) complementarity determining region (CDR) 1 (H-CDR1 ) comprising SEQ ID NO: 17, a H-CDR2 comprising SEQ ID NO: 18, and an H-CDR3 comprising SEQ ID NO: 19; and a light chain variable region (LCVR) comprising a light chain (LC) complementarity determining region (CDR) 1 (L-CDR1) comprising SEQ ID NO: 20, a L-CDR2 comprising the amino acid sequence WAS, and a L-CDR3 comprising SEQ ID NO: 22, wherein the sequences are numbered according to IMGT numbering scheme. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) comprising a heavy chain (HC) complementarity determining region (CDR) 1 (H-CDR1 ) comprising SEQ ID NO: 41 , a H-CDR2 comprising SEQ ID NO: 42, and an H-CDR3 comprising SEQ ID NO:43; and a light chain variable region (LCVR) comprising a light chain (LC) complementarity determining region (CDR) 1 (L-CDR1) comprising SEQ ID NO: 44, a L-CDR2 comprising SEQ ID NO: 45, and a L-CDR3 comprising SEQ ID NO: 46, wherein the sequences are numbered according to Kabat numbering scheme. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) comprising a heavy chain (HC) complementarity determining region (CDR) 1 (H-CDR1) comprising SEQ ID NO: 65, a H-CDR2 comprising SEQ ID NO: 66, and an H-CDR3 comprising SEQ ID NO:67; and a light chain variable region (LCVR) comprising a light chain (LC) complementarity determining region (CDR) 1 (L-CDR1) comprising SEQ ID NO: 68, a L-CDR2 comprising the amino acid sequence WAS, and a L-CDR3 comprising SEQ ID NO: 70, wherein the sequences are numbered according to Chothia numbering scheme.

[0058]

[0035] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) comprising a heavy chain (HC) complementarity determining region (CDR) 1 (H-CDR1 ) comprising SEQ ID NO: 23, a H-CDR2 comprising SEQ ID NO: 24, and an H-CDR3 comprising SEQ ID NO: 25; and a light chain variable region (LCVR) comprising a light chain (LC) complementarity determining region (CDR) 1 (L-CDR1) comprising SEQ ID NO: 26, a L-CDR2 comprising the amino acid sequence LAS, and a L-CDR3 comprising SEQ ID NO: 28, wherein the sequences are numbered according to IMGT numbering scheme. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) comprising a heavy chain (HC) complementarity determining region (CDR) 1 (H-CDR1) comprising SEQ ID NO: 47, a H-CDR2 comprising SEQ ID NO: 48, and an H-CDR3 comprising SEQ ID NO: 49; and a light chain variable region (LCVR) comprising a light chain (LC) complementarity determining region (CDR) 1 (L-CDR1 ) comprising SEQ ID NO: 50, a L-CDR2 comprising SEQ ID NO: 51 , and a L-CDR3 comprising SEQ ID NO: 52, wherein the sequences are numbered according to Kabat numbering scheme. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) comprising a heavy chain (HC) complementarity determining region (CDR) 1 (H-CDR1 ) comprising SEQ ID NO: 71 , a H-CDR2 comprising SEQ ID NO: 72, and an H-CDR3 comprising SEQ ID NO: 73; and a light chain variable region (LCVR) comprising a light chain (LC) complementarity determining region (CDR) 1 (L-CDR1) comprising SEQ ID NO: 74, a L-CDR2

[0059] 7

[0060] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0061] comprising the amino acid sequence LAS, and a L-CDR3 comprising SEQ ID NO: 76, wherein the sequences are numbered according to Chothia numbering scheme.

[0062]

[0036] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) comprising a heavy chain (HC) complementarity determining region (CDR) 1 (H-CDR1 ) comprising SEQ ID NO: 29, a H-CDR2 comprising SEQ ID NO: 30, and an H-CDR3 comprising SEQ ID NO: 31; and a light chain variable region (LCVR) comprising a light chain (LC) complementarity determining region (CDR) 1 (L-CDR1) comprising SEQ ID NO: 32, a L-CDR2 comprising the amino acid sequence GTS, and a L-CDR3 comprising SEQ ID NO: 34, wherein the sequences are numbered according to IMGT numbering scheme. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) comprising a heavy chain (HC) complementarity determining region (CDR) 1 (H-CDR1) comprising SEQ ID NO: 53, a H-CDR2 comprising SEQ ID NO: 54, and an H-CDR3 comprising SEQ ID NO: 55; and a light chain variable region (LCVR) comprising a light chain (LC) complementarity determining region (CDR) 1 (L-CDR1) comprising SEQ ID NO: 56, a L-CDR2 comprising SEQ ID NO: 57, and a L-CDR3 comprising SEQ ID NO: 58, wherein the sequences are numbered according to Kabat numbering scheme. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) comprising a heavy chain (HC) complementarity determining region (CDR) 1 (H-CDR1) comprising SEQ ID NO: 77, a H-CDR2 comprising SEQ ID NO: 78, and an H-CDR3 comprising SEQ ID NO: 79; and a light chain variable region (LCVR) comprising a light chain (LC) complementarity determining region (CDR) 1 (L-CDR1) comprising SEQ ID NO: 80, a L-CDR2 comprising the amino acid sequence GTS, and a L-CDR3 comprising SEQ ID NO: 82, wherein the sequences are numbered according to Chothia numbering scheme.

[0063]

[0037] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) comprising a heavy chain (HC) complementarity determining region (CDR) 1 (H-CDR1 ) comprising SEQ ID NO: 35, a H-CDR2 comprising SEQ ID NO: 36, and an H-CDR3 comprising SEQ ID NO: 37; and a light chain variable region (LCVR) comprising a light chain (LC) complementarity determining region (CDR) 1 (L-CDR1) comprising SEQ ID NO: 38, a L-CDR2 comprising the amino acid sequence AGY, and a L-CDR3 comprising SEQ ID NO: 40, wherein the sequences are numbered according to IMGT numbering scheme. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) comprising a heavy chain (HC) complementarity determining region (CDR) 1 (H-CDR1) comprising SEQ ID NO: 59, a H-CDR2 comprising SEQ ID NO: 60, and an H-CDR3 comprising SEQ ID NO: 61; and a light chain variable region (LCVR) comprising a light chain (LC) complementarity determining region (CDR) 1 (L-CDR1) comprising SEQ ID NO: 62, a L-CDR2 comprising SEQ ID NO: 63, and a L-CDR3 comprising SEQ ID NO: 64, wherein the sequences are numbered according to Kabat numbering scheme. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) comprising a heavy chain (HC) complementarity determining region (CDR) 1 (H-CDR1) comprising SEQ ID NO: 83, a H-CDR2 comprising SEQ ID NO: 84, and an H-CDR3 comprising SEQ ID NO: 85; and a light chain variable region (LCVR) comprising a light chain (LC) complementarity determining region (CDR) 1 (L-CDR1) comprising SEQ ID NO: 86, a L-CDR2

[0064] 8

[0065] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0066] comprising the amino acid sequence AGY, and a L-CDR3 comprising SEQ ID NO: 88, wherein the sequences are numbered according to Chothia numbering scheme.

[0067]

[0038] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) comprising a heavy chain (HC) complementarity determining region (CDR) 1 (H-CDR1 ) comprising SEQ ID NO: 207, a H-CDR2 comprising SEQ ID NO: 208, and an H-CDR3 comprising SEQ ID NO: 55; and a light chain variable region (LCVR) comprising a light chain (LC) complementarity determining region (CDR) 1 (L-CDR1) comprising SEQ ID NO: 56, a L-CDR2 comprising SEQ ID NO: 57, and a L-CDR3 comprising SEQ ID NO: 58, wherein the sequences are numbered according to Kabat numbering scheme.

[0068]

[0039] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) comprising a heavy chain (HC) complementarity determining region (CDR) 1 (H-CDR1 ) comprising SEQ ID NO: 209, a H-CDR2 comprising SEQ ID NO: 60, and an H-CDR3 comprising SEQ ID NO: 61; and a light chain variable region (LCVR) comprising a light chain (LC) complementarity determining region (CDR) 1 (L-CDR1) comprising SEQ ID NO: 62, a L-CDR2 comprising SEQ ID NO: 63, and a L-CDR3 comprising SEQ ID NO: 64, wherein the sequences are numbered according to Kabat numbering scheme.

[0069]

[0040] In some embodiments, the antibody or antigen-binding fragment comprises a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 89; and a LCVR having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 90. In some embodiments, the antibody or antigen-binding fragment comprises a HCVR comprising SEQ ID NO: 89; and a LCVR comprising SEQ ID NO: 90.

[0070]

[0041] In some embodiments, the antibody or antigen-binding fragment comprises a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 91; and a LCVR having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 92. In some embodiments, the antibody or antigen-binding fragment comprises a HCVR comprising SEQ ID NO: 91; and a LCVR comprising SEQ ID NO: 92.

[0071]

[0042] In some embodiments, the antibody or antigen-binding fragment comprises a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 93; and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 94. In some embodiments, the antibody or antigen-binding fragment comprises a HCVR comprising SEQ ID NO: 93; and a LCVR comprising SEQ ID NO: 94.

[0072]

[0043] In some embodiments, the antibody or antigen-binding fragment comprises a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 95; and a LCVR having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 96. In some embodiments, the antibody or antigen-binding fragment comprises a HCVR comprising SEQ ID NO: 95; and a LCVR comprising SEQ ID NO: 96.

[0073] 9

[0074] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0075]

[0044] In some embodiments, the antibody or antigen-binding fragment comprises a HCVR having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 210; and a LCVR having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 211. In some embodiments, the antibody or antigen-binding fragment comprises a HCVR comprising SEQ ID NO: 210; and a LCVR comprising SEQ ID NO: 211.

[0076]

[0045] In some embodiments, the antibody or antigen-binding fragment comprises a HCVR having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 212; and a LCVR having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 213. In some embodiments, the antibody or antigen-binding fragment comprises a HCVR comprising SEQ ID NO: 212; and a LCVR comprising SEQ ID NO: 213.

[0077]

[0046] In some embodiments, any of the novel MuSK agonist antibodies or antigen-binding fragments disclosed herein is a human or a humanized antibody or antigen-binding fragment. In some embodiments, the human or humanized antibody or antigen-binding fragment is a human IgG 1 or human lgG4 isotype. In some embodiments, the antibody or antigen-binding fragment of human lgG4 isotype comprises a Ser-to-Pro (S228P) hinge substitution.

[0078]

[0047] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain (HC) comprising SEQ ID NO: 105; and a light chain (LC) comprising SEQ ID NO: 107; a heavy chain (HC) comprising SEQ ID NO: 109; and a light chain (LC) comprising SEQ ID NO: 111; a heavy chain (HC) comprising SEQ ID NO: 113; and a light chain (LC) comprising SEQ ID NO: 115; or a heavy chain (HC) comprising SEQ ID NO: 117; and a light chain (LC) comprising SEQ ID NO: 119.

[0079]

[0048] Also provided is an antibody or antigen-binding fragment (e.g. as described herein) that crosscompetes for binding to human MuSK with an antibody, or antigen-binding fragment, selected from Ab1, Ab2, Ab3, Ab4, Ab5 or Ab6, or with an antibody or antigen-binding fragment having the HCVR and LCVR pair of any of the antibodies described herein, and particularly those of an antibody selected from Ab1 , Ab2, Ab3, Ab4, Ab5 or Ab6 (and more particularly those of Ab3, Ab4, Ab5 or Ab6). Preferably, the antibody or antigen-binding fragment cross-competes with an antibody or antigenbinding fragment having the HCVR and LCVR pair of Ab5 {e.g. cross-competes with Ab5). The antibody or antigen-binding fragment may be a high-affinity binder as described herein (e.g. capable of binding to human MuSK with a KD of 5 nM, as measured by an in vitro binding assay, such as SPR). In another aspect, the antibody or antigen-binding fragment competes for binding to human MuSK. In some embodiments, the cross-competing antibody or antigen-binding fragment binds and activates human MuSK, and is not capable of binding to a MuSK fragment consisting of SEQ ID NO: 8. In some embodiments, the antibody or antigen-binding fragment is capable of binding to a MuSK fragment comprising or consisting of SEQ ID NO: 7. In some embodiments, the antibody or antigenbinding fragment is capable of binding to a MuSK fragment comprising or consisting of SEQ ID NO: 173.

[0080] 10

[0081] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0082]

[0049] In some embodiments, competition for binding is measured using solution equilibrium titration, e.g., BLI-based assay. In some embodiments, the competition for binding is assessed by binding to the human MuSK extracellular domain.

[0083]

[0050] In another aspect, the present invention provides a nucleic acid encoding the heavy chain variable region of the antibody, or antigen-binding fragment thereof as disclosed herein. In another aspect, the present invention provides a nucleic acid encoding the light chain variable region of the antibody, or antigen-binding fragment thereof as disclosed herein. In another aspect, the present invention provides a nucleic acid encoding the heavy chain variable region and the light chain variable region of the antibody, or antigen-binding fragment thereof as disclosed herein. In some embodiments, a kit comprising the nucleic acids is provided. In some embodiments, the present invention provides a vector comprising any of the nucleic acids disclosed herein. In some embodiments, the present invention provides a cell comprising any nucleic acid or vector disclosed herein, or a cell expressing the antibody or antigen-binding fragment disclosed herein.

[0084]

[0051] In some embodiments, the present invention provides a composition comprising the antibody or antigen-binding fragment disclosed herein. In some embodiments, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment disclosed herein and a pharmaceutically acceptable excipient.

[0085]

[0052] In another aspect, the disclosure provides a method for making a composition comprising an antibody or antigen-binding fragment described herein, the method comprising:

[0086] i) providing at least one human MuSK protein fragment consisting of SEQ ID NO: 7, and providing a human MuSK protein fragment consisting of SEQ ID NO: 8;

[0087] ii) selecting a first pool of antibodies or antigen-binding fragments that specifically bind the human MuSK protein fragment consisting of SEQ ID NO: 7, but do not bind the human MuSK protein fragment consisting of SEQ ID NO: 8; and

[0088] iii) selecting from the first pool, a second pool of antibodies or antigen-binding fragments, or an antibody or antigen-binding fragment, which shows at least one of the following properties:

[0089] - bind to human MuSK with a bivalent binding affinity of < 1 nM as measured in an in vitro binding assay, and / or

[0090] - do not interfere with activation of MuSK by agrin in an in vitro AChR clustering assay, and further optionally:

[0091] - elicit a maximum muscle force at 150 Hz stimulation in an Agrn nmf380 mouse model of at least 25 mN (e.g. at least 30 mN)

[0092] and / or

[0093] - increase maximum muscle force at 150 Hz stimulation in an Agrn nmf380 mouse model by at least 20 mN as compared to a control;

[0094] wherein the in vitro binding assay is a BLI- or SPR-based binding assay, and wherein the control for measurement of maximum muscle force is an isotype matched control IgG, optionally wherein the isotype matched control IgG is a murine lgG4. The method may further comprise a step of iv) formulating an antibody or antigen-binding fragment selected in step iii) into a pharmaceutical composition.

[0095] 11

[0096] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0097]

[0053] In one aspect, the disclosure provides a method for identifying a MuSK agonist antibody or antigen-binding fragment thereof, the method comprising the steps of:

[0098] i) screening for antibodies or antigen-binding fragments for the ability to specifically bind a recombinant antigen comprising a human MuSK Truncated Tail sequence as set forth in SEQ ID NO: 9,

[0099] ii) carrying out an AChR clustering assay to select from step (i) one or more antibodies or antigenbinding fragments capable of inducing AChR clustering with an EC50 of 1 nM or less, thereby identifying a MuSK agonist antibody or an antigen-binding fragment thereof.

[0100]

[0054] In some embodiments, the method further comprises identifying an antibody or an antigen-binding fragment binds the recombinant antigen with a KD of 1 nM or less as measured in an in vitro binding assay. In some embodiments, the in vitro binding assay comprises a BLI-based technique or SPR-based technique.

[0101]

[0055] In some embodiments, the recombinant antigen used in a method for identifying a MuSK agonist antibody or antigen-binding fragment as disclosed herein further comprises a human Fz-like domain sequence of SEQ ID NO: 8 or a portion thereof. In some embodiments, the recombinant antigen is a fragment of human MuSK as set forth in SEQ ID NO: 173 or a portion thereof. In some embodiments, the recombinant antigen is a fragment of human MuSK as set forth in SEQ ID NO: 7. In some embodiments, the recombinant antigen does not comprise an Ig1 domain sequence, an Ig2 domain sequence, and an Ig3 domain sequence of human MuSK. In some embodiments, the AChR clustering assay of step comprises a cell-based in vitro potency assay. In some embodiments, the cell-based potency assay comprises cultured myoblasts or myotubes. In some embodiments, the methods disclosed herein further comprise carrying out an in vivo study in a preclinical model that manifests a defect in neuromuscular junctions (NMJs) to evaluate the ability of the antibody, relative to control: a) to alleviate or rescue the defect in NMJs; b) to have enhanced body weight; c) to have greater muscle weight at study end; d) to have enhanced muscle or motor function; and / or e) for improved survival. In some embodiments, the method further comprises determining thermostability of the antibody or a fragment thereof in a differential scanning fluorimetry (DSF) assay.

[0102]

[0056] In a further aspect, the antibody or antigen-binding fragment described herein, or composition {e.g. pharmaceutical composition) of the same described herein is used in a method of activating MuSK at a neuromuscular junction (NMJ). In another aspect, the antibody or antigen-binding fragment described herein, or composition {e.g. pharmaceutical composition) of the same described herein may be used in a method of treating a subject having a neuromuscular disease or disorder. The method may comprise administering to the subject an effective amount of the antibody or antigen-binding fragment. The neuromuscular disease or disorder may include, but is not limited to, Amyotrophic lateral sclerosis (ALS), Botulism, Charcot Marie Tooth type 2 (CMT2), Congenital myasthenic syndromes, Congenital myopathies, Cramp-fasciculation syndrome, Elevated creatine kinase, Inclusion-body myositis, Lambert-Eaton syndrome, Mitochondrial myopathy, Motor neuron disease, Muscular dystrophy, Myasthenia gravis, Myotonic dystrophy, Neuromyotonia, Peripheral neuropathy, Polymyositis, Sarcopenia, spinal muscular atrophy (SMA), Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), or a Dok7-congenital myasthenic syndrome. In some

[0103] 12

[0104] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0105] embodiments, the neuromuscular disease or disorder is myasthenia gravis. In some embodiments, the myasthenia gravis is MuSK myasthenia gravis (MuSK MG) or LRP4 myasthenia gravis (LRP4 MG). In some embodiments, the ALS is selected from the group consisting of: sporadic ALS, familial ALS, limb-onset ALS, bulbar-onset ALS, primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), juvenile ALS, and adult-onset ALS. In some embodiments, the subject has juvenile ALS, optionally wherein the subject carries a mutation in FUS and / or SOD1 genes. In some embodiments, the subject has MuSK lgG4 autoantibodies.

[0106]

[0057] In some embodiments, the administration of the antibody, or antigen-binding fragment thereof, results in increased body weight of the subject. In some embodiments, the administration of the antibody, or antigen-binding fragment thereof, results in increased muscle mass of the subject. In some embodiments, the administration of the antibody, or antigen-binding fragment thereof, results in increased muscle force of the subject. In some embodiments, the administration of the antibody, or antigen-binding fragment thereof, results in increased maximum muscle force of the subject.

[0107]

[0058] The antibodies or antigen-binding fragments described herein may be capable of promoting BMP-induced activation of Smad1 / 5 / 8. In some embodiments, the antibody or antigen-binding fragment used in a method described herein is administered to a subject in an amount sufficient to induce BMP-dependent activation of Smad1 / 5 / 8 activation.

[0108]

[0059] Also provided is an antibody or antigen-binding fragment thereof that binds an extracellular domain of MuSK for use in the treatment of a neuromuscular disease in a subject, wherein the antibody or antigen-binding fragment binds an extracellular domain fragment of MuSK that contains SEQ ID NO: 9 but does not bind and an extracellular domain fragment of MuSK that does not contain SEQ ID NO:9, wherein the antibody or fragment is capable of promoting BMP-induced activation of Smad1 / 5 / 8.

[0109]

[0060] Also provided is a MuSK antibody for use in the treatment of a neuromuscular disease in a subject, wherein the treatment comprises administration of an antibody that binds human MuSK in an amount sufficient to treat the disease, wherein the subject is further treated with an additional therapy for the disease. In some embodiments, the antibody is selected from the group consisting of antibodies listed in Table 6 herein or 3B2G2. In some embodiments, the neuromuscular disease is selected from the group consisting of Amyotrophic lateral sclerosis (ALS), Botulism, Charcot Marie Tooth type 2 (CMT2), Congenital myasthenic syndromes, Congenital myopathies, Cramp-fasciculation syndrome, Elevated creatine kinase, Inclusion-body myositis, Lambert-Eaton syndrome, Mitochondrial myopathy, Motor neuron disease, Muscular dystrophy, Myasthenia gravis, Myotonic dystrophy, Neuromyotonia, Peripheral neuropathy, Polymyositis, Sarcopenia, spinal muscular atrophy (SMA), Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), and a Dok7-congenital myasthenic syndrome, wherein optionally the additional therapy is a myostatin inhibitor, wherein further optionally the myostatin inhibitor is a myostatin-selective inhibitor. In some embodiments, the subject is treated with a corrector therapy aimed to correct or restore a genetic defect associated with the disease.

[0110] 13

[0111] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0112] BRIEF DESCRIPTION OF THE FIGURES

[0113]

[0061] FIG. 1A is a set of graphs that show evaluation of binding of Fabs and Mabs of comparator 3B2G2 to human and mouse MuSK ECD as measured by Biacore.

[0114]

[0062] FIG. 1 B is a set of graphs that show evaluation of binding of Fabs and Mabs of comparator mAb13 to human and mouse MuSK ECD as measured by Biacore.

[0115]

[0063] FIG. 1C is a set of graphs that show evaluation of binding of Fabs and Mabs of MuSK antibody Ab1 to human and mouse MuSK ECD as measured by Biacore.

[0116]

[0064] FIG. 1D is a set of graphs that show evaluation of binding of Fabs and Mabs of MuSK antibody Ab2 to human and mouse MuSK ECD as measured by Biacore.

[0117]

[0065] FIG. 1E is a set of graphs that show evaluation of binding of Fabs and Mabs of MuSK antibody Ab3 to human and mouse MuSK ECD as measured by Biacore.

[0118]

[0066] FIG. 1F is a set of graphs that show evaluation of binding of Fabs and Mabs of MuSK antibody Ab4 to human and mouse MuSK ECD as measured by Biacore.

[0119]

[0067] FIG. 2A is a schematic showing the experimental set up for an epitope binning experiment to assess binding of Ab1 , Ab2, Ab3, Ab4 and 3B2G2 and / or mAb13 Fabs to biotinylated human and mouse MuSK Frizzled-like constructs on Octet® Red384 using streptavidin biosensors.

[0120]

[0068] FIG. 2B is a table of epitope binning results showing assessment of binding of Ab1 , Ab2, Ab3, Ab4 and 3B2G2 Fabs to biotinylated human and mouse MuSK Frizzled-like constructs relative to each other.

[0121]

[0069] FIG. 2C is a table of epitope binning results showing assessment of binding of Ab1 , Ab2, Ab3, Ab4, 3B2G2 and mAb13 Fabs to biotinylated human and mouse MuSK Frizzled-like constructs relative to each other.

[0122]

[0070] FIG.3A is a schematic showing the workflow for an AChR clustering assay for in vitro assessment of MuSK antibody functional activity, using C2C12 myotubes and high content imaging analysis.

[0123]

[0071] FIG. 3B is a graph quantifying functional activity, AChR clustering, upon treatment with a concentration range of comparator mAbs. Data represented as ratio of total AChR area to total myotube area and as averages normalized against 10nM of control (3B2G2).

[0124]

[0072] FIG. 3C is a graph showing assessment of functional activity of mAb of MuSK Ig 1 domain binding antibody Ab-ILD1 in an AChR clustering assay. Data represented as ratio of total AChR area to total myotube area and as averages normalized against 10nM of control (3B2G2)..

[0125]

[0073] FIG.3D is a graph showing assessment of functional activity of mAb of MuSK Ig2 domain binding antibody Ab-ILD2 in an AChR clustering assay. Data represented as ratio of total AChR area to total myotube area and as averages normalized against 10nM of control (3B2G2)..

[0126]

[0074] FIG. 3E is a graph showing assessment of functional activity of mAb of MuSK Ig3 domain binding antibody Ab-ILD3 in an AChR clustering assay. Data represented as ratio of total AChR area to total myotube area and as averages normalized against 10nM of control (3B2G2)

[0127] 14

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[0129]

[0075] FIG. 3F is a graph showing assessment of functional activity of mAbs of MuSK antibodies Ab1, Ab2, Ab3 and Ab4 in an AChR clustering assay. Data represented as ratio of total AChR area to total myotube area and as averages normalized against 10nM of control (3B2G2)..

[0130]

[0076] FIG. 3G is a graph showing assessment of potential interference of Fabs of comparator antibodies 4D3, 3B2G2 and mAb13 in an AChR clustering assay. Data are shown as ratio of total AChR area to total myotube area, normalized to % of that for Agrin at 1 nM.

[0131]

[0077] FIG. 3H is a graph showing assessment of potential interference of Fab of MuSK Ig1 domain binding antibody Ab-ILD1 in an AChR clustering assay. Data are shown as ratio of total AChR area to total myotube area, normalized to % of that for Agrin at 1 nM.

[0132]

[0078] FIG. 3I is a graph showing assessment of potential interference of Fab of MuSK Ig2 domain binding antibody Ab-ILD2 in an AChR clustering assay. Data are shown as ratio of total AChR area to total myotube area, normalized to % of that for Agrin at 1 nM.

[0133]

[0079] FIG. 3J is a graph showing assessment of potential interference of Fab of MuSK Ig3 domain binding antibody Ab-ILD3 in an AChR clustering assay. Data are shown as ratio of total AChR area to total myotube area, normalized to % of that for Agrin at 1 nM.

[0134]

[0080] FIG. 3K a graph showing assessment of potential interference of Fabs of MuSK antibodies Ab1 , Ab2, Ab3 and Ab4 in an AChR clustering assay. Data are shown as ratio of total AChR area to total myotube area, normalized to % of that for Agrin at 1 nM.

[0135]

[0081] FIG. 4A is a set of graphs that show flow cytometry data for binding of control and comparator antibodies HuNeg, mAb13 and 3B2G2 to cells ectopically expressing human and mouse MuSK.

[0136]

[0082] FIG. 4B is a set of graphs that show flow cytometry data for binding of MuSK antibodies Ab1 , Ab2, Ab3 and Ab4 to cells ectopically expressing human MuSK.

[0137]

[0083] FIG. 4C is a set of graphs that show flow cytometry data for binding of MuSK antibodies Ab1 , Ab2, Ab3 and Ab4 to cells ectopically expressing mouse MuSK.

[0138]

[0084] FIG.4D is a table of flow cytometry results of binding of MuSK antibodies Ab1 , Ab2, Ab3 and Ab4 and comparator antibodies 3B2G2, mAb13 and control HuNeg, to cells ectopically expressing human and mouse MuSK.

[0139]

[0085] FIG. 5A provides a graph of terminal body weight (in grams) of wild-type (WT) or agrin nmf380 (Mutant) mice treated with MuSK antibodies Ab-ILD1 , Ab-ILD2, Ab-ILD3, Ab1, Ab2, Ab3, Ab4, comparator 3B2G2, or HuNeg control.

[0140]

[0086] FIG.5B provides a graph of isolated gastrocnemius (Gastroc) mass (in grams) of wild-type (WT) or agrin nmf380 (Mutant) mice treated with MuSK antibodies Ab-ILD1, Ab-ILD2, Ab-ILD3, Ab1 , Ab2, Ab3, Ab4, comparator 3B2G2, or HuNeg control.

[0141]

[0087] FIG. 5C provides a graph of isolated tibialis anterior (TA) mass (in grams) of wild-type (WT) or agrin nmf380 (Mutant) mice treated with MuSK antibodies Ab-ILD1 , Ab-ILD2, Ab-ILD3, Ab1 , Ab2, Ab3, Ab4, comparator 3B2G2, or HuNeg control.

[0142] 15

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[0144]

[0088] FIG. 5D provides graphs of muscle force and maximum force (Max force) assessments in wildtype (WT) or agrin nmf380 (Mutant) mice treated with MuSK antibodies Ab-ILD1 , Ab-ILD2, Ab-ILD3, Ab1 , Ab2, Ab3, Ab4, comparator 3B2G2, or HuNeg control.

[0145]

[0089] FIG.6A is a schematic showing experimental set up for an Octet® kinetics assessment of binding of MuSK antibodies to human or mouse MuSK ECD constructs.

[0146]

[0090] FIG. 6B is a schematic showing MuSK extracellular domain (ECD) constructs used in Octet® kinetics assessment of binding of MuSK antibodies. Length in amino acids of tail portions indicated (45aa, 7aa Truncated Tail or No Tail).

[0147]

[0091] FIG. 6C is a set of in vitro binding data showing Octet® traces of the antibodies 3B2G2, mAb13, Ab1 , Ab2, Ab3 and Ab4 binding to 45 amino acid Tail, 7 amino acid Truncated Tail and No Tail constructs (45aaT, 7aaT and No Tail, respectively).

[0148]

[0092] FIG. 6D is a schematic showing the 45 amino acid Tail, 7 amino acid Truncated Tail and No Tail constructs and a table providing an overview of the binding results for 3B2G2, mAb13, Ab1 , Ab2, Ab3 and Ab4 determined by Octet® analysis.

[0149]

[0093] FIGs. 7A and 7B provide a cryo-EM reconstruction model in two orientations, showing an Fab fragment of Ab5 and an Fab fragment of 3B2G2 bound to a human MuSK antigen in a ternary complex.

[0150]

[0094] FIG. 8A provides data showing terminal body weight of mice treated with Ab5, Ab6, 3B2G2 or isotype-matched IgG control in an agrin-deficient mouse model.

[0151]

[0095] FIG. 8B provides data showing gastroc muscle weight isolated from mice treated with Ab5, Ab6, 3B2G2 or isotype-matched IgG control in an agrin-deficient mouse model.

[0152]

[0096] FIG. 8C provides data showing TA muscle weight isolated from mice treated with Ab5, Ab6, 3B2G2 or isotype-matched IgG control in an agrin- deficient mouse model.

[0153]

[0097] FIG. 8D provides data showing maximum force measured in plantarflexor of mice treated with Ab5, Ab6, 3B2G2 or isotype-matched IgG control in an agrin- deficient mouse model

[0154]

[0098] FIG. 9A provides a representative data set showing the effects of MuSK agonist antibody treatment on mean area of AChR clusters in primary human myotube.

[0155]

[0099] FIG. 9B provides a representative data set showing the effects of MuSK agonist antibody treatment on large AChR cluster coverage in primary human myotube.

[0156] DETAILED DESCRIPTION

[0157]

[0100] Disclosed herein are novel, highly potent MuSK agonist antibodies capable of binding to and activating MuSK. The antibodies and antigen-binding fragments thereof provided herein bind to the extracellular domain of MuSK. The antibodies and antigen-binding fragments thereof of this novel class bind to MuSK, activate MuSK, and also bind to a fragment of MuSK comprising or consisting of the MuSK frizzled- like domain (Fz-like domain) and a tail (including a ‘Truncated Tail’ of 7 amino acids) that are immediately C-terminal to the Fz-like domain. The antibodies and antigen-binding fragments of the disclosure require the presence of the Truncated tail C-terminal to the Fz-like domain in order to bind

[0158] 16

[0159] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0160] to MuSK, and are not capable of binding to a MuSK protein, or fragment thereof, that lacks the tail C-terminal to the Fz-like domain. Notably, the novel class of antibodies do not compete for antigen binding with the prior art anti-MuSK antibodies, such as 3B2G2 and mab#13, indicating that the novel antibodies bind an epitope distinct from the known epitope of MuSK agonist antibodies.

[0161] Definitions

[0162]

[0101] In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.

[0163]

[0102] Affinity. Affinity is the strength of binding of a molecule (such as an antibody) to its ligand (such as an antigen). It is typically measured and reported by the equilibrium dissociation constant (KD). In the context of antibody-antigen interactions, KD is the ratio of the antibody dissociation rate (“off rate” or Koff or Kdis), how quickly it dissociates from its antigen, to the antibody association rate (“on rate” or Kon) of the antibody, how quickly it binds to its antigen. For example, an antibody with an affinity of 5 nM has a KD value that is 5 nM or lower ( / '.e., 5 nM or higher affinity) determined by a suitable in vitro binding assay. Suitable in vitro assays can be used to measure KD values of an antibody for its antigen, such as Biolayer Interferometry (BLI), Surface Plasmon Resonance (SPR) and Solution Equilibrium Titration (e.g., MSD-SET). In some embodiments, an antibody, or antigenbinding portion thereof, binds with high affinity to a target, e.g., MuSK, if the antibody has a KD for the target of at least about 107M, 10‘8M, 10‘9M, 10‘10M, 10‘11M, or less. In some embodiments, the terms “binding with high affinity to an epitope of MuSK”, “binds with high affinity to an epitope of MuSK”, “high affinity binding to MuSK”, or “binds with high affinity to MuSK” as used herein, refer to an antibody, or antigen-binding portion thereof, that binds to MuSK and has a dissociation constant (KD) of 1.0 x 10-7M or less, as determined by suitable in vitro binding assays. In one embodiment, an antibody, or antigen-binding portion thereof, can bind with high affinity to both human and a nonhuman (e.g., mouse) orthologue of MuSK.

[0164]

[0103] Antibody. The term “antibody” encompasses any naturally-occurring, recombinant, modified or engineered immunoglobulin or immunoglobulin-like structure or antigen-binding fragment or portion thereof, or derivative thereof, as further described elsewhere herein. Unless specified to the contrary, the term “antibody” as used herein shall encompass antigen-binding fragments and functional variants thereof. Thus, the term refers to an immunoglobulin molecule that specifically binds to a target antigen, and includes, for instance, chimeric, humanized, fully human, and bispecific antibodies. An intact antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but in some instances can include fewer chains such as antibodies naturally occurring in camelids which can comprise only heavy chains. Antibodies can be derived solely from a single source, or can be “chimeric,” that is, different portions of the antibody can be derived from two different antibodies. Antibodies, or antigen-binding portions thereof, can be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. The term antibodies, as used herein, includes monoclonal antibodies, bispecific antibodies, minibodies, domain

[0165] 17

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[0167] antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), respectively. In some embodiments, the term also encompasses peptibodies.

[0168]

[0104] Antigen'. The term “antigen” broadly includes any molecules comprising an antigenic determinant within a binding region(s) to which an antibody or a binding-fragment specifically binds. An antigen can be a single-unit molecule (such as a protein monomer or a fragment) or a complex comprised of multiple components. An antigen provides an epitope, e.g., a molecule or a portion of a molecule, or a complex of molecules or portions of molecules, capable of being bound by a selective binding agent, such as an antigen-binding protein (including, e.g., an antibody). Thus, a selective binding agent may specifically bind to an antigen that is formed by two or more components in a complex. In some embodiments, the antigen is capable of being used in an animal (e.g., host) to produce antibodies capable of binding to that antigen. An antigen can possess one or more binding regions (e.g., epitopes) that are capable of interacting with different antigen-binding proteins, e.g., antibodies. In the context of the present disclosure, a suitable antigen is the extra-cellular domain of the MuSK protein or a portion thereof. More specifically, the antigen is a MuSK protein, or fragment thereof, comprising at least a portion of the Frizzled-like domain and the Truncated tail C-terminal to the Fz-like domain. In preferred embodiments, the antigen is or comprises the amino acid sequence: DNKGYCAQYRGEVCNAVLAKDALVFLNTSYADPEEAQELLVHTAWNELKVVSPVCRPAAEALLCNHI FQECSPGVVPTPIPICREYCLAVKELFCAKEWLVMEEKTHRGLYRSEMHLLSVPECSKLPSMHWDPT ACARLPHLDYNK (SEQ ID NO: 7), in which the bold portion corresponds to the 7 amino acid “Truncated Tail” immediately C-terminus to the Fz-like domain of human MuSK.

[0169]

[0105] Antigen-binding portion / fragment: The terms “antigen-binding portion” or “antigen-binding fragment” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., MuSK). Antigen-binding portions include, but are not limited to, any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. In some embodiments, an antigen-binding portion of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Non-limiting examples of antigen-binding portions include: (i) Fab fragments, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of the VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody; (v) single-chain Fv (scFv) molecules (see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Nat’l. Acad. Sci. USA 85:5879-5883); (vi) dAb fragments (see, e.g., Ward et al., (1989) Nature 341 : 544-546); and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR)). Other forms of single chain antibodies, such as diabodies are also encompassed. The term antigen-binding portion of an antibody includes a “single chain Fab fragment” otherwise known as an “scFab,” comprising an

[0170] 18

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[0172] antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1 ), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1 -linker- VL-CL, b) VL-CL-linker-VH-CH1 , c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; and wherein said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids.

[0173]

[0106] Binding region'. As used herein, a “binding region” is a portion of an antigen (e.g., an antigen complex) that, when bound to an antibody or a fragment thereof, can form an interface of the antibody-antigen interaction. Upon antibody binding, a binding region becomes “protected” from surface exposure, which can be detected by suitable techniques, such as HDX-MS. Antibody-antigen interaction may be mediated via multiple (e.g., two or more) binding regions. A binding region can comprise an antigenic determinant, or epitope.

[0174]

[0107] Clinical benefit'. As used herein, the term “clinical benefits” is intended to include both efficacy and safety of a therapy. Thus, therapeutic treatment that achieves a desirable clinical benefit is both efficacious and safe (e.g., with tolerable or acceptable toxicities or adverse events).

[0175]

[0108] Combination therapy. “Combination therapy” refers to treatment regimens for a clinical indication that comprise two or more therapeutic agents. Thus, the term refers to a therapeutic regimen in which a first therapy comprising a first composition (e.g., active ingredient) is administered in conjunction with a second therapy comprising a second composition (active ingredient) to a patient, intended to treat the same or overlapping disease or clinical condition. The first and second compositions may both act on the same cellular target, or discrete cellular targets. The phrase “in conjunction with,” in the context of combination therapies, means that therapeutic effects of a first therapy overlaps temporarily and / or spatially with therapeutic effects of a second therapy in the subject receiving the combination therapy. Thus, the combination therapies may be formulated as a single formulation for concurrent administration, or as separate formulations, for sequential administration of the therapies. When a subject who has been treated with a first therapy for the treatment of a disease is administered with a second therapy to treat the same disease, the second therapy may be referred to as an “add-on therapy” or “adjunct therapy."

[0176]

[0109] Combinatory or combinatorial epitope'. A combinatorial epitope is an epitope that is recognized and bound by a combinatorial antibody at a site (i.e., antigenic determinant) formed by noncontiguous portions of a component or components of an antigen, which, in a three-dimensional structure, come together in close proximity to form the epitope. Thus, antibodies of the invention may bind an epitope formed by two or more components (e.g., portions or segments) of MuSK. A combinatory epitope may comprise amino acid residue(s) from a first component of the antigen, and amino acid residue(s) from a second component of the antigen, and so on. Each component may be of a single protein or of two or more proteins of an antigenic complex. A combinatory epitope is formed with structural contributions from two or more components (e.g., portions or segments, such as amino acid residues) of an antigen or antigen complex.

[0177]

[0110] Compete or cross-compete'. The term “compete”, as used herein with regard to an antibody, means that a first antibody binds to an epitope (e.g., an epitope of a MuSK protein, or fragment

[0178] 19

[0179] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0180] thereof, comprising a f rizzled-like domain and a Truncated tail C-terminal to the Fz-like domain) in a manner sufficiently similar to or overlapping with the binding of a second antibody, such that the result of binding of the first antibody with its epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, can, but need not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of the other antibody with its epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to “cross-compete” with each other for binding of their respective epitope(s). Both competing and cross-competing antibodies are within the scope of this disclosure. Regardless of the mechanism by which such competition or cross-competition occurs {e.g., steric hindrance, conformational change, or binding to a common epitope, or portion thereof), the skilled artisan would appreciate that such competing and / or cross-competing antibodies are encompassed and can be useful for the methods and / or compositions provided herein. The terms “cross-blocking" and “crosscompeting” may be used interchangeably.

[0181]

[0111] Two different monoclonal antibodies (or antigen-binding fragments) that bind the same antigen may be able to simultaneously bind to the antigen if the binding sites are sufficiently further apart in the three-dimensional space such that each binding does not interfere with the other binding. By contrast, two different monoclonal antibodies may have binding regions of an antigen that are the same or overlapping, in which case, binding of the first antibody may prevent the second antibody from being able to bind the antigen, or vice versa. “Cross-block” or “cross-compete” means that binding of the first antibody to an antigen detectably inhibits {e.g. prevents) binding of the second antibody to the same antigen, and similarly, binding of the second antibody to an antigen detectably inhibits e.g. prevents) binding of the first antibody to the same antigen.

[0182]

[0112] Competition between antigen-binding proteins for the same epitope may be determined by an assay in which the antigen-binding protein being tested prevents or inhibits {e.g., reduces) specific binding of a reference antigen-binding protein to a common antigen {e.g., MuSK or a fragment thereof). Numerous types of competitive binding assays can be used to determine if one antigenbinding protein competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay; solid phase direct biotin-avidin EIA; solid phase direct labeled assay, and solid phase direct labeled sandwich assay. Usually, when a competing antigen-binding protein is present in excess, it will inhibit {e.g., reduce) specific binding of a reference antigen-binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or 75% or more. In some instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more. In some instances, binding is inhibited by at least 80-90%, at least 85%-95%, at least 95-99%. In some instances, binding is inhibited by at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some embodiments, a first antibody or antigen-binding portion thereof and a second antibody or antigen-binding portion thereof cross-block with each other with respect to the same antigen, for example, as assayed by SPR or BLI (such as BIACORE® or OCTET®, respectively),

[0183] 20

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[0185] using standard test conditions, e.g., according to the manufacturer’s instructions (e.g., binding assayed at room temperature, ~20-25°C). In some embodiments, the first antibody or fragment thereof and the second antibody or fragment thereof may have the same epitope. In other embodiments, the first antibody or fragment thereof and the second antibody or fragment thereof may have non-identical but overlapping epitopes. In yet further embodiments, the first antibody or fragment thereof and the second antibody or fragment thereof may have separate (different) epitopes which are in close proximity in a three-dimensional space, such that antibody binding is cross-blocked via steric hindrance.

[0186]

[0113] Complementarity determining region (CDR): As used herein, the term "CDR" refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1 , CDR2 and CDR3, for each of the variable regions. The term “CDR set” as used herein refers to a group of three CDRs that occur in a single variable region (either VH or VL) that contribute to antigen binding. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., (1987; 1991) Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; and Chothia et al., (1989) Nature 342: 877-883) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as L-CDR1 , L-CDR2 and L-CDR3 or H-CDR1 , H-CDR2 and H-CDR3, where the "L" and the "H" designate the light chain and the heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (1995) FASEB J. 9: 133-139 and MacCallum (1996) J. Mol. Biol. 262(5): 732-45. Still other CDR boundary definitions may not strictly follow one of the herein systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen-binding (see, for example: Lu X et al., MAbs. 2019 Jan;11 (1 ):45-57). The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat-, Chothia- or IMGT-defined CDRs.

[0187]

[0114] Conformational epitope'. A conformational epitope is an epitope that is recognized and bound by a conformational antibody in a three-dimensional conformation, but not in an unfolded peptide of the same amino acid sequence. A conformational epitope may be referred to as a conformationspecific epitope, conformation-dependent epitope, or conformation-sensitive epitope. A corresponding antibody or fragment thereof that specifically binds such an epitope may be referred to as conformation-specific antibody, conformation-selective antibody, or conformation-dependent antibody. Binding of an antigen to a conformational epitope depends on the three-dimensional structure (conformation) of the antigen or antigen complex.

[0188] 21

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[0190]

[0115] Constant region / domain: An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art.

[0191]

[0116] Dissociation rate: The term dissociation rate as used herein has the meaning understood by the skilled artisan in the pertinent art {e.g., antibody technology) as refers to a kinetics parameter measured by how fast / slow a ligand {e.g., antibody or fragment) dissociates from its binding target {e.g., antigen, e.g., MuSK). Dissociation rate is also referred to as the “off” rate (“kopp”). Relative on / off rates between an antibody and its antigen (i.e., koN and kopp) determine the overall strength of the interaction, or affinity, typically expressed as a dissociation constant, or KD. Therefore, equivalent affinities {e.g., KD values) may be achieved by having fast association (high koN), slow dissociation (low kopp), or contribution from both factors. Monovalent interactions may be measured by the use of monovalent antigen-binding molecules / fragments, such as fAb (Fab), while divalent interactions may be measured by the use of divalent antigen-binding molecules such as whole immunoglobulins {e.g., IgGs).

[0192]

[0117] Effective amount: The terms “effective” and “therapeutically effective” refer to the ability or an amount to sufficiently produce a detectable change in a parameter of a disease, e.g., a slowing, pausing, reversing, diminution, or amelioration in a symptom or downstream effect of the disease. The term encompasses but does not require the use of an amount that completely cures a disease. According to some embodiments, an “effective amount” (or therapeutically effective amount, or therapeutic dose) is a dosage, concentration, or dosing regimen that achieves statistically significant clinical benefits {e.g., efficacy) in a patient population. For example, for an antibody that has been shown to be efficacious at doses between 3 mg / kg and 30 mg / kg in preclinical models, the effective amount can be said to be between about 3-30 mg / kg.

[0193]

[0118] Epitope: The term “epitope” refers to an antigenic determinant capable of being bound to an antibody. Epitope may be also referred to as an antigenic determinant, and is a molecular determinant {e.g., polypeptide determinant) that is capable of being specifically bound by a binding agent, immunoglobulin or T-cell receptor. Epitope determinants include chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three- dimensional structural characteristics, and / or specific charge characteristics. An epitope recognized by an antibody or an antigen-binding fragment of an antibody is a structural element of an antigen that interacts with CDRs {e.g., the complementary site) of the antibody or the fragment. An epitope may be formed by contributions from several amino acid residues, which interact with the CDRs of the antibody to produce specificity. An antigenic fragment can contain more than one epitope. In certain embodiments, an antibody specifically binds an antigen when it recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0194]

[0119] Frizzled-like domain: The term “Frizzled-like domain” (or “Fz-like domain”) as used herein refers to the domain on MuSK ECD that is C-terminal to Ig-like domains 1 , 2 and 3, and comprises a cysteine-rich domain. The Fz-like domain is not required for MuSK binding to the Agrin-LRP4 complex. However, the Fz-like domain is involved in other signaling pathways and binds to Wnt morphogens. In some embodiments, the MuSK is human MuSK and the Fz-like domain comprises:

[0195] 22

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[0197] DNKGYCAQYRGEVCNAVLAKDALVFLNTSYADPEEAQELLVHTAWNELKVVSPVCRPAAEALLCNHIF QECSPGVVPTPIPICREYCLAVKELFCAKEWLVMEEKTHRGLYRSEMHLLSVPECSKLPSMHWDPTA CARL (SEQ ID NO: 8). The human Fz-like domain corresponds to amino acid positions 312-450 of the human MuSK protein. Herein, amino acid positions in human MuSK are numbered according to the human MuSK protein sequence set out in SEQ ID NO: 1. In some embodiments, the MuSK is mouse MuSK and the Fz-like domain comprises:

[0198] DSQGYCAQYRGEVCDAVLAKDALVFFNTSYRDPEDAQELLIHTAWNELKAVSPLCRPAAEALLCNHLF QECSPGVVPTPMPICREYCLAVKELFCAKEWQAMEGKAHRGLYRSGMHLLPVPECSKLPSMHRDPT ACTRL (SEQ ID NO: 14).

[0199]

[0120] Human antibody. The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0200]

[0121] Humanized antibody. The term “humanized antibody” refers to antibodies, which comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and / or VL sequence has been altered to be more “human-like,” i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which non-human CDR sequences are introduced into human VH and VL framework sequences to replace the corresponding non-human framework sequences. Also "humanized antibody" is an antibody, or a variant, derivative, analog or fragment thereof, which immune-specifically binds to an antigen of interest and which comprises a framework region (FR) having substantially the amino acid sequence of a human antibody and a CDR region having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. In an embodiment a humanized antibody also comprises at least a portion of an immunoglobulin Fc region, typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1 , hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody only contains a humanized light chain. In some embodiments, a humanized antibody only contains a humanized heavy chain. In specific embodiments, a humanized antibody only contains a humanized variable domain of a light chain and / or humanized heavy chain.

[0201] 23

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[0203]

[0122] Hydrogen / deuterium exchange mass spectrometry (HDX-MS): HDX-MS is a well-known technique employed to interrogate protein confirmation and protein-protein interactions in solution by measuring the degree of solvent accessibility. See, for example, Wei et al., (2014) Drug Discov Today 19(1): 95-102. “Hydrogen / deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications.” The HDX-MS technique may be employed to determine a region or regions of an antigen bound by an antibody ( / .e., “binding region(s)”). Thus, such binding region(s) may contain or form an epitope.

[0204]

[0123] Isolated: An “isolated” antibody as used herein, refers to an antibody that is substantially free of other antibodies having different antigenic specificities. In some embodiments, an isolated antibody is substantially free of other unintended cellular material and / or chemicals.

[0205]

[0124] Maximally tolerated dose (MTD): The term MTD generally refers to, in the context of safety / toxicology considerations, the highest amount of a test article (such as a MuSK agonist) evaluated with no observed adverse effect level (NOAEL).

[0206]

[0125] Meso-Scale Discovery: “Meso-Scale Discovery” or “MSD” is a type of immunoassay that employs high binding carbon electrodes to capture proteins (e.g., antibodies). The antibodies can be incubated with particular antigens, which binding can be detected with secondary antibodies that are conjugated to electrochemiluminescent labels. Upon an electrical signal, light intensity can be measured to quantify analytes in the sample.

[0207]

[0126] Muscle force: Muscle performance in a subject or in an animal model is assessed by measuring the muscle force. The term “muscle force” as used herein refers to the measurement of force generated by a muscle. The terms “maximum force” and “max (muscle) force” refer to the maximum force generated by a muscle. In some embodiments, muscle force and “maximum force” are measured in a mouse model (e.g., an Agrin nmf380 mouse model). In some embodiments, maximum force is measured for the plantarflexor muscle group. In some embodiments, maximum force is determined by percutaneous electrical stimulation (e.g. of the sciatic nerve). In some embodiments, the maximum force is determined at 150 Hz stimulation. In some embodiments, maximum force is determined at post-natal day 28 (PND28) following weekly administration of the test antibody from birth (e.g. at 20 mg / kg). In some embodiments, treatment of an agrin mutant mouse model with a MuSK agonist antibody of the disclosure increases maximum muscle force as compared to a control. In some embodiments, treatment of an agrin mutant mouse model with a MuSK agonist antibody of the disclosure increases maximum muscle force by at least 5 mN, 10 mN, 20 mN, 25 mN or 30 mN compared to a control. In some embodiments, treatment of an agrin mutant mouse model with a MuSK agonist antibody of the disclosure increases maximum muscle force by at least 1.5-fold, 2-fold, 3-fold, 5-fold or 10-fold compared to a control. In some embodiments, the control is measured prior to treatment with the MuSK-agonist antibody. In some embodiments, the control is a mouse model that has received isotype matched control IgG, optionally wherein the isotype matched control IgG is a murine IgG 1. In some embodiments, the control is a mouse model treated with a control / comparator antibody.

[0208]

[0127] Muscle weight: The term “muscle weight” or “muscle mass,” as used herein refers to the weight of an isolated muscle. In some embodiments, the weight of the gastrocnemius (gastroc) muscle or the

[0209] 24

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[0211] tibialis anterior (TA) is measured. In some embodiments, treatment of an agrin mutant mouse model with a MuSK agonist antibody of the disclosure increases muscle weight of an isolated muscle as compared to a control. In some embodiments, treatment of an agrin mutant mouse model with a MuSK agonist antibody of the disclosure increases muscle weight of an isolated muscle by at least 5 mg, 10 mg, 20 mg, or 50 mg compared to a control. In some embodiments, treatment of an agrin mutant mouse model with a MuSK agonist antibody of the disclosure increases muscle weight by at least 1.5-fold, 2-fold, 3-fold, 5-fold or 10-fold compared to a control. In some embodiments, the control is measured prior to treatment with the MuSK-agonist antibody. In some embodiments, the control is a mouse model that has received isotype matched control IgG, optionally wherein the isotype matched control IgG is a murine lgG1. In some embodiments, the control is a mouse model treated with a control / comparator antibody.

[0212]

[0128] Off rate (kopp): The off rate is a kinetic parameter of how fast or how slowly an antibody (such as mAb) or antigen-binding fragment (such as fAb) dissociates from its antigen and may be also referred to as the dissociation rate. Dissociation rates can be experimentally measured in suitable in vitro binding assays, such as OCTET®- and BIACORE®-based systems.

[0213]

[0129] Phosphorylation: The term “phosphorylation” refers to the attachment of a phosphate group to a molecule. Phosphorylation of residues on the MuSK protein may be determined through suitable in vitro phosphorylation assays including, for e.g., kinase activity assays, mass spectrometry, use of phosphorylation-specific antibodies in techniques such as Western blotting, ELISA, flow cytometry, etc. In some embodiments, phosphorylation of MuSK is used to assess MuSK activation. In some embodiments, phosphorylation of MuSK is assessed by western blot, immunoprecipitation (IP) western blot, ELISA, or MSD on samples from cells (e.g., murine C2C12 myotubes or cells expressing human or mouse MuSK), with anti-phosphotyrosine antibodies and / or available anti-MuSK antibodies.

[0214]

[0130] Potency: The term “potency” as used herein refers to activity of a drug, such as an antibody (or fragment) having agonist activity, with respect to concentration or amount of the drug to produce a defined effect. For example, an antibody capable of producing certain effects at a given dosage is more potent than another antibody that requires twice the amount (dosage) to produce equivalent effects. Potency may be measured in in vitro or cell-based assays. Typically, among those antibodies capable of binding to the same or overlapping binding regions of an antigen (e.g., cross-blocking antibodies), antibodies with higher affinities (lower KD values) tend to show higher potency than antibodies with lower affinities (greater KD values). The potency of an antibody may be measured in suitable in vitro assays. In some embodiment, the potency of a MuSK agonist antibody may be determined by an AChR clustering assay for measuring MuSK activation (e.g., a high-content imaging-based AChR clustering assays described herein). In some embodiments, the potency of a MuSK agonist antibody is expressed as the ECso of the antibody, calculated based on in v / 'tro AChR clustering assays for measuring MuSK activation. In some embodiments, MuSK-agonist antibodies have an ECso of 10 nM or less (i.e. , < 10 nM). In some embodiments, the antibodies have an ECso of 8 nM or less (i.e., < 8 nM). In some embodiments, the antibodies have an ECso of 4 nM or less (i.e., < 4 nM). In some embodiments, the antibodies have an EC50 of 2.5 nM or less (i.e., < 2.5 nM). In some embodiments, the antibodies have an EC50 of less than 2.5 nM (i.e., < 2.5 nM). In some embodiments, the antibodies have an ECso of 1 nM or less (i.e., 2 1 nM). In some embodiments, the

[0215] 25

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[0217] antibodies have an ECso of 0.50 nM or less (i.e., 0.5 nM). In some embodiments, the antibodies have an ECso of 0.20 nM or less (i.e., < 0.2 nM). In some embodiments, the antibodies have an ECso of 0.10 nM or less (i.e., < 0.1 nM).

[0218]

[0131] Predictive biomarker: Predictive biomarkers provide information on the probability or likelihood of response to a particular therapy. Typically, a predictive biomarker is measured before and after treatment, and the changes or relative levels of the marker in samples collected from the subject indicates or predicts therapeutic benefit.

[0219]

[0132] Protection (from solvent exposure): In the context of HDX-MS-based assessment of proteinprotein interactions, such as antibody-antigen binding, the degree by which a protein (e.g., a region of a protein containing an epitope) is exposed to a solvent, thereby allowing proton exchange to occur, inversely correlates with the degree of binding / interaction. Therefore, when an antibody described herein binds to a region of an antigen, the binding region is “protected” from being exposed to the solvent because the protein-protein interaction precludes the binding region from being accessible by the surrounding solvent. Thus, the protected region is indicative of a site of interaction. Typically, suitable solvents are physiological buffers.

[0220]

[0133] Solution Equilibrium Titration (SET): The SET is an assay whereby binding between two molecules (such as an antigen and an antibody that binds the antigen) can be measured at equilibrium in a solution. For example, Meso-Scale Discovery (“MSD”)-based SET, or MSD-SET, is a useful mode of determining dissociation constants for particularly high-affinity protein-protein interactions at equilibrium, such as picomolar-affinity antibodies binding to their antigens (see, for example: Ducata et al., (2015) J Biomolecular Screening 20(10): 1256-1267). The SET-based assays are particularly useful for determining KD values of antibodies with sub-nanomolar (e.g., picomolar) affinities.

[0221]

[0134] Specific binding: As used herein, the term “specific binding” or “specifically binds” means that the interaction of the antibody, or antigen-binding portion thereof, with an antigen or amino acid residue is dependent upon the presence of a specific amino acid sequence and / or unique structure (e.g., an antigenic determinant or epitope). For example, the antibody, or antigen-binding portion thereof, binds to a specific protein rather than to proteins generally.

[0222]

[0135] Subject: The term “subject” in the context of therapeutic applications refers to an individual who receives clinical care or intervention, such as treatment, diagnosis, etc. Suitable subjects include vertebrates, including but not limited to mammals (e.g., human and non-human mammals). Where the subject is a human subject, the term “patient” may be used interchangeably. In a clinical context, the term “a patient population” or “patient subpopulation” is used to refer to a group of individuals that falls within a set of criteria, such as clinical criteria (e.g., disease presentations, disease stages, susceptibility to certain conditions, responsiveness to therapy, etc.), medical history, health status, gender, age group, genetic criteria (e.g., carrier of certain mutation, polymorphism, gene duplications, DNA sequence repeats, etc.) and lifestyle factors (e.g., diet, smoking, alcohol consumption, exercise, etc.).

[0223]

[0136] Tail: The term “tail,” “C-terminal tail” or “tail region” as used herein refers to the 45 amino acids of human MuSK or 44 amino acids of mouse MuSK that are C-terminal to the Frizzled-I ike domain. In

[0224] 26

[0225] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0226] some embodiments, the MuSK is human MuSK and the tail region consists of the sequence PHLDYNKENLKTFPPMTSSKPSVDIPNLPSSSSSSFSVSPTYSMT (SEQ ID NO: 175). In some embodiments, the MuSK is a mouse MuSK and the tail region of a mouse MuSK consists of the 44 amino acid sequence PYLDYKKENITTFPSITSSRPSADIPNLPASTSSFAVSPAYSMT (SEQ ID NO: 176).

[0227]

[0137] Truncated Tait. The term “truncated tail," “C-terminal truncated tail,” or “truncated C-terminal tail," as used herein refers to the 7 amino acids of MuSK that are C-terminal to the Frizzled-I ike domain. In some preferred embodiments, the MuSK is human MuSK and the truncated tail region consists of the sequence PHLDYNK (SEQ ID NO: 9). This Truncated Tail sequence corresponds to amino acid positions 451 -457 of the human MuSK protein. In some embodiments, the MuSK is mouse MuSK and the truncated tail region consists of the sequence PYLDYKK (SEQ ID NO: 15). In some embodiments, the MuSK is cynomolgus MuSK and the truncated tail region consists of the amino acid sequence PHLDYNK (SEQ ID NO: 9). In some embodiments, the MuSK is rat MuSK and the truncated tail region consists of the amino acid sequence PYLDYKK (SEQ ID NO: 15).

[0228]

[0138] Target engagement: As used herein, the term target engagement refers to the ability of a molecule (e.g., MuSK agonist) to bind to its intended target in vivo (e.g., endogenous MuSK).

[0229]

[0139] Therapeutic window. The term “therapeutic window” refers to a range of doses / concentrations that produces therapeutic response without causing significant / observable / unacceptable adverse effect (e.g., within adverse effects that are acceptable or tolerable) in subjects. Therapeutic window may be calculated as a ratio between minimum effective concentrations (MEG) to the minimum toxic concentrations (MTC). To illustrate, a MuSK agonist that achieves in vivo efficacy at 10 mg / kg and shows tolerability or acceptable toxicities at 100 mg / kg provides at least a 10-fold (e.g., 10x) therapeutic window. By contrast, a comparator agonist of MuSK that is efficacious at 10 mg / kg but causes adverse effects at 5 mg / kg is said to have “dose-limiting toxicities.”

[0230]

[0140] Toxicity: As used herein, the term “toxicity” or “toxicities” refers to unwanted in vivo effects in subjects (e.g., patients) associated with a therapy administered to the subjects (e.g., patients), such as undesirable side effects and adverse events. “Tolerability” refers to a level of toxicities associated with a therapy or therapeutic regimen, which can be reasonably tolerated by patients, without discontinuing the therapy due to the toxicities. Typically, toxicity / toxicology studies are carried out in one or more preclinical models prior to clinical development to assess safety profiles of a drug candidate (e.g., monoclonal antibody therapy). Toxicity / toxicology studies may help determine the “no observed adverse effect level (NOAEL)" and the “maximally tolerated dose (MTD)" of a test article, based on which a therapeutic window may be deduced. Preferably, a species that is shown to be sensitive to the particular intervention should be chosen as a preclinical animal model in which safety / toxicity study is to be carried out. Suitable species include rats, mice, and cynos.

[0231]

[0141] Treat / treatment: The term “treat” or “treatment” includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Thus the term is intended to broadly mean: causing therapeutic benefits in a patient by, for example, slowing disease progression, reversing certain disease features, normalizing gene expression, enhancing or boosting the body’s immunity; reducing or reversing immune suppression;

[0232] 27

[0233] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0234] reducing, removing or eradicating harmful cells or substances from the body; reducing disease burden (e.g., neuromuscular diseases and disorders); preventing recurrence or relapse; prolonging a refractory period, and / or otherwise improving survival. The term includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors. In the context of combination therapy, the term may also refer to: I) the ability of a second therapeutic to reduce the effective dosage of a first therapeutic so as to reduce side effects and increase tolerability; ii) the ability of a second therapy to render the patient more responsive to a first therapy; and / or iii) the ability to effectuate additive or synergistic clinical benefits.

[0235]

[0142] Variable region: The term “variable region” or “variable domain” refers to a portion of the light and / or heavy chains of an antibody, typically including approximately the amino-terminal 120 to 130 amino acids in the heavy chain and about 100 to 110 amino terminal amino acids in the light chain. In certain embodiments, variable regions of different antibodies differ extensively in amino acid sequence even among antibodies of the same species. The variable region of an antibody typically determines specificity of a particular antibody for its target.

[0236]

[0143] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages can mean ±1 %.

[0237]

[0144] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0238]

[0145] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0239]

[0146] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than

[0240] 28

[0241] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0242] one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0243]

[0147] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0244]

[0148] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 1 , 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50, e.g., 10-20, 1-10, 30-40, etc.

[0245] Neuromuscular Junction (NMJ)

[0246]

[0149] A motor unit is comprised of a motor neuron and a target muscle it innervates. The neuromuscular junction (NMJ) is a synaptic connection formed between a motor neuron terminal (presynaptic) and a muscle fiber (postsynaptic). NMJs are essential for all voluntary movements and physical mobility since muscle contraction is initiated by synaptic transmissions at the NMJ. Deficits in NMJ formation and function thus cause several neuromuscular diseases / disorders, {e.g. Amyotrophic lateral sclerosis (ALS), Botulism, Congenital myasthenic syndromes, Congenital myopathies, Cramp-fasciculation syndrome, Elevated creatine kinase, Inclusion-body myositis, Lambert-Eaton syndrome, Mitochondrial myopathy, Motor neuron disease e.g., spinal muscular atrophy (SMA)), Muscular dystrophy {e.g., Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD)), Myasthenia gravis, Myotonic dystrophy, Neuromyotonia, Peripheral neuropathy, Polymyositis)

[0247]

[0150] An NMJ comprises a presynaptic terminal on a motor neuron and a postsynaptic muscle membrane separated by a synaptic cleft. Synaptic transmission at the NMJ begins with the arrival of an action potential at the presynaptic terminal of the motor neuron, upon which the motor neuron terminals release the neurotransmitter acetylcholine (ACh). ACh activates ACh receptors (AChRs) on the postsynaptic muscle membrane which in turn triggers the opening of ion channels such as voltage-gated Na+channels, to depolarize the muscle and initiate muscle contraction.

[0248]

[0151] Proper synaptic transmission requires a high density of receptors localized on the post-synaptic muscle membrane which is achieved through the clustering of ACh receptors on the membrane. In vertebrate NMJs, AChRs are typically clustered to an estimated density of ~10,000 / pm2. AChR clustering is thus the hallmark of a normally functioning NMJ. The clustering of AChRs on the postsynaptic membrane occurs during the formation of the NMJ and is the result of a complex signaling

[0249] 29

[0250] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0251] process involving the proteins Agrin, Low-density lipoprotein receptor-related protein 4 (LRP4) and Muscle-Specific Kinase (MuSK), collectively referred to as the agrin-LRP4-MuSK axis.

[0252] Muscle-Specific Kinase (MuSK)

[0253]

[0152] MuSK is a receptor tyrosine kinase that is required for the formation, organization and maintenance of the NMJ. It is highly expressed in skeletal muscles and is concentrated at NMJs as a postsynaptic integral membrane protein. MuSK plays an important role in the formation and stabilization of NMJs. Before innervation of the muscle fiber, MuSK initiates post-synaptic differentiation, thereby priming the muscle for synapse formation. After innervation, MuSK is responsible for efficient neurotransmission. In addition, MuSK promotes retrograde signaling that stimulates pre-synaptic differentiation and stabilization of motor nerve terminals.

[0254]

[0153] MuSK is critical for AChR clustering and neuromuscular transmission at the NMJ. In particular, MuSK activation at the NMJ organizes Acetylcholine Receptor (AChR) clusters on the muscle necessary for neuro-transmission. The canonical pathway for MuSK activation involves the proteins Agrin and LRP4. Agrin, a large heparan sulphate proteoglycan, is secreted by motor neurons into the synaptic cleft and localizes to basal laminae. Agrin binds to the extracellular domain of its receptor LRP4 which is a transmembrane protein on the postsynaptic membrane. The Agrin-LRP4 complex subsequently binds to the extracellular domain of MuSK, which induces MuSK dimerization. This in turn leads to the autophosphorylation of a tyrosine residue (Y554 in human MuSK, Y553 in mouse MuSK) present on the intracellular domain of MuSK, which promotes the recruitment of the protein Dok7 via its binding to the Y553 residue. Dok7 binding induces Dok7 dimerization, which then causes the trans autophosphorylation of the kinase domain of MuSK, at tyrosine residues present on the kinase activation loop (Y751 , S752, Y755 and Y756 in human MuSK; Y750, S751 , Y754 and Y755 in mouse MuSK). This event relieves the autoinhibition of the kinase domain, and activates and stabilizes MuSK. Subsequently, the kinase domain of activated MuSK phosphorylates Dok7, which triggers a signaling cascade that ultimately induces rapsyn-dependent clustering of AChRs on the postsynaptic muscle membrane. Thus, MuSK activation leads to the signaling involved in AChR regulation, including AChR expression, transport, stabilization and clustering. The neurotransmitter acetylcholine, secreted by motor neurons, binds to the clustered AChRs and initiates muscle contraction.

[0255]

[0154] Human MuSK is an 869 amino acid-long Type 1 membrane protein with a size of ~97 kDa. Murine MuSK is an 868 amino acid-long protein. MuSK is a transmembrane receptor protein comprising an extracellular domain (ECD), a transmembrane region and an intracellular domain, starting from the N-terminus. The ECD of human MuSK is 472 amino acids long (471 amino acids in mouse MuSK) with a molecular weight of 52 kDa, and contains two N-linked glycosylation sites at N222 & N338. The ECD contains of three Ig-like domains (Ig-LD) in tandem, followed by a fourth domain called a Frizzled-like domain (Fz-like domain) and a tail region C-terminal to the Fz-like domain (this region itself comprising a Truncated Tail region of 7 amino acids). The ECD is followed by a transmembrane region which connects to an intracellular domain. The intracellular domain of MuSK comprises a juxtamembrane region followed by the tyrosine kinase domain. The dimerization of

[0256] 30

[0257] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0258] MuSK upon Agrin-LRP4 binding leads to the phosphorylation of several residues. In human MuSK, these residues include: Y554 (present on the juxtamembrane region), and Y751 , S752, Y755 and Y756 (found on the activation loop of the kinase domain). In mouse MuSK, these residues correspond to: Y553, Y750, S751, Y754 and Y755, respectively. Table 1 provides the amino acid sequences of exemplary MuSK proteins. Preferably, the MuSK protein or MuSK protein domain bound by antibodies or antigen-binding fragments of the disclosure is a human MuSK protein or protein domain. The antibodies or antigen-binding fragments are preferably also cross-reactive with mouse MuSK.

[0259] Table 1. Exemplary MuSK protein amino acid sequences

[0260]

[0261] 31

[0262] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0263]

[0264]

[0155] The MuSK sequence is highly conserved among mammalian species. Table 2 lists the percent (%) sequence identity between human MuSK and other species homologs.

[0265] Table 2. Percent sequence identity between human MuSK and species homologs.

[0266]

[0267] 32

[0268] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0269]

[0156] Table 3 below provides the sequences of select protein domains / modules of human and mouse MuSK proteins.

[0270] Table 3. Select protein domains / modules of MuSK proteins

[0271]

[0272] 33

[0273] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0274]

[0275] 34

[0276] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0277]

[0278]

[0157] Disruptions of the MuSK pathway lead to impaired neuromuscular transmission and motor function causing muscle weakness and / or atrophy. Deficiencies in the pathway have been implicated in several neuromuscular diseases or disorders including, but not limited to Amyotrophic lateral sclerosis (ALS), Botulism, Congenital myasthenic syndromes, Congenital myopathies, Cramp-fasciculation syndrome, Elevated creatine kinase, Inclusion-body myositis, Lambert-Eaton syndrome, Mitochondrial myopathy, Motor neuron disease, Muscular dystrophy, Myasthenia gravis (MG), Myotonic dystrophy, Neuromyotonia, Peripheral neuropathy, Polymyositis. Most forms of MG are associated with autoantibodies to components of the MuSK pathway. Congenital Myasthenia, which occurs more rarely in the overall population, is caused by loss-of-function mutations in Agrin, MuSK or related proteins.

[0279] Comparator anti-MuSK agonist antibodies (“comparator antibodies”)

[0280]

[0158] Agonist antibodies that bind to the F rizzled - 1 ike domain of MuSK have been shown to exhibit some preclinical therapeutic potential in animal models of congenital myasthenia and amyotrophic

[0281] 35

[0282] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0283] lateral sclerosis (ALS). Some of these antibodies were not selective to MuSK and / or did not crossreact with human MuSK.

[0284] mAb13

[0285]

[0159] In the context of MuSK agonist antibodies, previously Xie et al. (Nature Biotechnology 1997, Vol 15: August, 768-771 ) produced scFvs that bind MuSK, by engineering fusion proteins comprising ECD of MuSK fused to intracellular domain of c-mpl (thrombopoietin receptor) as a reporter assay for binding (using proliferation as readout). Antibodies disclosed included agonist anti-MuSK antibody and ScFv (referred to as “lgG#13” and corresponding ScFv as “ScFv#13”).

[0286]

[0160] Subsequently, Cantor et al. (ELIFE vol.7, 20 Feb 2018, pp. 1-19) further studied this antibody #13 on a murine lgG2a backbone that also lacked effector function in SOD1-G93A mice, a model of ALS, and “re-engineered as human lgG1 molecules.” The authors found both this “reverse chimera” and the human engineered version to bind and stimulate MuSK and induce AChR clustering. In addition, Cantor et al. determined that antibody #13 bound to the Frizzled- like domain “(D312 to K456)” from mouse MuSK.

[0287]

[0161] United States Patent No. 6,342,220 (entitled “Multispecific molecules that bind to myeloproliferative leukemia (MPL) protein and uses thereof”) disclosed the same data with respect to MuSK antibody lgG#13 as Xie et al, despite having claims directed to c-mpl antibodies.

[0288]

[0162] United States Patent No. 9,329,182 (published as US2015 / 0050289, entitled “Method of Treating Motor Neuron Disease with an Antibody that Antagonizes MuSK”) discloses antibody #13 as a MuSK agonist antibody, and the same sequences as United States Patent No. US 6,342,220. The ‘182 patent discloses that antibody #13 “binds MuSK” and “increases MuSK activity upon binding.”

[0163] Notably, antibody #13 is not cross-reactive to human MuSK. This lack of human reactivity limits its clinical utility, while serving as a useful tool for preclinical research and discovery. Antibody #13 (mAb13) sequences are disclosed in Sengupta-Ghosh et al. (2019, Neurobiology of Disease, Vol. 124, 340-352). The VH and VL sequences from antibody #13, along with CDR sequences determined according to IMGT, are as follows:

[0289]

[0164] VH: (SEQ ID NO: 177) EVQLVESGGGVVQPGGSLRLSCAASGFTFSSFGMHWVRQAPDKGLEWVSSISGSGGSTYYADSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWERWLPQGAFDYWGRGTLVTVSS

[0290]

[0165] VL: (SEQ ID NO: 178) DIQMTQSPSTLSASIGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRFSGSGSG TDFTLTISSLQPDDFATYYCQQYSNYPLTFGGGTKLEIK

[0291]

[0166] Antibody #13 CDR-H1 : GFTFSSFG (SEQ ID NO: 179)

[0292]

[0167] Antibody #13 CDR-H2: ISGSGGST(SEQ ID NO: 180)

[0293]

[0168] Antibody #13 CDR-H3: AKGWERWLPQGAFDY (SEQ ID NO: 181 )

[0294]

[0169] Antibody #13 CDR-L1 : EGIYHW (SEQ ID NO: 182)

[0295] 36

[0296] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0297]

[0170] Antibody #13 CDR-L2: KAS

[0298]

[0171] Antibody #13 CDR-L3: QQYSNYPLT (SEQ ID NO: 184)

[0299] 3B2G2

[0300]

[0172] An anti-MuSK agonist antibody termed 3B2G2 and related antibodies are disclosed in United States Patent No. 11,492,401 (corresponding PCT publication: WO2021 / 212053, entitled “Therapeutic musk antibodies”). Disclosed is an anti-MuSK agonist antibody that binds the Frizzled- like domain of human MuSK (defined as SEQ ID NO: 130 in WO2021 / 212053; SEQ ID NO: 8 herein):

[0301] DNKGYCAQYRGEVCNAVLAKDALVFLNTSYADPEEAQELLVHTAWNELKVVSPVCRPAAEALLCNHIF QECSPGVVPTPIPICREYCLAVKELFCAKEWLVMEEKTHRGLYRSEMHLLSVPECSKLPSMHWDPTA CARL.As disclosed in the aforementioned PCT publication, the anti-MuSK antibody, 3B2g2, binds an epitope contained within the Fz-like domain defined as SEQ ID NO: 130 according to WO 2021 / 21053 of human MuSK. 3B2G2 solves the shortcoming of mab#13 in that 3B2G2 cross-reacts with both murine and human MuSK. In addition, mab#13 and 3B2G2 cross-block one another, indicating that they are classified into the same epitope bin and share the mechanism of action. As shown in the Example section herein, neither mab#13 nor 3B2G2 requires the presence of a tail sequence (e.g., 7AA Truncated Tail C-terminus to the Fz-like domain) for antigen binding. Rather, each binds an epitope contained within the Fz-like domain.

[0302]

[0173] As disclosed in WO2021 / 212053, antibody 3B2G2m1 has the following sequences:

[0303]

[0174] 3B2G2m1 VH (SEQ 234 in WO2021 / 212053); (SEQ ID NO: 185 herein):

[0304] EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYGMSWVRQAPGKGLEWVSAIPWSGGSTYYKESVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRSGRIAFGALDAWGQGTLVTVSS

[0305]

[0175] 3B2G2m1 VL (SEQ 235 in WO2021 / 212053); (SEQ ID NO: 186 herein):

[0306] QTVVTQEPSFSVSPGGTVTLTCGLSSGSVTSSNYPDWYQQTPGQAPRTLIYSTDSRHSGVPDRFSG SILGNKAALTITGAQADDESDYYCGLYSYSGSKNYVFGGGTKLTVL

[0307]

[0176] The CDR sequences for antibody 3B2G2m1 in the disclosure of WO2021 / 212053 are as follows:

[0308]

[0177] 3B2G2m1 CDR-H1 : DYGMS (SEQ ID NO: 187 herein)

[0309]

[0178] 3B2G2m1 CDR-H2: AIPWSGGSTYYKESVKG (SEQ ID NO: 188 herein)

[0310]

[0179] 3B2G2m1 CDR-H3: RSGRIAFGALDA (SEQ ID NO: 189 herein)

[0311]

[0180] 3B2G2m1 CDR-L1 : GLSSGSVTSSNYPD (SEQ ID NO: 190 herein)

[0312]

[0181] 3B2G2m1 CDR-L2: STDSRHS (SEQ ID NO: 191 herein)

[0313]

[0182] 3B2G2m1 CDR-L3: GLYSYSGSKNYV (SEQ ID NO: 192 herein)

[0314]

[0183] ARGX-119 is a humanized agonist monoclonal antibody to MuSK that binds the Frizzled-like domain of human and mouse MuSK (Vanhauwaert et al., Sci Transl Med, 2024 Sep 18; 16(765): 7189), which was derived from 3B2G2. ARGX-119 is currently in clinical development byArgenx.

[0315] 37

[0316] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0317] Vanhaurwaert et al. references PCT Publication No. WO2023 / 147489. WO2023 / 147489 discloses an antibody described as “3B2g2m1 -h IgG 1 ” and “3B2g2m1 -h IgG 1 LALAdelk” and having the same CDR sequences 3B2G2m1 in WO2021 / 212053.

[0318]

[0184] The CDR sequences of antibody 3B2g2m1 (derived according to IMGT numbering from VH and VL sequences SEQ ID NO: 234 and SEQ ID NO: 235 in WO2023 / 147489) are the following: 3B2g2m1 CDR-H1 : GFTFSDYG (SEQ ID NO: 193 herein)

[0319] 3B2g2m1 CDR-H2: IPWSGGST (SEQ ID NO: 194 herein)

[0320] 3B2g2m1 CDR-H3: AKRSGRIAFGALDA (SEQ ID NO: 195 herein)

[0321] 3B2g2m1 CDR-L1 : SGSVTSSNY (SEQ ID NO: 196 herein)

[0322] 3B2g2m1 CDR-L2: STD (SEQ ID NO: 197 herein)

[0323] 3B2g2m1 CDR-L3: GLYSYSGSKNYV (SEQ ID NO: 198 herein)

[0324] 4D3

[0325]

[0185] In contrast to the above-identified comparator antibodies (which bind to the Fz-like domain of MuSK), a previous anti-MuSK antibody is included as a comparator (in this case as a non-Fz-like domain binder, that instead binds to the Ig-like domain 1 (lg-1 ) of MuSK). 4D3 is an antibody described in Lim et al. (Nature Sci Reports 2023 13:7478) as binding to the Ig-like domain 1 (lg-1) of MuSK, from a MuSK MG patient. In addition, sequences of antibody 4D3 are disclosed in PCT Publication No. WO2021 / 180676.

[0326] 4D3a VH and VL sequences:

[0327]

[0186] 4D3 VH SEQ 38 in WO2021 / 180676; SEQ ID NO: 199 herein:

[0328] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYTMTWVRQAPGKGLEWVSSISSGGHYIYYTDSLKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARERLLRLGVGFDFWGQGTLVTVSS

[0329]

[0187] 4D3 VK SEQ 58 in WO2021 / 180676; SEQ ID NO: 200 herein:

[0330] DIQMTQSPSSLSASVGDRVTITCRASQSISGYLNWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQSYSALYTFGQGTKLEIK

[0331]

[0188] The 4D3 CDRs determined by IMGT numbering based on the disclosure of WO2021 / 180676 4D3 HCDR1; GFTFSSYT (SEQ ID NO: 201 herein)

[0332] 4D3 HCDR2; ISSGGHYI (SEQ ID NO: 202 herein)

[0333] 4D3 HCDR3; ARERLLRLGVGFDF (SEQ ID NO: 203 herein)

[0334] 4D3 LCDR1; QSISGY (SEQ ID NO: 204 herein)

[0335] 4D3 LCDR2; AAS (SEQ ID NO: 205 herein)

[0336] 38

[0337] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0338] 4D3 LCDR3: QQSYSALYT (SEQ ID NO: 206 herein)

[0339] Novel and potent MuSK agonist antibodies

[0340]

[0189] The present disclosure provides a class of novel monoclonal antibodies and antigen-binding fragments thereof capable of binding the MuSK protein. The novel antibodies and the antigen-binding fragments thereof disclosed in the present application are capable of binding to and activating MuSK. In some embodiments, the novel antibodies of the disclosure promote the dimerization of MuSK, leading to phosphorylation and activation of the MuSK signaling pathway. The MuSK agonist antibodies or antigen-binding fragments of the disclosure are capable of stabilizing the NMJ structure and increasing motor unit strength / muscle function in neuromuscular diseases.

[0341]

[0190] The present disclosure provides a novel class of MuSK agonist antibodies and antigenfragments thereof. This class of antibodies is characterized by binding to a novel epitope(s) within the ECD of MuSK and activating MuSK. In some embodiments, antibodies and antigen-binding fragments of the disclosure require the presence of the Truncated tail C-terminal to the Fz-like domain in order to bind to MuSK. In some embodiments, the antibodies and antigen-binding fragments of the disclosure are not capable of binding to a MuSK protein, or fragment thereof, that lacks the tail C-terminal to the Fz-like domain. More particularly, the antibodies may not be capable of binding to a MuSK protein, or fragment thereof, that lacks the Truncated tail C-terminal to the Fz-like domain. The MuSK antibodies or antigen-binding fragments may bind a conformational epitope. In some embodiments, the presence of the Truncated Tail region of MuSK is required to form the conformational epitope to which the antibody binds. In some embodiments, the antibodies or antigenbinding fragments thereof bind the Frizzled-I ike domain of MuSK to the extent that the MuSK comprises the 7 amino acid long Truncated tail C-terminal to the Fz-like domain. In some embodiments, the antibodies or antigen-binding fragments thereof bind a conformational epitope that comprises one or more amino acid residues of the Fz-like domain and one or more amino acid residues of the Truncated tail C-terminal to the Fz-like domain. In other embodiments, the antibodies or antigen-binding fragments thereof bind an epitope within the tail C-terminal to the Fz-like domain. In some embodiments, the antibodies or antigen-binding fragments thereof bind an epitope within the Truncated tail C-terminal to the Fz-like domain. In any of the embodiments, the novel class of MuSK agonists require the Truncated tail C-terminal to the Fz-like domain for binding to MuSK.

[0342]

[0191] In some embodiments, the antibodies and antigen-binding fragments bind MuSK with high affinity (e.g., below 1 nM KD). In some embodiments, the binding affinity is measured, for example, by biolayer interferometry (BLI), surface plasmon resonance (SPR) or Solution Equilibrium Titration (e.g., MSD-SET).

[0343]

[0192] The MuSK agonist antibody or the antigen-binding fragment thereof comprises an H-CDR1 , an H-CDR2, and H-CDR3, an L-CDR1, an L-CDR2 and an L-CDR3. In some embodiments, an antibody or an antigen-binding fragment thereof according to the present disclosure has the six CDRs of Ab1 , Ab2, Ab3, Ab4, Ab5 or Ab6. For instance, in some embodiments, an antibody or an antigen-binding fragment thereof according to the present disclosure comprises an H-CDR1 , an H-CDR2, and H-CDR3, an L-CDR1, an L-CDR2 and an L-CDR3, wherein: the H-CDR1 comprises GGTFSDQT (SEQ

[0344] 39

[0345] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0346] ID NO: 17), the H-CDR2 comprises ISGYSGIT (SEQ ID NO: 18), the H-CDR3 comprises ARDRYGYFDY (SEQ ID NO: 19); and the L-CDR1 comprises RDLGGW (SEQ ID NO: 20), the L-CDR2 comprises WAS, the L-CDR3 comprises QQTDRLPLT (SEQ ID NO: 22) as determined using the IMGT numbering scheme. In some embodiments, the antibody or an antigen-binding fragment thereof according to the present disclosure comprises an H-CDR1 , an H-CDR2, and H-CDR3, an L-CDR1, an L-CDR2 and an L-CDR3, wherein: the H-CDR1 comprises GLTVSTSD (SEQ ID NO: 23), the H-CDR2 comprises IAGSGFGT (SEQ ID NO: 24), the H-CDR3 comprises ARDIGGDYGHYYGMDV (SEQ ID NO: 25); and the L-CDR1 comprises QSLLHSDRYNY (SEQ ID NO: 26), the L-CDR2 comprises LAS, the L-CDR3 comprises MQALGLPRT (SEQ ID NO: 28) as determined using the IMGT numbering scheme. In some embodiments, the antibody or an antigenbinding fragment thereof according to the present disclosure comprises an H-CDR1 , an H-CDR2, and H-CDR3, an L-CDR1 , an L-CDR2 and an L-CDR3, wherein: the H-CDR1 comprises GYSFSLYW (SEQ ID NO: 29), the H-CDR2 comprises ISPGHTAT (SEQ ID NO: 30), the H-CDR3 comprises ATGGVVAATAFDI (SEQ ID NO: 31); and the L-CDR1 comprises QSVDRY (SEQ ID NO: 32), the L-CDR2 comprises GTS, the L-CDR3 comprises QQYNQLPIT (SEQ ID NO: 34) as determined using the IMGT numbering scheme. In some embodiments, the antibody or an antigen-binding fragment thereof according to the present disclosure comprises an H-CDR1 , an H-CDR2, and H-CDR3, an L-CDR1, an L-CDR2 and an L-CDR3, wherein: the H-CDR1 comprises GGTFSSHA (SEQ ID NO: 35), the H-CDR2 comprises I IPISGTI (SEQ ID NO: 36), the H-CDR3 comprises AKHFRWSMDV (SEQ ID NO: 37); and the L-CDR1 comprises QSIRDY (SEQ ID NO: 38), the L-CDR2 comprises AGY, the L-CDR3 comprises QQTYNIPLT (SEQ ID NO: 40) as determined using the IMGT numbering scheme. In particular embodiments, the antibody or antigen-binding fragment thereof has the six CDRs of Ab3 or Ab4. In particular embodiments, the antibody or antigen-binding fragment thereof has the six CDRs of Ab5 or Ab6.

[0347] Table 4. Complementarity determining regions (CDR) of the heavy chain of exemplary antibodies, as determined using the IMGT numbering scheme.

[0348]

[0349] Table 5. Complementarity determining regions (CDR) of the light chain of exemplary antibodies, as determined using the IMGT numbering scheme.

[0350]

[0351] 40

[0352] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0353]

[0354]

[0193] Determination of CDR sequences within an antibody depends on the particular numbering scheme being employed. Commonly used systems include but are not limited to: Kabat numbering system, IMTG numbering system, Chothia numbering system, and others such as the numbering scheme described by Lu et al. (Lu X et al., MAbs. 2019 Jan;11(1):45-57). To illustrate, the 6 CDR sequences of exemplary antibodies as defined by three different numbering systems are exemplified below. Any art-recognized CDR numbering systems may be used to define CDR sequences of the antibodies of the present disclosure.

[0355] Table 6. Six CDRs of exemplary antibodies based on three numbering schemes

[0356]

[0357] 41

[0358] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0359]

[0360] 42

[0361] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0362]

[0363]

[0194] Amino acid sequences of the heavy chain variable domain and the light chain variable domain of exemplary antibodies of the present disclosure are provided in Table 7. Thus, in some embodiments, the MuSKagonist antibody of the present disclosure may be an antibody or an antigen-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the H and the VL sequences are selected from any one of the sets of VH and VL sequences listed in Table 7 below.

[0364] Table 7. Heavy chain variable domains and light chain variable domains of exemplary antibodies

[0365]

[0366] 43

[0367] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0368]

[0369]

[0195] Thus, the invention provides an antibody or an antigen-binding fragment thereof that comprises a heavy chain variable domain and a light chain variable domain, wherein, the heavy chain variable domain has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99% and 100%) sequence identity with any one of the heavy chain variable domain sequences selected from the group consisting of: Ab1 , Ab2, Ab3, Ab4, Ab5 and Ab6; and, wherein the light chain variable domain has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99% and 100%) sequence identity with any one of the light chain variable domain sequences of Ab1 , Ab2, Ab3, Ab4, Ab5, or Ab6, wherein, optionally, the heavy chain variable domain has at least 95% sequence identity, and / or, the light chain variable domain has at least 95% (e.g., at least 95%, 96%, 97%, 98% 99% and 100%) sequence identity. In some embodiments, the heavy chain variable domain of the antibody or the fragment has at least 90% sequence identity with any one of SEQ ID NOs: 89, 91 , 93, 95, 210 or 212, and optionally, the light chain variable domain of the antibody or the fragment has at least 90% sequence identity with any one of SEQ ID NOs: 90, 92, 94, 96, 211 or 213. In some embodiments, the heavy chain variable domain of the antibody or the fragment has at least 95% sequence identity with any one of SEQ ID NOs: 89, 91 , 93, 95, 210 or 212, and optionally, the light chain variable domain of the antibody or the fragment has at least 95% sequence identity with any one of SEQ ID NOs: 90, 92, 94, 96, 211 or 213. In some embodiments, the heavy chain variable domain of the antibody or the fragment has at least 98% sequence identity with any one of SEQ ID NOs: 89, 91 , 93, 95, 210 or 212, and optionally, the light chain variable domain of the antibody or the fragment has at least 98% sequence identity with any one of SEQ ID NOs: 90, 92, 94, 96, 211 or 213. In some embodiments, the heavy chain variable domain of the antibody or the fragment has 100% sequence identity with any one of SEQ ID NOs: 89, 91 , 93, 95, 210 or 212, and optionally, the light chain variable domain of the antibody or the fragment has 100% sequence identity with any one of SEQ ID NOs: 90, 92, 94, 96, 211 or 213. In further embodiments, the heavy chain variable domain and light chain variable domain have at least 90% (e.g. at least 95%, or at least 98%) identity to or are identical to the corresponding pair of heavy and light chain variable domains of Ab1 , Ab2, Ab3, Ab4, Ab5 or Ab6. In particular embodiments, the heavy chain variable domain and light chain variable

[0370] 44

[0371] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0372] domain have at least 90% (e.g. at least 95%, or at least 98%) identity to or are identical to the corresponding pair of heavy and light chain variable domains of Ab3, Ab4, Ab5 or Ab6.

[0373]

[0196] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable domain and / or a light chain variable domain wherein the N-terminal glutamine of the heavy chain variable domain amino acid sequence is a pyroglutamate and / or the N-terminal glutamine of the light chain variable domain amino acid sequence is a pyroglutamate. In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable domain comprising an N-terminal pyroglutamate and / or a light chain variable domain comprising an N-terminal pyroglutamate. In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable domain and / or a light chain variable domain wherein the N-terminal glutamine of the heavy chain variable domain amino acid sequence is expected to be cyclized to pyroglutamate and / or the N-terminal glutamine of the light chain variable domain amino acid sequence is expected to be cyclized to a pyroglutamate.

[0374]

[0197] In some embodiments, the antibody selected from Ab1 , Ab4 and Ab6 or antigen-binding portion thereof comprises a heavy chain variable domain comprising an N-terminal pyroglutamate. In some embodiments, the antibody Ab1 or antigen-binding portion thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 89, wherein the N-terminal glutamine of SEQ ID NO: 89 is a pyroglutamate. In some embodiments, the antibody Ab4 or antigen-binding portion thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 95, wherein the N-terminal glutamine of SEQ ID NO: 95 is a pyroglutamate. In some embodiments, the antibody Ab6 or antigen-binding portion thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 212, wherein the N-terminal glutamine of SEQ ID NO: 212 is a pyroglutamate.

[0375]

[0198] In some examples, any of the antibodies of the disclosure that specifically bind to a MuSK protein, or fragment thereof, comprising a frizzled-like domain and a truncated tail C-terminal to the f rizzled-like domain, but are not capable of binding to a frizzled-like domain without the truncated tail C-terminal to the frizzled-like domain, and activate MuSK include any antibody (including antigen binding portions thereof) having one or more CDR (e.g., CDRH or CDRL) sequences substantially similar to CDRH1 , CDRH2, CDRH3, CDRL1 , CDRL2, and / or CDRL3. For example, the antibodies may include one or more CDR sequences as shown in Table 6 containing up to 5, 4, 3, 2, or 1 amino acid residue variations as compared to the corresponding CDR region in any one of SEQ ID NOs: 17-20, 22-26, 28-32, 34-38, 40-68, 70-74, 76-80, 82-86 or 88. In some embodiments, one or more of the six CDR sequences contain up to three (3) amino acid changes as compared to the sequences provided in Table 6. Such antibody variants comprising up to 3 amino acid changes per CDR are encompassed by the present invention. In some embodiments, such variant antibodies are generated by the process of optimization, such as affinity maturation. The complete amino acid sequences for the heavy chain variable region and light chain variable region of the antibodies listed in Table 7 (e.g., Ab1 , Ab2, Ab3, Ab4, Ab5, Ab6), as well as nucleic acid sequences encoding the heavy chain and light chain of certain antibodies are provided below:

[0376] 45

[0377] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0378] Ab1 - Heavy chain variable region amino acid sequence (SEQ ID NO: 89) QVQLVQSGAEVKKPGASVKVSCKVSGGTFSDQTIHWVRQAPGKGLEWMGGISGYSGITIYAQKFQG RVTMTEDTSTDTAYMELSSLKSEDTAVYYCARDRYGYFDYWGQGTLVTVSS

[0379] Ab1 - Light chain variable region amino acid sequence (SEQ ID NO: 90) DIQMTQSPSSVSASVGDRVTITCRASRDLGGWLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSG SGTDFTLTISSLQPEDFANYYCQQTDRLPLTFGGGTKVEIK

[0380] Ab2 - Heavy chain variable region amino acid sequence (SEQ ID NO: 91) EVQLVESGGGLVQPGGSLRLSCAASGLTVSTSDMSWVRQAPGKGLELVASIAGSGFGTYYPDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDIGGDYGHYYGMDVWGQGTTVTVSS

[0381] Ab2 - Light chain variable region amino acid sequence (SEQ ID NO: 92) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDRYNYLHWYLQKPGQSPQLLIYLASIRASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCMQALGLPRTFGGGTKVEIK

[0382] Ab3 - Heavy chain variable region amino acid sequence (SEQ ID NO: 93) EVQLVQSGAEVKKPGESLKISCKGSGYSFSLYWMNWVRQVPGKGLEWMGNISPGHTATTYNRKFKG QVTISADKSISTAYLQWSSLKASDTAIYYCATGGVVAATAFDIWGQGTMVTVSS

[0383] Ab3 - Light chain variable region amino acid sequence (SEQ ID NO: 94) EIVLTQSPATLSLSPGERATLSCRASQSVDRYLAWYQQKPGQAPRLLIYGTSVRATGIPARFSGSGSG TDFTLTISSLEPEDFAVYYCQQYNQLPITFGGGTKVEIK

[0384] Ab4 - Heavy chain variable region amino acid sequence (SEQ ID NO: 95) QVQLVQSGAEVKKPGASVKVSCKVSGGTFSSHAIHWVRQAPGKGLEWMGGIIPISGTIIYAQKFQGR VTMTEDTSTDTAYMELSSLKSEDTAVYYCAKHFRWSMDVWGQGTTVTVSS

[0385] Ab4 - Light chain variable region amino acid sequence (SEQ ID NO: 96) DIQMTQSPSSVSASVGDRVTITCRASQSIRDYLAWYQQKPGKAPKLLIYAGYSLQNGVPSRFSGSGS GTDFTLTISSLQPEDFANYYCQQTYNIPLTFGGGTKVEIK

[0386] Ab1 - Heavy chain amino acid sequence (SEQ ID NO: 105) QVQLVQSGAEVKKPGASVKVSCKVSGGTFSDQTIHWVRQAPGKGLEWMGGISGYSGITIYAQKFQG RVTMTEDTSTDTAYMELSSLKSEDTAVYYCARDRYGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSR STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0387] Ab1 - Heavy chain nucleotide sequence (SEQ ID NO: 106) CAGGTTCAGTTGGTACAAAGTGGTGCAGAAGTGAAGAAACCAGGAGCCTCTGTGAAAGTTAGTT GCAAAGTATCAGGAGGAACATTTTCAGACCAGACGATACATTGGGTTAGGCAAGCACCCGGAAAA

[0388] 46

[0389] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0390] GGCCTGGAATGGATGGGGGGGATATCTGGGTATAGCGGTATTACAATCTATGCACAAAAGTTTCAG GGGAGAGTTACGATGACGGAGGATACTAGCACAGATACCGCATATATGGAGTTGAGTAGCTTGAA GAGTGAAGATACGGCTGTTTATTATTGTGCTCGCGACCGCTACGGTTATTTTGATTATTGGGGACA GGGAACCCTCGTGACAGTGTCTAGCGCGTCGACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCC TTGCTCCCGGTCCACCTCCGAGTCCACCGCCGCTCTGGGCTGTCTGGTGAAGGACTACTTCCCT GAGCCTGTGACCGTGAGCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCC GTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCTTCCTCCTCCCTGG GCACCAAGACCTACACCTGCAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGT GGAGTCCAAGTACGGCCCTCCTTGCCCTCCCTGCCCTGCCCCTGAGTTCCTGGGCGGACCCTC CGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAGGTGACCT GCGTGGTGGTGGACGTGTCCCAGGAAGATCCTGAGGTCCAGTTCAATTGGTACGTGGATGGCGT GGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTACCGGGTGGT GTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCAG CAACAAGGGCCTGCCCTCCTCCATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTCGCGA GCCTCAGGTGTACACCCTGCCTCCTAGCCAGGAAGAGATGACCAAGAATCAGGTGTCCCTGACA TGCCTGGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCA GAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAG GCTGACCGTGGACAAGTCCCGGTGGCAGGAAGGCAACGTCTTTTCCTGCTCCGTGATGCACGA GGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCTGGGC

[0391] Ab1 - Light chain amino acid sequence (SEQ ID NO: 107) DIQMTQSPSSVSASVGDRVTITCRASRDLGGWLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSG SGTDFTLTISSLQPEDFANYYCQQTDRLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC

[0392] Ab1 - Light chain nucleotide sequence (SEQ ID NO: 108) GACATACAAATGACTCAATCCCCAAGCAGCGTGTCCGCCTCAGTAGGAGACCGAGTGACAATTAC TTGCAGAGCTAGTAGAGACCTGGGTGGCTGGTTGGCTTGGTATCAGCAGAAACCTGGTAAAGCC CCAAAACTCCTCATTTACTGGGCAAGCACCCGAGAGTCCGGCGTTCCCTCTCGATTTTCTGGAAG TGGTTCAGGAACGGATTTTACCCTGACGATAAGCAGTCTGCAACCCGAAGATTTCGCAAATTACTA CTGTCAACAAACGGACCGACTCCCTCTCACTTTCGGCGGAGGTACAAAGGTAGAAATAAAACGTA CGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCC TCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAA CGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTAC AGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCG AAGTCACCCATCAGGGCGTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0393] Ab2 - Heavy chain amino acid sequence (SEQ ID NO: 109) EVQLVESGGGLVQPGGSLRLSCAASGLTVSTSDMSWVRQAPGKGLELVASIAGSGFGTYYPDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDIGGDYGHYYGMDVWGQGTTVTVSSASTKGPSVF PLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG

[0394] 47

[0395] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0396] TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0397] Ab2- Heavy chain nucleotide sequence (SEQ ID NO: 110) GAAGTGCAGCTCGTTGAATCAGGTGGGGGACTCGTTCAGCCCGGCGGCTCTCTTAGACTCTCCT GTGCGGCATCCGGATTGACCGTGAGTACCAGCGACATGAGTTGGGTCAGACAGGCACCAGGAAA AGGGCTGGAATTGGTAGCTAGCATTGCAGGATCTGGATTTGGAACATACTACCCTGACTCCGTCA AAGGGCGATTTACAATTTCTCGGGACAACGCAAAAAATAGTTTGTATCTGCAAATGAATTCACTTAG GGCAGAGGATACTGCAGTCTACTACTGTGCAAGAGATATAGGAGGTGATTATGGACACTATTACGG CATGGATGTATGGGGACAGGGCACGACAGTTACAGTCAGTTCCGCGTCGACCAAGGGCCCTTCC GTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCCGAGTCCACCGCCGCTCTGGGCTGTCTG GTGAAGGACTACTTCCCTGAGCCTGTGACCGTGAGCTGGAACTCTGGCGCCCTGACCTCCGGC GTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCG TGCCTTCCTCCTCCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAAGCCTTCCAACAC CAAGGTGGACAAGCGGGTGGAGTCCAAGTACGGCCCTCCTTGCGCTCCCTGCCCTGCCCCTGA GTTCCTGGGCGGACCCTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCC GGACCCCTGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAAGATCCTGAGGTCCAGTTCA ATTGGTACGTGGATGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTTCAA CTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGA ATACAAGTGCAAGGTCAGCAACAAGGGCCTGCCCTCCTCCATCGAGAAAACCATCTCCAAGGCC AAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCTAGCCAGGAAGAGATGACCAAG AATCAGGTGTCCCTGACATGCCTGGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGG AGAGCAACGGCCAGCCAGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCT CCTTCTTCCTGTACTCCAGGCTGACCGTGGACAAGTCCCGGTGGCAGGAAGGCAACGTCTTTTC CTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCTG GGC

[0398] Ab2 - Light chain amino acid sequence (SEQ ID NO: 111) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDRYNYLHWYLQKPGQSPQLLIYLASIRASGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCMQALGLPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC

[0399] Ab2 - Light chain nucleotide sequence (SEQ ID NO: 112) GATATTGTTATGACCCAATCACCCCTCTCACTGCCCGTTACACCCGGCGAGCCAGCTTCCATATCA TGCCGCTCATCTCAGTCACTCCTTCATTCTGACCGCTATAACTATCTTCATTGGTACCTTCAAAAAC CTGGCCAATCCCCTCAACTCCTCATTTATCTCGCTTCAATTCGCGCCAGCGGCGTTCCTGACAGA TTCTCTGGCTCCGGATCCGGCACGGATTTCACACTTAAGATTTCACGCGTAGAAGCAGAAGATGT CGGCGTCTACTACTGCATGCAAGCACTCGGACTTCCTCGCACATTCGGCGGAGGTACAAAAGTTG AAATTAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT

[0400] 48

[0401] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0402] CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGG AAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGG ACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGT CTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGG AGAGTGT

[0403] Ab3 - Heavy chain amino acid sequence (SEQ ID NO: 113) EVQLVQSGAEVKKPGESLKISCKGSGYSFSLYWMNWVRQVPGKGLEWMGNISPGHTATTYNRKFKG QVTISADKSISTAYLQWSSLKASDTAIYYCATGGVVAATAFDIWGQGTMVTVSSASTKGPSVFPLAPCS RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0404] Ab3 - Heavy chain nucleotide sequence (SEQ ID NO: 114) GAAGTGCAATTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGAGAGTCTCTCAAAATTTCCTG TAAGGGGTCTGGATATTCTTTTTCTCTGTATTGGATGAACTGGGTCCGGCAAGTTCCCGGCAAGG GATTGGAATGGATGGGAAATATCAGCCCAGGACACACCGCCACCACGTATAATAGAAAATTCAAAG GTCAAGTTACAATCTCTGCCGATAAATCAATTAGTACTGCCTACTTGCAATGGTCATCCCTGAAAGC CTCTGACACTGCAATTTATTATTGTGCAACTGGAGGTGTCGTAGCTGCCACGGCATTTGATATTTG GGGACAAGGTACAATGGTTACCGTCTCTTCTGCGTCGACCAAGGGCCCTTCCGTGTTCCCTCTG GCCCCTTGCTCCCGGTCCACCTCCGAGTCCACCGCCGCTGTGGGCTGTCTGGTGAAGGACTAC TTCCCTGAGCCTGTGACCGTGAGCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTC CCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCTTCCTCCT CCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAA GCGGGTGGAGTCCAAGTACGGCCCTCCTTGCCCTCCCTGCCCTGCCCCTGAGTTCCTGGGCGG ACCCTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAGG TGACCTGCGTGGTGGTGGACGTGTCCCAGGAAGATCCTGAGGTCCAGTTCAATTGGTACGTGGA TGGCGTGGAGGTGCACAACGCCAAGACCAAGGCTCGGGAGGAACAGTTGAACTCCACCTACCG GGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAA GGTCAGCAACAAGGGCCTGCCCTCCTCCATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCT CGCGAGCCTCAGGTGTACACCCTGCCTCCTAGCCAGGAAGAGATGACCAAGAATCAGGTGTCCC TGACATGCCTGGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGAGCAACGGCCA GCCAGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACT CCAGGCTGACCGTGGACAAGTCCCGGTGGCAGGAAGGCAACGTCTTTTCCTGCTCCGTGATGC ACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCTGGGC

[0405] Ab3 - Light chain amino acid sequence (SEQ ID NO: 115) EIVLTQSPATLSLSPGERATLSCRASQSVDRYLAWYQQKPGQAPRLLIYGTSVRATGIPARFSGSGSG TDFTLTISSLEPEDFAVYYCQQYNQLPITFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN

[0406] 49

[0407] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0408] FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC

[0409] Ab3 - Light chain nucleotide sequence (SEQ ID NO: 116) GAGATCGTACTCACTCAGAGCCCTGCAACCCTTTCACTCTCCCCTGGGGAACGAGCAACCCTTTC ATGTCGCGCATCCCAATCAGTTGATCGATATCTCGCTTGGTATCAACAAAAACCTGGCCAGGCTCC TAGACTTCTTATCTACGGCACATCAGTTCGGGCCACCGGCATTCCCGCTAGATTTTCCGGATCCG GTAGTGGTACCGACTTCACCCTGACCATCTCCTCTCTCGAGCCAGAAGATTTCGCCGTTTATTACT GCCAACAGTATAACCAGCTCCCAATTACATTCGGCGGAGGCACGAAAGTAGAAATCAAACGTACG GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTC TGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAG CCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0410] Ab4 - Heavy chain amino acid sequence (SEQ ID NO: 117) QVQLVQSGAEVKKPGASVKVSCKVSGGTFSSHAIHWVRQAPGKGLEWMGGIIPISGTIIYAQKFQGR VTMTEDTSTDTAYMELSSLKSEDTAVYYCAKHFRWSMDVWGQGTTVTVSSASTKGPSVFPLAPCSR STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0411] Ab4 - Heavy chain nucleotide sequence (SEQ ID NO: 118) CAAGTACAGCTCGTTCAAAGCGGGGCTGAGGTGAAAAAGCCAGGCGCTTCAGTAAAGGTTAGCT GCAAAGTCTCAGGAGGCACTTTCTCATCTCACGCTATACATTGGGTTCGACAAGCTCCAGGAAAA GGACTCGAGTGGATGGGAGGCATTATTCCTATAAGTGGCACTATTATATATGCCCAGAAATTTCAGG GGAGAGTCACAATGACAGAAGATACAAGCACCGATACCGCCTATATGGAACTTTCCTCTCTGAAAT CTGAAGATACAGCCGTCTATTATTGTGCTAAACACTTTAGGTGGTCAATGGATGTGTGGGGACAGG GTACTACCGTCACTGTTAGTAGTGCGTCGACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGC TCCCGGTCCACCTCCGAGTCCACCGCCGCTCTGGGCTGTCTGGTGAAGGACTACTTCCCTGAGC CTGTGACCGTGAGCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGC TGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCTTCCTCCTCCCTGGGCAC CAAGACCTACACCTGCAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGA GTCCAAGTACGGCCCTCGTTGGCCTCCCTGCCCTGCCCCTGAGTTCCTGGGCGGACCCTCCGT GTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAGGTGACCTGC GTGGTGGTGGACGTGTCCCAGGAAGATCCTGAGGTCCAGTTCAATTGGTACGTGGATGGCGTGG AGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTACCGGGTGGTGT CTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCAGCAA CAAGGGCCTGCCCTCCTCCATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCC TCAGGTGTACACCCTGCCTCCTAGCCAGGAAGAGATGACCAAGAATCAGGTGTCCCTGACATGC

[0412] 50

[0413] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0414] CTGGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCAGAG AACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAGGCT GACCGTGGACAAGTCCCGGTGGCAGGAAGGCAACGTCTTTTCCTGCTCCGTGATGCACGAGGC CCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCTGGGC

[0415] Ab4 - Light chain amino acid sequence (SEQ ID NO: 119) DIQMTQSPSSVSASVGDRVTITCRASQSIRDYLAWYQQKPGKAPKLLIYAGYSLQNGVPSRFSGSGS GTDFTLTISSLQPEDFANYYCQQTYNIPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC

[0416] Ab4 - Light chain nucleotide sequence (SEQ ID NO: 120) GATATTCAAATGACTCAATCCCCATCCAGTGTCTCCGCGTCAGTCGGCGATCGCGTCACCATCACCTGCAGAGCCT CTCAGTCAATCAG G G ACTATCTTG CCTGGTATCAG CAAAAACCCG G AAAAG CACCAAAACTCCTCATCTATG CCG GTTATTCTCTG CAAAATG G CGTTCCATCTCG GTTCTCAG G CTCCG G AAG CG G CACAG ATTTCACACTCACAATTT CATCCTTGCAACCTGAAGATTTCGCTAATTACTATTGTCAACAAACTTATAACATACCACTTACATTTGGGGGAGG AACCAAGGTAGAAATTAAGCGTACGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAA GGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTAC AG CCTCAG CAG CACCCTG ACGCTG AG CAAAG CAG ACTACG AG AAACACAAAGTCTACGCCTG CG AAGTCACCC ATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0417]

[0199] In some embodiments, the “percent identity” of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin andAltschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990.

[0418] BLAST protein searches can be performed with the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0419]

[0200] In any of the antibodies or antigen-binding fragments described herein, one or more conservative mutations can be introduced into the CDRs or framework sequences at positions where the residues are not likely to be involved in an antibody-antigen interaction. In some embodiments, such conservative mutation(s) can be introduced into the CDRs or framework sequences at position(s) where the residues are not likely to be involved in interacting with a MuSK protein. In some embodiments, likely interface (e.g., residues involved in an antigen-antibody interaction) may be deduced from crystal structures or cryo-EM derived structures or structural models or known structural information on another antigen sharing structural similarities.

[0420] 51

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[0422]

[0201] As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D

[0423]

[0202] In some embodiments, the antibodies provided herein comprise mutations that confer desirable properties to the antibodies. For example, to avoid potential complications due to Fab-arm exchange, which is known to occur with native lgG4 mAbs, the antibodies provided herein may comprise a stabilizing ‘Adair’ mutation (Angal et al., “A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (lgG4) antibody,” Mol Immunol 30, 105-108; 1993), where serine 228 (ELI numbering; residue 241 Kabat numbering) is converted to proline resulting in an lgG1 -like (CPPCP (SEQ ID NO: 54)) hinge sequence. Accordingly, any of the antibodies may include a stabilizing ‘Adair’ mutation or the amino acid sequence CPPCP (SEQ ID NO: 121).

[0424]

[0203] MuSK agonists of the present disclosure may optionally comprise antibody constant regions or parts thereof. For example, a VL domain may be attached at its C-terminal end to a light chain constant domain like CK or CK. Similarly, a VH domain or portion thereof may be attached to all or part of a heavy chain like IgA, IgD, IgE, IgG, and IgM, and any isotype subclass. Antibodies may include suitable constant regions (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md.

[0425] (1991)). Therefore, antibodies within the scope of this disclosure may include VH and VL domains, or an antigen binding portion thereof, combined with any suitable constant regions.

[0426]

[0204] Additionally or alternatively, such antibodies may or may not include the framework regions of the heavy or light chain variable regions of SEQ ID NOs: 89-96 and 210-213. In some embodiments, antibodies that specifically bind to a MuSK protein, or fragment thereof, comprising a frizzled-like domain and a Truncated tail C-terminal to the Fz-like domain, and activate MuSK are murine antibodies and include murine framework region sequences.

[0427]

[0205] In some embodiments, such antibodies bind to the MuSK protein e.g. human MuSK) with relatively high affinity, e.g., with a KD less than 10'9M, 10'10M, 10'11M or lower. For example, such antibodies may bind a MuSK protein with an affinity between 5 pM and 1 nM, e.g., between 10 pM and 1 nM, e.g., between 10 pM and 100 pM. The disclosure also includes antibodies or antigen binding fragments that compete with any of the antibodies described herein for binding to a MuSK protein and that have a Ko value of 1 nM or lower {e.g., 1 nM or lower, 500 pM or lower, 100 pM or lower). The affinity and binding kinetics of the antibodies that specifically bind to a MuSK protein can be tested using any suitable method including but not limited to biosensor-based technology {e.g., OCTET® or BIACORE) and solution equilibrium titration-based technology {e.g., MSD-SET).

[0428] 52

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[0430]

[0206] In some embodiments. MuSK agonists according to the invention include antibodies or fragments thereof that specifically bind MuSK to induce clustering of ACh receptors (AChR). In some embodiments, the MuSK agonist antibodies or fragments thereof bind to a MuSK protein, or fragment thereof, comprising a frizzled-like domain and a Truncated tail C-terminal to the Fz-like domain, to induce MuSK dimerization. In some embodiments, the MuSK agonist antibodies or fragments thereof induce clustering of ACh receptors (AChR).

[0431] Antibodies competing with high-affinity MuSK agonists

[0432]

[0207] Aspects of the disclosure relate to antibodies that compete (e.g., cross-compete) with any of the antibodies provided herein. Antibody “binning” experiments are useful for classifying multiple antibodies that are made against the same antigen into various “bins” based on the relative crossblocking activities. Each “bin” therefore represents a discrete binding region(s) of the antigen.

[0433] Antibodies in the same bin by definition cross-block each other. Binning can be examined by standard in vitro binding assays, such as Biacore or Octet®, using standard test conditions, e.g., according to the manufacturer’s instructions, (e.g., binding assayed at room temperature, ~20-25°C)

[0208] Aspects of the disclosure relate to antibodies that compete or cross-compete with any of the specific antibodies, or antigen binding portions thereof, for binding to human MuSk, as provided herein. In some embodiments, the antibody or antigen binding portion cross-competes with an antibody having the six CDRs of any one of Ab1, Ab2, Ab3, Ab4, Ab5 or Ab6 (particularly Ab5 or Ab6). More preferably, the antibody or antigen binding portion cross-competes with an antibody having the heavy chain variable domain sequence and light chain variable domain sequence of any one of Ab1 , Ab2, Ab3, Ab4, Ab5 or Ab6 (particularly Ab5 or Ab6). For example, the antibody or antigen binding portion may cross-compete with any one of Ab1 , Ab2, Ab3, Ab4, Ab5 or Ab6 (particularly Ab5 or Ab6) as described herein. In some embodiments, an antibody, or antigen binding portion thereof, binds at or near the same epitope as any of the antibodies provided herein. In some embodiments, an antibody, or antigen binding portion thereof, binds near an epitope if it binds within 15 or fewer amino acid residues of the epitope. In some embodiments, any of the antibody, or antigen binding portion thereof, as provided herein, binds within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues of an epitope that is bound by any of the antibodies provided herein. In preferred embodiments, the antibody or antigen-binding fragment that competes or cross-competes with any of the specific antibodies or antigen binding portions thereof provided herein binds and activates human MuSK, and is not capable of binding to a MuSK fragment consisting of SEQ ID NO: 8. The antibody or antigen-binding fragment may additionally be capable of binding to a MuSK fragment consisting of SEQ ID NO: 7. Indeed, the antibody or antigen-binding fragment that competes or cross-competes with any of the specific antibodies or antigen binding portions provided herein may have one or more of the additional functional properties described in the various aspects and embodiments of the present disclosure.

[0434]

[0209] In another embodiment, provided herein is an antibody, or antigen binding portion thereof, competes or cross-competes for binding to any of the antigens provided herein (e.g., a MuSK protein, or fragment thereof, comprising a frizzled-like domain and a Truncated tail C-terminal to the Fz-like domain) with an equilibrium dissociation constant, KD, between the antibody and the protein of less

[0435] 53

[0436] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0437] than 10'8M. In other embodiments, an antibody competes or cross-competes for binding to any of the antigens provided herein with a Ko in a range from 10'12M to 10'9M. In some embodiments, provided herein is an anti-MuSK antibody, or antigen binding portion thereof that competes for binding with an antibody, or antigen binding portion thereof, described herein. In some embodiments, provided herein is an anti-MuSK antibody, or antigen binding portion thereof, that binds to the same epitope as an antibody, or antigen binding portion thereof, described herein.

[0438]

[0210] Any of the antibodies provided herein can be characterized using any suitable methods. For example, one method is to identify the epitope to which the antigen binds, or “epitope mapping.” There are many suitable methods for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. In an additional example, epitope mapping can be used to determine the sequence to which an antibody binds. The epitope can be a linear epitope, i.e., contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that may not necessarily be contained in a single stretch (primary structure linear sequence). In some embodiments, the epitope is a MuSK epitope. In some embodiments, the epitope is contained in a frizzled-like domain of a MuSK protein. In some embodiments, the epitope is contained in a MuSK protein, or fragment thereof, comprising a frizzled-like domain and a Truncated tail C-terminal to the Fz-like domain. Peptides of varying lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used for binding assays with an antibody. In another example, the epitope to which the antibody binds can be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody. According to the gene fragment expression assays, the open reading frame encoding the target antigen is fragmented either randomly or by specific genetic constructions and the reactivity of the expressed fragments of the antigen with the antibody to be tested is determined. The gene fragments may, for example, be produced by PCR and then transcribed and translated into protein in vitro, in the presence of radioactive amino acids. The binding of the antibody to the radioactively labeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in simple binding assays. In an additional example, mutagenesis of an antigen binding domain, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and / or necessary for epitope binding.

[0439]

[0211] Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine whether an antibody binds to the same epitope as the other antibodies. Competition assays are well known to those of skill in the art.

[0440]

[0212] In some embodiments, the invention includes antibodies (e.g., immunoglobulins, antigenbinding fragments, etc.) that cross-block (cross-compete) with any one of the antibodies described

[0441] 54

[0442] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0443] herein. Thus, in some embodiments, a pharmaceutical composition may be made by the process comprising a step of: selecting an antibody or antigen-binding fragment thereof, which crosscompetes with an antibody disclosed herein; and, formulating the antibody into a pharmaceutical composition.

[0444]

[0213] In some embodiments, a pharmaceutical composition may be made by the process comprising a step of: selecting an antibody or antigen-binding fragment thereof, which cross-competes with the antibody selected from the group consisting of Ab1 , Ab2, Ab3, Ab4, Ab5, Ab6; and, formulating into a pharmaceutical composition.

[0445]

[0214] In some embodiments, the antibody selected by the process is a high-affinity binder characterized in that the antibody or the antigen-binding fragment is capable of binding to human MuSK with a KD of < 5 nM, as measured by an in vitro binding assay. Preferably, the antibody selected by the process is a high-affinity binder characterized in that the antibody or the antigenbinding fragment is capable of binding to a MuSK protein, or fragment thereof, comprising a frizzled-like domain and a 7 amino acid long Truncated tail C-terminal to the Fz-like domain, with a KD of < 5 nM, as measured by an in vitro binding assay. In some embodiments, the antibody selected by the process is a high-affinity binder characterized in that the antibody or the antigen-binding fragment is capable of binding to a MuSK protein, or fragment thereof, comprising a frizzled-like domain and a tail C-terminal to the Fz-like domain, with a KD of < 5 nM, as measured by an in vitro binding assay. Such cross-competing antibodies may be used in the treatment of MuSK-related indications a subject in accordance with the present disclosure.

[0446] Modifications and Variations of Antibodies

[0447]

[0215] Non-limiting variations, modifications, and features of any of the antibodies or antigen-binding fragments thereof encompassed by the present disclosure are briefly discussed below. Embodiments of related analytical methods are also provided.

[0448]

[0216] Naturally-occurring antibody structural units typically comprise a tetramer. Each such tetramer typically is composed of two identical pairs of polypeptide chains, each pair having one full-length “light” (in certain embodiments, about 25 kDa) and one full-length “heavy” chain (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain typically includes a variable region of about 100 to 110 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that can be responsible for effector function. Human antibody light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the isotype of the antibody. An antibody can be of any type (e.g., IgM, IgD, IgG, IgA, IgY, and IgE) and class (e.g., IgGi, lgG2, IgGa, lgG4, IgM-i, IgMa, IgAi, and IgAa). Within full-length light and heavy chains, typically, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids (see, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety)). The variable regions of each light / heavy chain pair typically form the antigen binding site.

[0449] 55

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[0451]

[0217] The variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which can enable binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions typically comprise the domains FR1 , CDR1 , FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or IMGT ( created in 1989 by Marie-Pauie Lefranc (University of Montpellier and CNRS) ). The CDRs of a light chain can also be referred to as CDR-L1 , CDR-L2, and CDR-L3, and the CDRs of a heavy chain can also be referred to as CDR-H1 , CDR-H2, and CDR-H3. In some embodiments, an antibody can comprise a small number of amino acid deletions from the carboxy end of the heavy chain(s). In some embodiments, an antibody comprises a heavy chain having 1 -5 amino acid deletions in the carboxy end of the heavy chain. In certain embodiments, definitive delineation of a CDR and identification of residues comprising the binding site of an antibody is accomplished by solving the structure of the antibody and / or solving the structure of the antibodyligand complex. In certain embodiments, that can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In some embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the IMGT definition, AbM definition, the definition described by Lu et al (see above), and the contact definition.

[0452]

[0218] An "affinity matured" antibody is an antibody with one or more alterations in one or more CDRs thereof, which result in an improvement in the affinity of the antibody for antigen compared to a parent antibody, which does not possess those alteration(s). Exemplary affinity matured antibodies will have nanomolar or even picomolar affinities (e.g., KD of ~109M-1012M range) for the target antigen.

[0453] Affinity matured antibodies are produced by procedures known in the art. Marks et al. (1992) Bio / Technology 10: 779-783 describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described by Barbas, et al. (1994) Proc Nat. Acad. Sci. USA 91 : 3809-3813; Schier et al. (1995) Gene 169: 147- 155; Yelton et al., (1995) J Immunol. 155: 1994-2004; Jackson et al. (1995) J. Immunol. 154(7): 3310-9; and Hawkins et al. (1992) J. Mol. Biol. 226: 889-896; and selective mutation at selective mutagenesis positions, contact or hypermutation positions with an activity enhancing amino acid residue is described in U.S. Patent No.

[0454] 6,914,128. Typically, a parent antibody and its affinity-matured progeny (e.g., derivatives) retain the same binding region within an antigen, although certain interactions at the molecular level may be altered due to amino acid residue alternation(s) introduced by affinity maturation.

[0455]

[0219] The term “CDR-grafted antibody” refers to antibodies in which non-human CDR sequences are introduced into human VH and VL framework sequences to replace the corresponding nonhuman framework sequences.

[0456]

[0220] The term “chimeric antibody” refers to antibodies, which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.

[0457] 56

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[0459]

[0221] As used herein, the term "framework" (FR) or "framework sequence" refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1 , -L2, and -L3 of light chain and CDR-H1 , -H2, and -H3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1 , FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FR1 , FR2, FR3 or FR4, a framework region, as referred by others, represents the combined FR's within the variable region of a single, naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region.

[0460]

[0222] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 1 (H-FR1 ) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes: QVQLVQSGAEVKKPGASVKVSCKVS (SEQ ID NO: 122). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 1 (H-FR1 ) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 123). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 1 (H-FR1 ) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes:

[0461] EVQLVQSGAEVKKPGESLKISCKGS (SEQ ID NO: 124).

[0462]

[0223] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 2 (H-FR2) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes: IHWVRQAPGKGLEWMGG (SEQ ID NO: 125). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 2 (H-FR2) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes:

[0463] MSWVRQAPGKGLELVAS (SEQ ID NO: 126). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 2 (H-FR2) having the following amino acid sequence with optionally 1, 2 or 3 amino acid changes: MNWVRQVPGKGLEWMGN (SEQ ID NO: 127). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 2 (H-FR2) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes: IGWVRQMPGKGLEWMGI ( SEQ ID NO: 214). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 2 (H-FR2) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes:

[0464] MHWVRQAPGKGLEWMGG (SEQ ID NO: 216).

[0465]

[0224] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 3 (H-FR3) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes: IYAQKFQGRVTMTEDTSTDTAYMELSSLKSEDTAVYYC (SEQ ID NO: 128). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 3 (H-FR3) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes: YYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC (SEQ ID NO: 129). In some

[0466] 57

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[0468] embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 3 (H-FR3) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes: TYNRKFKGQVTISADKSISTAYLQWSSLKASDTAIYYC (SEQ ID NO: 130). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 3 (H-FR3) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes: RYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYC (SEQ ID NO: 215). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 3 (H-FR3) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes: IYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYC (SEQ ID NO: 217).

[0469]

[0225] In some embodiments, the antibody or antigen-binding fragment thereof comprises a HEAVY chain framework region 4 (H-FR4) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes: WGQGTLVTVSS (SEQ ID NO: 131). In some embodiments, the antibody or antigen-binding fragment thereof comprises a HEAVY chain framework region 4 (H-FR4) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes: WGQGTMVTVSS (SEQ ID NO: 133). In some embodiments, the antibody or antigen-binding fragment thereof comprises a HEAVY chain framework region 4 (H-FR4) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes: WGQGTTVTVSS (SEQ ID NO: 132).

[0470]

[0226] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain framework region 1 (L-FR1 ) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes: DIQMTQSPSSVSASVGDRVTITCRAS (SEQ ID NO: 134). In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain framework region 1 (L-FR1 ) having the following amino acid sequence with optionally 1 , or 3 amino acid changes:

[0471] DIVMTQSPLSLPVTPGEPASISCRSS (SEQ ID NO: 135). In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain framework region 1 (L-FR1 ) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes:

[0472] EIVLTQSPATLSLSPGERATLSCRAS (SEQ ID NO: 136).

[0473]

[0227] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain framework region 2 (L-FR2) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes: LAWYQQKPGKAPKLLIY (SEQ ID NO: 137). In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain framework region 2 (L-FR2) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes:

[0474] LHWYLQKPGQSPQLLIY(SEQ ID NO: 138). In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain framework region 2 (L-FR2) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes: LAWYQQKPGQAPRLLIY (SEQ ID NO: 139).

[0475]

[0228] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain framework region 3 (L-FR3) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes: GVPSRFSGSGSGTDFTLTISSLQPEDFANYYC (SEQ ID NO: 140). In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain framework region 3 (L-FR3) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes: GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO: 141). In some embodiments,

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[0478] the antibody or antigen-binding fragment thereof comprises a light chain framework region 3 (L-FR3) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes:

[0479] GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC (SEQ ID NO: 142). In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain framework region 3 (L-FR3) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes:

[0480] GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 218).

[0481]

[0229] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain framework region 4 (L-FR4) having the following amino acid sequence with optionally 1 , 2 or 3 amino acid changes: FGGGTKVEIK (SEQ ID NO: 144).

[0482]

[0230] In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain immunoglobulin constant domain of a human IgM constant domain, a human IgG constant domain, a human lgG1 constant domain, a human lgG2 constant domain, a human lgG2A constant domain, a human lgG2B constant domain, a human lgG2 constant domain, a human lgG3 constant domain, a human lgG3 constant domain, a human lgG4 constant domain, a human IgA constant domain, a human lgA1 constant domain, a human Ig A2 constant domain, a human IgD constant domain, or a human IgE constant domain. In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain immunoglobulin constant domain of a human lgG1 constant domain or a human lgG4 constant domain. In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain immunoglobulin constant domain of a human lgG4 constant domain.

[0483]

[0231] In some embodiments, the antibody, or antigen binding portion thereof, comprises a so-called Adair hinge mutation, in which a human lgG4 constant domain contains a backbone substitution of Ser to Pro (S228P) that produces an lgG1 -like hinge and permits formation of inter-chain disulfide bonds.

[0484]

[0232] In some embodiments, the antibody or antigen-binding portion thereof includes or is engineered to include a mutation or modification that causes an extended half-life of the antibody. In some embodiments, such mutations or modifications are within the Fc domain of the antibodies (e.g., Fc-modified antibodies), e.g., to promote circulating half-life or other PK properties. In some embodiments, the mutation is a YTE mutation e.g., M252Y / S254T / T256E) (see, e.g., Saunders, KO (2019) “Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life,” Frontiers in Immunology 10:1296 and U.S. Patent No. 7,083,784, each of which is herein incorporated by reference). In some embodiments, the mutation is an LS mutation {e.g., M428UN434S) (Zalevsky et al., Nat Biotechnol. 2010, 28:157-159). In some embodiments, the mutation is a QL mutation {e.g., T250Q / M428L) (Hinton et al., J Biol Chem. 2004, 279:6213-6216). In some embodiments, the mutation comprises V308P (Datta-Mannan et al., Drug Metab Dispos. 2012, 40:1545-1555). Thus, in some embodiments, a variant of any one of antibodies encompassed herein {e.g., Ab1 , Ab2, Ab3, Ab4, Ab5 and Ab6) may comprise one or more mutations in the Fc domain. In some embodiments, such mutation(s) include a YTE mutation, LS mutation, QL mutation, and / or V308P.

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[0487]

[0233] In some embodiments, mutations can be introduced to Fc regions, aimed at eliminating Fc binding to FcRn. Such mutations include, for example, so-called an AAA mutation {e.g., T307A / E380A / N434A) (Petkova et al., Int Immunol. 2006, 18:1759-1769)

[0488]

[0234] In some embodiments, the antibody or antigen binding portion thereof, further comprises a light chain immunoglobulin constant domain comprising a human Ig lambda constant domain or a human Ig kappa constant domain.

[0489]

[0235] In some embodiments, the antibody is an IgG having four polypeptide chains which are two heavy chains and two light chains.

[0490]

[0236] In some embodiments, the antibody is a humanized antibody, a diabody, or a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody comprises a framework having a human germline amino acid sequence.

[0491]

[0237] In some embodiments, the antigen binding portion is a Fab fragment, a F(ab')2 fragment, a scFab fragment, or an scFv fragment.

[0492]

[0238] As used herein, the term "germline antibody gene" or "gene fragment" refers to an immunoglobulin sequence encoded by non-lymphoid cells that have not undergone the maturation process that leads to genetic rearrangement and mutation for expression of a particular immunoglobulin (see, e.g., Shapiro et al. (2002) Crit. Rev. Immunol. 22(3): 183-200; Marchalonis et al (2001 ) Adv. Exp. Med. Biol. 484: 13-30). One of the advantages provided by various embodiments of the present disclosure stems from the recognition that germline antibody genes are more likely than mature antibody genes to conserve essential amino acid sequence structures characteristic of individuals in the species, hence less likely to be recognized as from a foreign source when used therapeutically in that species.

[0493]

[0239] As used herein, the term “neutralizing” refers to counteracting the biological activity of an antigen {e.g., target protein) when a binding protein specifically binds to the antigen. In an embodiment, the neutralizing binding protein binds to the antigen / target, e.g., cytokine, kinase, growth factor, cell surface protein, soluble protein, phosphatase, or receptor ligand, and reduces its biologically activity by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%. 96%, 97%. 98%, 99% or more.

[0494]

[0240] The term “binding protein” as used herein includes any polypeptide that specifically binds to an antigen {e.g., MuSK), including, but not limited to, an antibody, or antigen binding portions thereof, a DVD-lgTM, a TVD-lg, a RAb-lg, a bispecific antibody and a dual specific antibody.

[0495]

[0241] The term "monoclonal antibody" or “mAb” when used in a context of a composition comprising the same may refer to an antibody preparation obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. The

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[0498] modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method.

[0499]

[0242] The term "recombinant human antibody" as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further in Section II C, below), antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom, H.R. (1997) TIB Tech. 15: 62-70; Azzazy, H and Highsmith, W E (2002) Clin Biochem. 35: 425-445; Gavilondo, J.V. and Larrick, J.W. (2002) BioTechniques 29: 128-145;

[0500] Hoogenboom, H. and Chames, P. (2000) Immunol. Today 21 : 371-378, incorporated herein by reference), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, Taylor, L. D. et al. (1992) Nucl. Acids Res. 20: 6287-6295; Kellermann, S-A. and Green, L.L. (2002) Cur. Opin. in Biotechnol. 13: 593-597; Little, M. et al. (2000) Immunol. Today 21 : 364-370) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0501]

[0243] As used herein, “Dual Variable Domain Immunoglobulin” or “DVD-lgTM” and the like include binding proteins comprising a paired heavy chain DVD polypeptide and a light chain DVD polypeptide with each paired heavy and light chain providing two antigen binding sites. Each binding site includes a total of 6 CDRs involved in antigen binding per antigen binding site. A DVD-lgTM is typically has two arms bound to each other at least in part by dimerization of the CH3 domains, with each arm of the DVD being bispecific, providing an immunoglobulin with four binding sites. DVD-lgTM are provided in US Patent Publication Nos. 2010 / 0260668 and 2009 / 0304693, each of which are incorporated herein by reference including sequence listings.

[0502]

[0244] As used herein, “Triple Variable Domain Immunoglobulin” or “TVD-lg” and the like are binding proteins comprising a paired heavy chain TVD binding protein polypeptide and a light chain TVD binding protein polypeptide with each paired heavy and light chain providing three antigen binding sites. Each binding site includes a total of 6 CDRs involved in antigen binding per antigen binding site. A TVD binding protein may have two arms bound to each other at least in part by dimerization of the CH3 domains, with each arm of the TVD binding protein being trispecific, providing a binding protein with six binding sites.

[0503]

[0245] As used herein, “Receptor-Antibody Immunoglobulin” or “RAb-lg” and the like are binding proteins comprising a heavy chain RAb polypeptide, and a light chain RAb polypeptide, which together form three antigen binding sites in total. One antigen binding site is formed by the pairing of the heavy and light antibody variable domains present in each of the heavy chain RAb polypeptide and the light chain RAb polypeptide to form a single binding site with a total of 6 CDRs providing a

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[0506] first antigen binding site. Each the heavy chain RAb polypeptide and the light chain RAb polypeptide include a receptor sequence that independently binds a ligand providing the second and third “antigen” binding sites. A RAb-lg is typically has two arms bound to each other at least in part by dimerization of the CH3 domains, with each arm of the RAb-lg being trispecific, providing an immunoglobulin with six binding sites. RAb-lgs are described in US Patent Application Publication No. 2002 / 0127231, the entire contents of which including sequence listings are incorporated herein by reference).

[0507]

[0246] The term "bispecific antibody," as used herein, and as differentiated from a “bispecific half-lg binding protein” or “bispecific (half-lg) binding protein”, refers to full-length antibodies that are generated by quadroma technology (see Milstein, C. and Cuello, A.C. (1983) Nature 305(5934): p. 537-540), by chemical conjugation of two different monoclonal antibodies (see Staerz, U.D. et al. (1985) Nature 314(6012): 628-631), or by knob-into-hole or similar approaches, which introduce mutations in the Fc region that do not inhibit CH3-CH3 dimerization (see Holliger, P. et al. (1993) Proc. Natl. Acad. Sci USA 90(14): 6444-6448), resulting in multiple different immunoglobulin species of which only one is the functional bispecific antibody. By molecular function, a bispecific antibody binds one antigen (or epitope) on one of its two binding arms (one pair of HC / LC), and binds a different antigen (or epitope) on its second arm (a different pair of HC / LC). By this definition, a bispecific antibody has two distinct antigen binding arms (in both specificity and CDR sequences), and is monovalent for each antigen it binds to.

[0508]

[0247] The term "dual-specific antibody," as used herein, and as differentiated from a bispecific half-lg binding protein or bispecific binding protein, refers to full-length antibodies that can bind two different antigens (or epitopes) in each of its two binding arms (a pair of HC / LC) (see PCT Publication No. WO 02 / 02773). Accordingly, a dual-specific binding protein has two identical antigen binding arms, with identical specificity and identical CDR sequences, and is bivalent for each antigen to which it binds.

[0509]

[0248] The term "Kon,” as used herein, is intended to refer to the on rate constant for association of a binding protein (e.g., an antibody) to the antigen to form the, e.g., antibody / antigen complex as is known in the art. The “Kon” also is known by the terms “association rate constant,” or “ka,” as used interchangeably herein. This value indicating the binding rate of an antibody to its target antigen or the rate of complex formation between an antibody and antigen also is shown by the equation:

[0510] Antibody (“Ab”) + Antigen (“Ag”HAb-Ag.

[0511]

[0249] The term "Koff,” as used herein, is intended to refer to the off rate constant for dissociation of a binding protein (e.g., an antibody) from the, e.g., antibody / antigen complex as is known in the art. The “Koff” also is known by the terms “dissociation rate constant” or “kd” as used interchangeably herein. This value indicates the dissociation rate of an antibody from its target antigen or separation of Ab-Ag complex over time into free antibody and antigen as shown by the equation: Ab + Ag«-Ab-Ag.

[0512]

[0250] The terms “equilibrium dissociation constant” or “Ko,” as used interchangeably herein, refer to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (koff) by the association rate constant (kon). The association rate constant, the dissociation rate constant, and the equilibrium dissociation constant are used to represent the binding affinity of a

[0513] 62

[0514] MEl\59597320.vlSR64-WO-PCT / 127036-05220

[0515] binding protein, e.g., antibody, to an antigen. Methods for determining association and dissociation rate constants are well known in the art. Using fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental approaches and instruments, such as a BIAcore® (biomolecular interaction analysis) assay, can be used {e.g., instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). Additionally, an OCTET® Biolayer Interferometry (BLI) assay can be used. Additionally, a KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Idaho), can also be used.

[0516]

[0251] The terms “crystal” and “crystallized” as used herein, refer to a binding protein {e.g., an antibody), or antigen binding portion thereof, that exists in the form of a crystal. Crystals are one form of the solid state of matter, which is distinct from other forms such as the amorphous solid state or the liquid crystalline state. Crystals are composed of regular, repeating, three-dimensional arrays of atoms, ions, molecules {e.g., proteins such as antibodies), or molecular assemblies {e.g., antigen / antibody complexes). These three-dimensional arrays are arranged according to specific mathematical relationships that are well-understood in the field. The fundamental unit, or building block, that is repeated in a crystal is called the asymmetric unit. Repetition of the asymmetric unit in an arrangement that conforms to a given, well-defined crystallographic symmetry provides the "unit cell" of the crystal. Repetition of the unit cell by regular translations in all three dimensions provides the crystal. See Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ea., pp. 201-16, Oxford University Press, New York, New York, (1999). The term “linker” is used to denote polypeptides comprising two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Such linker polypeptides are well known in the art (see, e.g., Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, R.J. et al. (1994) Structure 2:1121-1123). Exemplary linkers include, but are not limited to, ASTKGPSVFPLAP (SEQ ID NO: 145), ASTKGP (SEQ ID NO: 146); TVAAPSVFIFPP (SEQ ID NO: 147); TVAAP (SEQ ID NO: 148); AKTTPKLEEGEFSEAR (SEQ ID NO: 149);

[0517] AKTTPKLEEGEFSEARV (SEQ ID NO: 150); AKTTPKLGG (SEQ ID NO: 151); SAKTTPKLGG (SEQ ID NO: 152); SAKTTP (SEQ ID NO: 153); RADAAP (SEQ ID NO: 154); RADAAPTVS (SEQ ID NO: 155); RADAAAAGGPGS (SEQ ID NO: 156); RADAAAA(G4S)4 (SEQ ID NO: 157);

[0518] SAKTTPKLEEGEFSEARV (SEQ ID NO: 158); ADAAP (SEQ ID NO: 159); ADAAPTVSIFPP (SEQ ID NO: 160); QPKAAP (SEQ ID NO: 161); QPKAAPSVTLFPP (SEQ ID NO: 162); AKTTPP (SEQ ID NO: 163); AKTTPPSVTPLAP (SEQ ID NO: 164); AKTTAP (SEQ ID NO: 165); AKTTAPSVYPLAP (SEQ ID NO: 166); GGGGSGGGGSGGGGS (SEQ ID NO: 167); GENKVEYAPALMALS (SEQ ID NO: 168); GPAKELTPLKEAKVS (SEQ ID NO: 169); GHEAAAVMQVQYPAS (SEQ ID NO: 170);

[0519] TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID NO: 171); and ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 172).

[0520]

[0252] “Label” and “detectable label” or “detectable moiety” mean a moiety attached to a specific binding partner, such as an antibody or an analyte, e.g., to render the reaction between members of a specific binding pair, such as an antibody and an analyte, detectable, and the specific binding partner, e.g., antibody or analyte, so labeled is referred to as “delectably labeled.” Thus, the term “labeled binding protein” as used herein, refers to a protein with a label incorporated that provides for the

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[0523] identification of the binding protein. In an embodiment, the label is a detectable marker that can produce a signal that is detectable by visual or instrumental means, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g.,18F.11C,13N,150,68Ga,18F,89Zr,3H,14C,35S,90Y, "Tc,111In,125l,1311,177Lu,166Ho, and153Sm); chromogens; fluorescent labels (e.g., FITC, rhodamine, and lanthanide phosphors); enzymatic labels (e.g., horseradish peroxidase, luciferase, and alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, and epitope tags); and magnetic agents, such as gadolinium chelates. Representative examples of labels commonly employed for immunoassays include moieties that produce light, e.g., acridinium compounds, and moieties that produce fluorescence, e.g., fluorescein. Other labels are described herein. In this regard, the moiety itself may not be detectably labeled but may become detectable upon reaction with yet another moiety. Use of “detectably labeled” is intended to encompass the latter type of detectable labeling.

[0524]

[0253] In some embodiments, the binding affinity of an antibody, or antigen binding portion thereof, to an antigen (e.g., protein complex), such as MuSK, is determined using an Octet assay. In some embodiments, an Octet assay is an assay that determines one or more kinetic parameters indicative of binding between an antibody and antigen. In some embodiments, an Octet® system (ForteBio, Menlo Park, CA) is used to determine the binding affinity of an antibody, or antigen binding portion thereof, to MuSK protein. In some embodiments, an Octet® system (ForteBio, Menlo Park, CA) is used to determine the binding affinity of an antibody, or antigen binding portion thereof, to a MuSK protein, or fragment thereof, comprising a frizzled-like domain and a Truncated tail C-terminal to the Fz-like domain. For example, binding affinities of antibodies may be determined using the ForteBio Octet QKe dip and read label free assay system utilizing bio-layer interferometry. In some embodiments, antigens are immobilized to biosensors (e.g., Anti-human Fc coated biosensors or streptavidin-coated biosensors) and the antibodies and complexes (e.g., biotinylated or nonbiotinylated MuSK proteins, or MuSK Fz-like domain constructs) are presented in solution at high concentration (50 pg / mL) to measure binding interactions. In some embodiments, the binding affinity of an antibody, or antigen binding portion thereof, to a MuSK protein is determined using the protocol outlined herein.

[0525] Characterization of the Novel, High-Affinity, MuSK-agonist antibodies

[0526] Binding profiles

[0527]

[0254] Antibodies disclosed herein have enhanced binding activities. Included are a class of antibodies capable of binding to MuSK with high affinity, and activating MuSK to induce AChR clustering. Included are a class of antibodies capable of binding to a MuSK protein, or fragment thereof, comprising the Frizzled-like domain and the Truncated tail C-terminal to the Fz-like domain with high affinity and inducing AChR clustering. Antibodies disclosed herein are not capable of binding to a MuSK protein, or fragment thereof, when the tail region or truncated tail C-terminal to the Fz-like domain region is not

[0528] 64

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[0530] present. As presented below, many antibodies encompassed by the invention have KD values in a sub-nanomolar range.

[0531]

[0255] Thus, the antibodies are capable of specifically binding to MuSK or fragments thereof. Typically, recombinantly produced, purified protein complexes are used as antigens (e.g., antigen complexes) to evaluate or confirm the ability of an antibody to bind the antigen complexes in suitable in vitro binding assays. Such assays are well known in the art and include, but are not limited to Bio-Layer Interferometry (BLI)-based assays (such as Octet®), Surface Plasmon Resonance (SPR)-based assays (such as BIACORE®) and solution equilibrium titration-based assays (such as MSD-SET).

[0532]

[0256] BLI-based binding assays are widely used in the art for measuring affinities and kinetics of antibodies to antigens. It is a label-free technology in which biomolecular interactions are analyzed on the basis of optical interference. One of the proteins, for example, an antibody being tested, can be immobilized on the biosensor tip. When the other protein in solution, for example, an antigen, becomes bound to the immobilized antibody, it causes a shift in the interference pattern, which can be measured in real-time. This allows the monitoring of binding specificity, rates of association and dissociation, as well as concentration dependency. Thus, BLI is a kinetic measure that reveals the dynamics of the system. Due to its ease of use and fast results, BLI-based assays such as the Octet® system (available from ForteBio / Molecular Devices, Fremont California), are particularly convenient when used as an initial screening method to identify and separate a pool of “binders” from a pool of “non-binders” or “weak binders” in the screening process.

[0533]

[0257] BLI-based binding assays revealed that the novel antibodies are characterized high affinity binding to MuSK when binding affinity is measured by Octet®. Table 8 below provides non-limiting examples of high-affinity, MuSK-agonist antibodies encompassed by the present invention. The table provides representative results from in vitro binding assays, as measured by Octet®.

[0534]

[0258] Column (A) of the table lists monoclonal antibodies with discrete amino acid sequences.

[0535] Columns (B) and (C) provide affinities of each of the listed antibodies to the indicated MuSk construct, measured in KD (nM) Column (D) indicates the domain on MuSK that the antibody binds to.

[0536] Table 8. Non-limiting examples of high-affinity MuSK antibodies and KD values measured by BLI

[0537]

[0538] 65

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[0540]

[0541] Table 9. Non-limiting examples of high-affinity MuSK antibodies and summary of kinetic data from SPR experiments

[0542]

[0543] ** Limit of detection for experiment

[0544]

[0259] The invention provides a class of high-affinity, MuSK antibodies, each of which is capable of binding to MuSK. In some embodiments, the antibody binds to a MuSK protein, or fragment thereof, only when it comprises the Fz-like domain and the Truncated tail C-terminal to the Fz-like domain.

[0545] According to the invention, such antibody specifically binds a MuSK protein with an affinity

[0546] (determined by KD) of < 5 nM as measured by a suitable in vitro binding assay, such as Biolayer Interferometry and surface plasmon resonance. In some embodiments, the antibody or the fragment binds a human MuSK complex with an affinity of < 5 nM, 4 nM, 3 nM, 22 nM, 2 1 nM, 25 nM or 2

[0547] 0.5 nM. In some embodiments, the antibody or the fragment binds the MuSK protein, or fragment thereof, with an affinity of s 5 nM, s 4 nM, s 3 nM, 2 nM, 2 1 nM, s 5 nM or i 0.5 nM. In some embodiments, the antibody or the fragment binds MuSK Ig-like domains with an affinity of < 5 nM, < 4 nM, < 3 nM, < 2 nM, < 1 nM, < 5 nM or < 0.5 nM.

[0548]

[0260] In preferred embodiments, such antibody is human- and murine-cross-reactive. Thus, in some embodiments, the antibody or the fragment binds a murine MuSK with an affinity of 25 nM, 24 nM,

[0549] 3 nM, < 2 nM, < 1 nM, < 5 nM or < 0.5 nM. In some embodiments, the antibody or the fragment binds

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[0552] a murine MuSK protein, or fragment thereof, comprising a frizzled- like domain and a Truncated tail C-terminal to the Fz-like domain, with an affinity of < 5 nM, < 4 nM, < 3 nM, < 2 nM, < 1 nM or < 0.5 nM. In some embodiments, the antibody or the fragment binds murine MuSK Ig-like domains with an affinity of < 5 nM, < 4 nM, < 3 nM, < 2 nM, < 1 nM or < 0.5 nM.

[0553]

[0261] In some embodiments, the antibody or antigen-binding fragment thereof, is cross-reactive to human, cynomolgus, mouse and rat MuSK.

[0554] Binding Regions

[0555]

[0262] In the context of the present disclosure, “binding region(s)” of an antigen provides a structural basis for the antibody-antigen interaction. As used herein, a “binding region” refers to the areas of interface between the antibody and the antigen, such that, when bound to the MuSK protein (“antigen”) in a physiological solution, the antibody or the fragment protects the binding region from solvent exposure, as determined by suitable techniques, such as hydrogen-deuterium exchange mass spectrometry (HDX-MS). Identification of binding regions is useful in gaining insight into the antigen-antibody interaction and the mechanism of action for the particular antibody. Identification of additional antibodies with similar or overlapping binding regions may be facilitated by cross-blocking experiments that enable epitope binning. As shown herein, cryo-EM may also be used to characterize the binding region. Optionally, X-ray crystallography may be employed to identify the exact amino acid residues of the epitope that mediate antigen-antibody interactions.

[0556]

[0263] The art is familiar with HDX-MS, which is a widely used technique for exploring protein conformation or protein-protein interactions in solution. This method relies on the exchange of hydrogens in the protein backbone amide with deuterium present in the solution. By measuring hydrogen-deuterium exchange rates, one can obtain information on protein dynamics and conformation (reviewed in: Wei et al. (2014) “Hydrogen / deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications.” Drug Disco Today. 19(1): 95-102; incorporated by reference). The application of this technique is based on the premise that when an antibody-antigen complex forms, the interface between the binding partners may occlude solvent, thereby reducing or preventing the exchange rate due to steric exclusion of solvent.

[0557]

[0264] The present disclosure includes antibodies or antigen-binding fragments thereof that bind a MuSK protein, or fragment thereof, comprising at least portion of the f rizzled-l ike domain and a C-terminal extra amino acid residues within the “tail” region of the ECD. Preferably, the antibodies or antigen-binding fragments bind an epitope that comprises one or more amino acid residues of the 7AA Truncated tail C-terminal to the Fz-like domain. In some embodiments, the antibodies or the fragments bind an epitope that comprises one or more amino acid residues of the Truncated Tail C-terminal to the Fz-like domain, and one or more amino acid residues of the Fz-like domain. For example, the epitope may include one or more amino acid residues of the amino acid positions 363-365, 404-411 , and the residue Leucine at position 450 (based on the Fz-domain of human MuSK). In prefrred embodiments, the epitope further comprises two or more amino acid residues of the 7 AA truncated tail C-terminal to the Fz-like domain. In some embodiments, such amino acids include

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[0560] residue(s) P, L, D, Y, and / or K of the human sequence PHLDYNK. In some embodiments, the epitope comprises 3, 4, 5, or more amino acid residues of positions 441-457 (based on human MuSK).

[0561]

[0265] As shown in the data presented herein, the novel class of MuSK agonist antibodies disclosed herein requires the presence of the 7AA Truncated Tail motif C-terminal to the Fz-like domain, or at least a portion thereof, for stable interaction with the antigen. Cryo-EM-based reconstructions reveal that the tail motif which is immediately C-terminus to the Fz-like domain appears to “fold onto” a portion of the Fz-domain, presumably forming a unique epitope to which the antibodies can bind. This interpretation is consistent with the observation that any of the antibodies (e.g., Ab1 , Ab2, Ab3, Ab4, Ab5 and Ab6) is unable to bind MuSK / Fz-like domain unless the tail C-terminal to the Fz-like domain is present. Taken together, the tail motif (e.g., Truncated Tail C-terminal to the Fz-like domain) appears to directly participate in mediating the antibody-antigen interactions, in other words, forming at least part of the epitope. This common feature, together with the fact that they belong to the same epitope bin, indicates that these antibodies likely share the mechanism of action by which they induce activation of MuSK. By contrast, two of the prior art MuSK agonist antibodies, mab#13 and 3B2G2, are found to cross-block with one another (and not with the antibodies of the present disclosure), and they each bind to the Fz-like domain itself, without requiring the presence of the tail C-terminal to the Fz-like domain. From a structural point of view, the inventors found that the Fab fragment of 3B2G2 and the Fab fragment of Ab5 can simultaneously bind a single antigen molecule (see Examples herein), supporting the notion that these two classes of antibodies appear to bind MuSK in discrete orientations, allowing the formation of the ternary complex.

[0562]

[0266] The unique epitope disclosed herein which requires contribution from the tail motif C-terminal to the Fz-like domain may enable particularly stable binding, which may be in some cases reflected in slow off rate of the antibodies. Additionally or alternatively, this epitope may confer an antibodyantigen geometry that enables a particularly effective MuSK activation. It is contemplated that the antibodies of the novel class of MuSK agonists disclosed herein bind the antigen with a favorable orientation, which may confer advantageous mechanism of action.

[0563]

[0267] Accordingly, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds and induces activation (e.g., phosphorylation) of human MuSK, wherein the antibody or antigen-binding fragment binds an epitope in the extracellular domain of human MuSK comprising one or more amino acid residues of the Truncated Tail sequence PHLDYNK (SEQ ID NO: 9), C-terminal to the Fz-like domain. In some embodiments, the epitope further comprises one or more amino acid residues of the Fz-like domain, which is N-terminus to the Truncated Tail. Thus, the novel class of MuSK agonist antibodies can bind Fz-like domain in the presence of Truncated Tail C-terminal to the Fz-like domain, but not in the absence of Truncated Tail C-terminal to the Fz-like domain. In some embodiments, the antibody or the antigen-binding fragment comprises the six CDR sequences of Ab1 , Ab2, Ab3, Ab4, Ab5, Ab6. In some embodiments, the antibody or the antigenbinding fragment cross-competes with Ab1 , Ab2, Ab3, Ab4, Ab5 and / or Ab6. In some embodiments, such antibody or antigen-binding fragment is a variant (e.g. an affinity matured variant) of Ab1 , Ab2, Ab3, Ab4, Ab5 or Ab6.

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[0566]

[0268] It is contemplated that many potent activation agonists may bind this region of a MuSK protein in such a way that MuSK is dimerized and / or activated. It has been shown that MuSK activation is associated with phosphorylation of MuSK.

[0567]

[0269] The art is familiar with various techniques that may be employed to determine the structural aspects of MuSK agonist antibodies and their binding characteristics with MuSK. These include, without limitation, X-ray crystallography, Cryo-EM, and HDX-MS.

[0568]

[0270] Using the HDX-MS technique, binding regions of MuSK can be determined. Further techniques for epitope mapping are described above. In addition, as mentioned above, residues involved in an antigen-antibody interaction may also be determined by structural studies, e.g. using cryo-EM and / or X-ray crystallography. In some embodiments, a portion on MuSK identified to be important in binding an antibody or fragment includes at least a portion of the Fz-like domain and / or the Truncated tail C-terminal to the Fz-like domain. The tail may also contribute, directly or indirectly, to the high-affinity interaction and a particularly activating geometry of these antibodies disclosed herein. Among regions that may contribute to the antibody-antigen interaction, in some embodiments, the high-affinity antibody of the present disclosure may bind an epitope of human or cynomolgus MuSK that comprises at least one residue of the amino acid sequence PHLDYNK (7 amino acid Truncated tail C-terminal to the Fz-like domain) (SEQ ID NO:9).

[0569]

[0271] As explained above and without being bound by theory, the antibodies of the present disclosure may bind to an epitope that includes one or more residues on human MuSK within PHLDYNK (SEQ ID NO: 9), because they require the presence of PHLDYNK to bind to human MuSK. Accordingly, in some embodiments, the antibodies of the present disclosure bind an epitope on human MuSK comprising one or more residues within SEQ ID NO: 9. Additionally or alternatively, the antibodies may bind to a conformational epitope that is formed only in the presence of the Truncated Tail region of MuSK. The confirmational epitope may or may not include one or more amino acid residues within the Truncated Tail C-terminal to the Fz-like domain. In any of the embodiments, however, the antibodies or antigen-binding fragments require the Truncated tail C-terminal to the Fz-like domain for binding to MuSK.

[0570]

[0272] Thus, any of the antibody or antigen-binding fragment encompassed by the present disclosure, such as antibodies or fragments listed in Table 6 or 7 herein, may bind one or more of the binding regions disclosed herein. Such antibodies may be used in the treatment of a MuSK indication in a subject as described herein. Accordingly, selection of an antibody or antigen-binding fragment thereof suitable for therapeutic use in accordance with the present disclosure may include identifying or selecting an antibody or a fragment thereof that binds a MuSK protein, or fragment thereof, comprising or consisting of an Fz-like domain and a 7 amino acid Truncated tail , or identifying or selecting an antibody or a fragment thereof that binds a MuSK protein, or fragment thereof, comprising an Fz-like domain and a tail C-terminal to the Fz-like domain. The selection may further include identifying or selecting an antibody or a fragment thereof that does not bind in the absence of the Truncated tail C-terminal to the Fz-like domain or a portion thereof (e.g. that does not bind to the Fz-like domain alone).

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[0573]

[0273] Table 10 provides a summary of the MuSK agonist antibodies disclosed herein, as well as the prior art antibodies 3B2G2 and Mab#13. Binding affinities and related kinetics profiles shown in the table are as measured by SPR-based technique (e.g., Biacore).

[0574] Table 10. Binding Affinities and related kinetic profiles of antibodies 3B2G2, mab#13 and novel MuSK Agonist antibodies

[0575]

[0576] * Limit of detection for experiment

[0577] Frizzled: Frizzled-like domain

[0578] Truncated Tail: 7 amino acid truncated tail C-terminal to frizzled-like domain

[0579] Mechanism of Action

[0580]

[0274] Antibodies of the present invention that are useful as therapeutics are agonists of MuSK. Further, the antibodies are activators, that is, the antibodies promote the activation of MuSK signaling. The role of MuSK in orchestrating NMJ complexes that mediate synaptic formation and transmission has been extensively studied. The primary mode of action by which MuSK plays a role in this process is via agrin-

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[0583] dependent phosphorylation (e.g., autophosphorylation) in the intracellular domains of MuSK, which triggers AChR clustering.

[0584]

[0275] In some embodiments, antibodies or antigen-binding fragments thereof of the disclosure do not compete with agrin binding to MuSK. In some embodiments, antibodies or antigen-binding fragments thereof of the disclosure do not interfere with endogenous activation of MuSK by agrin. In some embodiments, activation of MuSK is indicated by MuSK phosphorylation, NMJ formation or maintenance, or AChR clustering. In some embodiments, antibodies or antigen-binding fragments thereof of the disclosure induce phosphorylation of human MuSK at one or more residues selected from Y554, Y577, Y600, Y751, S752, Y755 and / or Y756. In some embodiments, antibodies or antigenbinding fragments thereof of the disclosure induce phosphorylation of mouse MuSK at one or more residues selected from Y553, Y576, Y599, Y750, S751, Y754 and / or Y755. In some embodiments, phosphorylation of MuSK is used to assess MuSK activation. In some embodiments, phosphorylation of MuSK is assessed by western blot, immunoprecipitation-western blot, ELISA, or MSD on samples from treated cells {e.g., murine C2C12 myotubes or cells expressing human or mouse MuSK), with anti-phosphotyrosine antibodies and / or commercially-available anti-MuSK antibodies.

[0585]

[0276] Another possible mode of action by which MuSK may play a role in modulating muscle function is through regulating gene expression of proteins involved in muscle cell proliferation, maturation, regeneration and function. Evidence in literature points to possible involvement of BMP proteins in this process. Consistent with the notion, the Ig3 domain of MuSK is reported to contain a BMP binding site, establishing MuSK as a BMP co-receptor in myoblasts and myotubes. Upon BMP binding, MuSK, triggers downstream signal transduction pathway that includes Smad 1 / 5 / 8 phosphorylation (see, for example, Yilmaz et al. Science Signaling 9(444): ra97). This axis is thought to be independent of MuSK kinase activity. Jaime et al has demonstrated that the MuSK-BMP4 pathway is distinct from Agrin-Lrp4-induced MuSK phosphorylation pathway (Skeletal Muscle 14: 1 (2024). Moreover, MuSK in its role as a BMP co-receptor has been shown to regulate adult muscle stem cell quiescence (Madigan et al., eLife 13:RP101078).

[0586]

[0277] Accordingly, in some embodiments, the MuSK antibodies disclosed herein may be capable of promoting BMP-induced activation of Smad1 / 5 / 8 in a MuSK phosphorylation-independent manner. In some embodiments, the BMP is selected from BMP2, BMP4, BMP7 and BMP9. BMP-activation of the Smad1 / 5 / 8 pathway may promote muscle regeneration and by increasing the expression of muscle proteins. Evidence points to BMPs as positive regulators of muscle mass. In addition, BMPs may play a role in coordinating the differentiation of satellite cells from a proliferative to committed state during regeneration. Notably, ALS-derived myoblasts are unable to fully differentiate into myotubes (reviewed in Tsitkanou et al., (2016) Front. Physiol., 7: Article 403). Therefore, it is contemplated that targeting the BMP-MuSK axis with anti-MuSK antibodies may help restore BMP-dependent gene expression that regulate satellite cell differentiation.

[0587] Thermostability

[0588]

[0278] Immunoglobulins are naturally stable proteins. However, biophysical stability of recombinant monoclonal antibodies varies. Typically, many known monoclonal antibodies have a melting

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[0591] temperature (Tm) of around 70°C (e.g., ~65-75°C). Antibodies with relatively low Tm may begin to unfold or denature in certain sections when exposed to high temperatures, and this can render the antibody more susceptible to aggregation. By contrast, antibodies with relatively high Tm can resist structural disintegration (hence more stable).

[0592]

[0279] As noted above, one parameter for evaluating the stability of proteins such as antibodies is thermal stability. To assess thermal stability of a protein (e.g., antibody), melting temperature (Tm) can be measured via differential scanning fluorimetry (DSF). A PCR machine is commonly used to carry out DSF assays. As the protein melts and the interior hydrophobic sections are exposed, hydrophobic dye binds to the newly exposed protein parts. Tm is defined as the point at which 50% of the sample is melted and 50% is unmelted. The term “melting” in this context refers to disintegration or unfolding of the overall protein structure, which is reflected in changes in fluorescent signals as a function of temperature. The unfolding of different parts of an antibody can accelerate the aggregation events due to the exposure of the hydrophobic residues to the solvent. Therefore, it is desirable for a therapeutic antibody to have sufficient thermal stability. In some embodiments, certain fragments of antibodies are used to carry out DSF assays. In some embodiments, the fragment comprises CH3 of an antibody is used in DSF assays. For example, CH3-Fab may be used in a DSF assay to measure Tm.

[0593]

[0280] Whilst 3B2g2 is said to be a potent MuSK agonist antibody, it exhibits less-than-optimal thermostability as measured by melting temperature, posing a potential liability. For example, measured melting temperature for CH3-Fab 3B2G2 is about 61.2°C, as compared to approximately 76.0°C for both Ab5 and Ab6, providing about a 15°C differential. Advantageously, the novel class of MuSK agonist antibodies disclosed herein (e.g., Ab3, Ab4, Ab5, Ab6) all have relatively high melting temperatures (e.g., above 74°C), indicating that the novel antibodies of the present disclosure provide improved stability and hence less liabilities associated with manufacturability and developability as therapeutics.

[0594] Potency

[0595]

[0281] Antibodies disclosed herein may be broadly characterized as “functional antibodies” for their ability to activate MuSK signaling. As used herein, “a functional antibody” confers one or more biological activities by virtue of its ability to bind a target protein (e.g., antigen), in such a way as to modulate its function. Functional antibodies therefore broadly include those capable of modulating the activity / function of target molecules (i.e., antigen). Such modulating antibodies include inhibiting antibodies (or inhibitory antibodies) and activating antibodies. The present disclosure is drawn to antibodies which can activate a biological process mediated by MuSK signaling. The novel antibodies of the present disclosure have enhanced activation properties (potency).

[0596]

[0282] In some embodiments, potency of an activating antibody may be measured in suitable in vitro assays. In some embodiment, a high-content imaging-based AChR clustering assays described herein may be used. In some embodiments, mouse myotubes may be used for carrying out in vitro AChR clustering assays. Cultured cells such as mouse myoblast cell line (e.g. C2C12 cells) can be induced in the presence of low-serum media to differentiate into myotubes. Myotubes are treated with antibodies, and AChRs are visualized through the application of a-bungarotoxin (BTX) (e.g. Alexa

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[0599] Fluor 647-labeled BTX), and fixing the cells. High-throughput fluorescence microscopy is then used to image the cells and the area and intensity of AChR clusters are quantified through image analysis. Plots of the AChR cluster area and / or cluster intensity as a function of antibody concentration are used to calculate the in vitro functional ECso of the antibody. Such assays are exemplified in Example 4 herein. Examples of parameters (readouts) which can be employed to measure AChR clustering include, for example, cluster mean AChR intensity (AU) defined as Average of mean AChR staining intensity per cluster; myotube mean AChR intensity (AU) defined as Average of mean AChR staining intensity per myotube; cluster mean area (pm2) defined as Average of the area per cluster; cluster coverage (%) defined as Average of the area per cluster; and, cluster count per myotube defined as Count of clusters divided by count of myotubes (Readout for total, small and large clusters).

[0600]

[0283] In some embodiments, AChR clustering assay is performed using human cells. In some embodiments, the human cells are primary human cells. In some embodiments, the human cells are human myoblasts. In preferred embodiments, the human cells are human myotubes. Such cells are responsive to agrin stimulation,

[0601]

[0284] Thus, in some embodiments, the activation potency (ECso) of the novel antibodies of the present disclosure calculated based on in vitro AChR clustering assays for measuring MuSK activation, may be 10 nM or less. In some embodiments, the antibodies have an ECso of 5 nM or less (i.e., < 5 nM). In some embodiments, the antibodies have an ECso of 1 nM or less (i.e., < 1 nM). In some embodiments, the antibodies have an ECso of 0.50 nM or less (i.e., < 0.5 nM). For example, Ab3, Ab4, AB5 and Ab6 disclosed herein are capable of inducing AChR clustering with a sub-nanomolar ECso value as measured by cluster mean area in cultured myotubes.

[0602]

[0285] In some embodiments, MuSK-agonist antibodies of the disclosure do not interfere with AChR clustering induced by agrin. In some embodiments, MuSK-agonist antibodies of the disclosure do not interfere with endogenous activation of MuSk by agrin. In some embodiments, MuSK-agonist antibodies that do not interfere with agrin comprise monoclonal antibodies (mAb). In some embodiments, MuSK-agonist antibodies that do not interfere with agrin comprise Fab fragments. Interference of MuSK agonist antibodies with Agrin-induced clustering of AChRs may be assessed through suitable in vitro assays. In some embodiment, a high-content imaging-based AChR clustering assays described herein may be used. In some embodiments, mouse myotubes may be used for carrying out in vitro AChR clustering assays. Cultured cells such as mouse myoblast cell line (e.g. C2C12 cells) can be induced in the presence of low-serum media to differentiate into myotubes. AChR clustering in myotubes is subsequently induced through the addition of agrin in the presence or absence of MuSK agonist antibodies, and myotubes are stained with BTZ and fixed. Quantification of AChR cluster intensity and area can be used to assess interference of Agrin-induced AChR clustering.

[0603] Table 11. Activation potencies (in ECso) of select antibodies as measured by AChR clustering assays

[0604]

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[0607]

[0608]

[0286] In some embodiments, the selection process of an antibody or antigen-binding fragment thereof for therapeutic use may therefore include identifying an antibody or fragment that shows sufficient activation potency. For example, the selection process may include a step of carrying out an AChR clustering-based activation assay to measure potency (e.g., EC50) of one or more test antibodies or fragments thereof, and, selecting a candidate antibody or fragment thereof that shows desirable potency. In some embodiments, EC50 is 10 nM or less. In some embodiments, EC50 is 2 nM or less. The selected antibody or fragment may then be used in the treatment of a MuSK-related indication described herein.

[0609]

[0287] In some embodiments, NMJ morphology is assessed as a measure of MuSK activation and signaling in response to treatment with a MuSK agonist. In some embodiments, staining in whole mount muscle or in cells is used to visualize and quantify differences in NMJ morphology, e.g. between wild type, mutant and mutant treated mice and / or in treated and untreated cells. In some embodiments, assessment of effects of NMJ morphology include overlay of AChR area with synaptophysin area, size and shape of acetylcholine receptor (AChR) areas, and / or differences in neurofilament morphology. In some embodiments, MuSK activation and signaling is measured by assessment of morphology of motor axons, neurofilaments and / or nerve terminals in the NMJ by staining with antibodies to neurofilament and synaptophysin, and / or by staining of AChR with labeled bungarotoxin or antibodies to AChR.

[0610] In vivo Characterization of MuSK-agonist antibodies

[0611]

[0288] In some embodiments, potency of MuSK agonist antibodies may be evaluated in suitable in vivo models as a measure of efficacy and / or pharmacodynamics effects. For example, if the first antibody is efficacious in an in vivo model at a certain concentration, and the second antibody is equally efficacious at a lower concentration than the first in the same in vivo model, then, the second antibody can be said to me more potent than the first antibody. Any suitable disease models known in the art may be used to assess relative potencies of MuSK agonists, depending on the particular indication of interest, e.g., neuromuscular disease models. Disease models which show evidence of significant NMJ dysfunction include, but are not limited to, SMNA7 model, SOD1-G93A / TDP-43 model, Mdx mouse model, MuSK-MG model, Agrn nmf380 model. Preferably, multiple doses or concentrations of each test antibody are included in such studies. Similarly, pharmacodynamics (PD) effects may be measured to determine relative potencies of activating antibodies.

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[0614]

[0289] In vivo studies may be carried out in Agrin mutant mice (e.g. Agrn nmf380 mouse model, Bogdanik and Burgess, 2011, Human Mol Genetics, Vol 20, No 23: 4617-4633) that bear a point mutation in agrin (F1061S: changing a phenylalanine 1061 to serine in the SEA domain of agrin) that results in a partial loss of function of Agrin (effectively mimicking a phenotype of congenital myasthenic syndrome caused by mutations in Agrin). Agrin is required for MuSK activation and hence AChR clustering. An Agrn nmf380 mouse model mimics mutations found in rare congenital myasthenia. In these models, mutant agrin protein is processed differently from the wild-type protein with reduced glycosylation, changes in sensitivity to the protease neurotrypsin, and less efficient externalization and secretion. Agrin mutant mice have dysfunctional NMJs with decreased AchR density, synapses denervation and muscles atrophy. Further, agrin mutant mice are smaller than wild-type, with decreased hind limb motor control and atrophy. Treatment with a MuSK-agonist that induces MuSK activation can increase AChR clustering in these models. Additionally, MuSK agonists can lead to an improvement in body weight, and an increase in muscle mass which translates to an increase in muscle function (e.g., force generation).

[0615]

[0290] Administration of exemplary antibodies of the disclosure to agrin mutant mouse models leads to an increase in the levels of AChR clustering (e.g. as observed through immunohistochemistry). In some embodiments, treatment of exemplary MuSK-agonist antibodies disclosed herein leads to a significant increase in body weight, and muscle function as measured by force generation. In some embodiments, treatment of an agrin mutant mouse model with a MuSK agonist antibody of the disclosure increases maximum muscle force by at least 5 mN, 10 mN, 20 mN, 25 mN or 30 mN compared to a control. In some embodiments, treatment of an agrin mutant mouse model with a MuSK agonist antibody of the disclosure increases maximum muscle force by at least 1.5-fold, 2-fold, 3-fold, 5-fold or 10-fold compared to a control. In some embodiments, treatment of exemplary MuSK-agonist antibodies disclosed herein leads to a significant increase in the mass of Gastrocnemius and / or Tibialis anterior muscles. In some embodiments, improvements in body weight and muscle function are observed after treatment with MuSK agonists of the disclosure that bind to MuSK protein, or fragment thereof, comprising a frizzled-like domain and the Truncated tail C-terminal to the Fz-like domain. In some embodiments, treatment of an agrin mutant mouse model with a MuSK agonist antibody of the disclosure increases muscle weight of an isolated muscle by at least 5 mg, 10 mg, 20 mg, or 50 mg compared to a control. In some embodiments, treatment of an agrin mutant mouse model with a MuSK agonist antibody of the disclosure increases muscle weight by at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 5-fold or 10-fold compared to a control. In some embodiments, treatment with exemplary MuSK-agonist antibodies disclosed herein leads to a significant increase in body weight. In some embodiments, treatment of an agrin mutant mouse model with a MuSK agonist antibody of the disclosure increases body weight by at least 1 g, 2 g, 5 g, or 10 g compared to a control. In some embodiments, treatment of an agrin mutant mouse model with a MuSK agonist antibody of the disclosure increases body weight by at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 5-fold or 10-fold compared to a control. In some embodiments, the control is measured prior to treatment with the MuSK-agonist antibody. In some embodiments, the control is a wild-type mouse counterpart to the agrin mutant mouse model. In some embodiments, the control is a mouse model that has received isotype matched control IgG, optionally

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[0618] wherein the isotype matched control IgG is a murine lgG1. In some embodiments, the control mouse model is treated with a control / comparator antibody.

[0619] Synopsis of in vitro and in vivo properties of MuSK-agonist antibodies

[0620]

[0291] Antibodies of the present disclosure Ab1 , Ab2, Ab3, Ab4, Ab5, Ab6 and antigen-binding fragments thereof represent a new class of highly potent anti-MuSK agonists, distinct from previous antibodies such as mAb13 and 3B2G2, as demonstrated by their differential binding profiles to human and mouse MuSK fragments and unexpectedly superior effects.

[0621]

[0292] Anti-MuSK antibodies mAb13 and 3B2G2, which bind to the Fz-like domain of human MuSK and activate MuSK, were compared to the MuSK agonist antibodies of the present disclosure and antigen-binding fragments thereof in Examples 2-9 below. This section and Table 12 provide a synopsis of those comparisons.

[0622]

[0293] Prior antibodies mAb13 and 3B2G2 bound to fragments of human MuSK comprising the Fz-like domain of human MuSK, regardless of whether or not the 7 amino acid ‘Truncated Tail’ that follows C-terminal to the Frizzled-like domain was also present, and exhibited KDs of binding to human MuSK of 14.87 nM and 0.26 nM for mAbs of mAb13 and 3B2G2, respectively. These antibodies did show activity in inducing AChR clustering in C2C12 mouse myotubes in vitro, with EC50s of 0.107 nM and 0.016 nM, and activity in the agrin nmf380 mouse model to increase body weight, isolated muscle mass and muscle force.

[0623]

[0294] Unlike mAb13 and 3B2G2, antibodies Ab1 , Ab2, Ab3, Ab4 (and antigen-binding fragments thereof) were shown to bind to fragments of human MuSK comprising the Fz-like domain of human MuSK when the Truncated Tail C-terminal to the Fz-like domain is present, but not to the Fz-like domain where the Truncated Tail C-terminal to the Fz-like domain is absent. Similar to antibodies Ab1 , Ab2, Ab3 and Ab4, antibodies Ab5 and Ab6 bound to fragments of human MuSK comprising the Fz-like domain of human MuSK when the Truncated Tail C-terminal to the Fz-like domain was present, but not to the Fz-like domain where the Truncated Tail C-terminal to the Fz-like domain was absent (data not shown). Antibodies Ab1 , Ab2, Ab3, Ab4, Ab5 and Ab6 (and antigen-binding fragments thereof) were also found to bind to MuSK in a common epitope bin distinct from mAb13 and 3B2G2, both of which were in their own epitope bin (hence cross-blocked each other) distinct from the epitope bin for Abs 1 -6. Furthermore, mAb13 and 3B2G2 did not cross-block with any of Ab1 , Ab2, Ab3, Ab4, Ab5 and Ab6.

[0624]

[0295] Despite this distinct binding mechanism compared to MuSK activator antibodies mAb13 and 3B2G2, antibodies Ab1 , Ab2, Ab3 and Ab4 (and antigen-binding fragments thereof) not only bound specifically to both mouse and human MuSK, and induced AChR clustering in vitro at similar or greater potencies as mAb13 and 3B2G2 in a C2C12 murine cell model (and without interference with agrin activation of MuSK), Ab2, Ab3 and Ab4 also surprisingly showed in vivo effects on isolated muscle mass similar to or greater than 3B2G2, in particular leading to comparatively greater increases in muscle force and maximum muscle force (with a maximum force at 150 Hz of at least 30 mN or at least 50% of wild type control treated with HuNeg). In addition, Ab3 and Ab4 are further differentiated from the others by greater binding affinities (e.g. respective bivalent KDs for human MuSK ECD of 0.48 nM and 8.81 nM as determined by BLI-based assay, and respective monovalent

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[0627] KDs of 0.48 nM and 8.81 as determined by BLI-based assay), and by greater induction of AChR clustering in C2C12 myotubes compared to Ab1 and Ab2 (e.g. EC50 of 0.076 nM and 0.151 nM, respectively).

[0628] Table 12. Synopsis of Selected Results from Examples 2-9

[0629]

[0630]

[0296] Antibodies Ab1 , Ab2, Ab3, Ab4, Ab5 and Ab6 (and antigen-binding fragments thereof) therefore define an important new class of anti-MuSK agonists with unexpected and distinct binding features and in vitro and in vivo effects compared to previous anti-MuSK agonists such as the antibodies mAb13 and 3B2G2.

[0631] Determining Biological Effects of MuSK agonist antibodies

[0632]

[0297] Clinical effects of a MuSK activator, described herein can be monitored and / or evaluated for effectiveness by various means. Exemplary such biologically beneficial effects are provided herein. Beneficial biological effects in a subject can be achieved by administration of MuSK agonists. In some embodiments, the MuSK agonist is administered in an amount effective to cause one or more of the biological effects described below.

[0633]

[0298] The ability to assess functional scales that can be reliably measured in patients (e.g. MG patients) is crucial in tracking patients’ disease progression as well as effects of therapy over time.

[0634] While muscle function may be assessed by physiological measurements, such as muscle strength and force generation, motor functional scales monitor disease progression in ways that relate to

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[0637] patients’ functionality in everyday life and carries more meaning and relevance than a measure that quantitates strength perse. Provided below is a list of several known motor functional assessment tests that can be utilized to evaluate patients with neuromuscular disorders, while not intended to be limiting. Other tests include, but are not limited to, gross motor function measure (GMFM), the 6-minute walk test, the 10-meter walk / run test, the time to rise from floor test, the timed up and go test (TUG), and the stair climb test, the methods of which are well known to one of ordinary skill in the art.

[0638]

[0299] Activities of daily living, and disease progression, of patients with ALS can be assessed using Amyotrophic Lateral Sclerosis Functional Rating Scale (ALS-FRS; Arch Neurol. 1996;53(2): 141 -147). ALS-FRS is a tool used for assessing a person’s performance in 10 items of activities of daily living, speech, salivation and swallowing on a functional rating scale, where each item is scored from 0 (for poor performance) to 4 (normal performance). Items in the ALS-FRS scored are: speech, salivation, swallowing, handwriting, cutting food and handling utensils (with or without gastrostomy), dressing and hygiene, turning in bed and adjusting bed clothes, walking, climbing stairs, and breathing.

[0639] Minimum score is 0 and maximum score is 40. Higher scores indicate more function is retained.

[0640]

[0300] For patients with myasthenia gravis (MG), congenital myasthenia (CM) or congenital myasthenic syndrome (CMS), activities of daily living, quality of life, and disease progression, can be assessed with several tools. The Myasthenia Gravis Activities of Daily Living (MG-ADL) assessment tool is a patient-reported measure with 8 items measuring symptoms and functional impacts in bulbar, limb, ocular, and respiratory symptom areas (including talking, chewing, swallowing, breathing, impairment of ability to brush teeth or comb hair and to arise from a chair, double vision and eyelid droop). Items are rated on a scale from 0 (Normal) to 3 (Most Severe). The Myasthenia Gravis Foundation Quality of Life 15 (MG-QoL15r) assessment tool is a patient-reported assessment of wellbeing and independence with 15 items. Patients rank agreement with statements on limitations or difficulties in areas of QoL on a scale of 0 (Not at all) to 2 (Very Much). The Myasthenia Gravis Composite (MGC) assessment tool is a weighted assessment performed by a patient and physician in 10 items relating to bulbar, muscular, ocular and respiratory weaknesses. Patient and physician rank items in areas via physician examination or determination (ptosis (upward ease), double vision, eye closure, breathing, neck flexion or extension (weakest), shoulder abduction and hip flexion); and via patient history (talking, chewing, swallowing). Scores for each increase with severity or difficulty. The Quantitative Myasthenia Gravis (QMG) assessment tool is a physician-reported assessment with 13 items to evaluate axial, bulbar, facial, gross motor, ocular and respiratory weaknesses. Scores for each range from 0 (None) to 3 (Severe), and assess double vision on lateral gaze (in seconds), ptosis in upward gaze (in seconds), facial muscle weakness, swallowing difficulty, speech after counting aloud from 1 to 50 (onset of dysarthria), difficulty 90-degree sitting (with right or left arm outstretched), forced vital capacity, right and left hand grip strength (in kgW, scaled for men or women), time in 45-degree supine with head lifted (in seconds), and time in 45-degree supine with right or left leg outstretched (in seconds).

[0641] Expanded Hammersmith Functional Motor Scale

[0642]

[0301] The disease severity of a patient having a neuromuscular disease / disorder, both before treatment, during treatment, and after treatment with a MuSK agonist described herein, can be classified using many tests and assays well known to those of ordinary skill in the art. Hammersmith

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[0645] Functional Motor Scale, Expanded (HFMSE) is a validated endpoint for SMAtype II and nonambulatory type III, and is well known to those of ordinary skill in the art. The testing system comprises 33 items (e.g., motor tasks or activities) that assess motor function. Items that are primarily strength-driven motor activities of short duration that require type II fast-twitch fibers include: sitting without hand support for 3 seconds; sitting to lying down; rolling from back to abdomen; push body up for 3 seconds; kneeling to standing position; climbing up / down 4 steps of stairs; jump 12 inches forward, etc.

[0646]

[0302] In some embodiments, the subject has a baseline Expanded Hammersmith Functional Motor Scale score of < 66 prior to receiving a corrector or a MuSK agonist therapy (“baseline”), e.g., < 65, < 60 , < 55, < 50, < 40, < 35, < 30, < 25, < 20, etc. In one embodiment, the subject has a baseline Expanded Hammersmith Functional Motor Scale score of < 50 prior to receiving a MuSK agonist. In one embodiment, the subject has a baseline Expanded Hammersmith Functional Motor Scale score of 40 prior to receiving MuSK agonist. In one embodiment, the subject has a baseline Expanded Hammersmith Functional Motor Scale score of 235 prior to receiving MuSK agonist. In one embodiment, the subject has a baseline Expanded Hammersmith Functional Motor Scale score of 2 30 prior to receiving a MuSK agonist. In one embodiment, the subject has a baseline Expanded Hammersmith Functional Motor Scale score of < 25 prior to receiving a MuSK agonist. In one embodiment, the subject has a baseline Expanded Hammersmith Functional Motor Scale score of < 20 prior to receiving a MuSK agonist.

[0647]

[0303] In some embodiments, the subject has an increased (or “corrected”) Expanded Hammersmith Functional Motor Scale score following a MuSK agonist therapy. In some embodiments, the subject has improved the score by at least 3 points, at least 4 points, at least 5 points, at least 6 points, at least 7 points, at least 8 points, at least 9 points, at least 10 points, at least 11 points, at least 12 points, at least 13 points, at least 14 points, or at least 15 points, over the baseline, after receiving the MuSK agonist. In some embodiments, the subject has an increased Expanded Hammersmith Functional Motor Scale score following a MuSK agonist therapy. In some embodiments, the subject has improved the score by at least 3 points, at least 4 points, at least 5 points, at least 6 points, at least 7 points, at least 8 points, at least 9 points, at least 10 points, at least 11 points, at least 12 points, at least 13 points, at least 14 points, or at least 15 points.

[0648]

[0304] For example, many non-ambulatory SMA patients have the baseline Hammersmith score ranging from 15 to 30 points, out of 66 points total. As a result of a MuSK agonist therapy, such patients may improve the score on the average by 4-10 points over the respective baseline.

[0649]

[0305] For SMA patients, clinical significance of even a single point difference in various motor test scoring systems is noteworthy. To put this in perspective, examples are provided below for illustrative purposes, based on the test items from the standard HFMSE system for non-ambulatory SMA patients:

[0650]

[0306] Tasks 1 and 2 of the HFMSE test involve sitting up (without back support) for 3+ seconds. 2 points are given if the patient can sit using no hand support for a count of 3 or more; 1 point for maintaining balance for a count of 3 using one hand as support; and 0 point for requiring both hands to maintain balance. In a real life setting, the difference between the ability to sit without using a hand as support (2 point) vs. requiring to use one hand merely to maintain balance (1 point) is enormous, because in the former case the patient can use both hands for carrying out activities (such as holding

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[0653] an item) while sitting up. Task 3 of the test evaluates whether the patient, while sitting, is able to bring one hand up to touch head above ear level. The ability to perform this seemingly simple task may make a difference in being able to comb one’s own hair or putting on a hat without assistance. Motor Function Measure (MFM)

[0654]

[0307] The Motor Function Measure (MFM) test provides a genetic scale to assess various parameters of motor function in patients with neuromuscular disease of varying degrees of disease severity, including ambulatory and non-ambulatory children and adults aged about 6 and 62 years. There are multiple versions of MFM that are tailored toward different patient populations. For example, MFM32 is suitable for children who are older than 6 years, while MFM20, which is a modified version, has been validated for children under 6 years of age. MFM has been successfully employed in clinical trials to monitor or detect changes in motor function in patients, which reflect deterioration over time (see, for example, clinicaltrials.gov NCT02628743).

[0655]

[0308] In one embodiment, a subject having a neuromuscular disease and being administered a therapy or combination therapy described herein exhibits an increase in MFM score of at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold after administration of the therapy or combination therapy.

[0656] Upper Limb Module (ULM)

[0657]

[0309] The Upper Limb Module (ULM) test provides assessment of arm function which has been specifically designed as an add-on module. The ULM is intended to capture performance of activities of daily living not typically included in measures of gross motor function. The assessment includes 9 items of activities which can be reliably performed in children and takes ~10 minutes to complete. The ULM has been used in a multicentric setting and in clinical trials.

[0658]

[0310] In one embodiment, a subject having a neuromuscular disease / dsorder and being administered a therapy or combination therapy described herein exhibits an increase in ULM score of at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold after administration of the therapy or combination therapy. Revised Upper Limb Module

[0659]

[0311] Revised Upper Limb Module (RULM) allows assessment of arm function in patients with neuromuscular disease which has shown good validity and reliability, making it suitable for use in clinical research. The RULM included 20 items of activities that could be completed successfully by children as young as 30 months. These items include tasks such as bringing hands from lap to table, picking up small items, pushing buttons, tearing paper, opening a Ziploc container, bringing hands above shoulders, and lifting items of different weight to different heights. Outcome measures are tests used by researchers to assess whether a certain treatment under trial is having any effects on the patient. Using the right outcome measure is vital to make sure a trial can prove if a treatment works. According to SMA News Today, RULM effectively captured the progressive muscle weakness in the weak end of the spectrum.

[0660] Six-Minute Walk Test

[0661]

[0312] Six Minute Walk Test (6MWT) is reported to be a reliable and valid functional assessment in patients with neuromuscular disease, which is able to capture a fatigue element of the disease. For example, fatigue observed in patients reflected a 17% decrease in gait velocity from the first minute to the last minute during the 6MWT.

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[0664]

[0313] In one embodiment, a subject having a neuromuscular disease and being administered a therapy or combination therapy described herein exhibits an increase in RULM score of at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold after administration of the therapy or combination therapy. CHOP INTEND Score

[0665]

[0314] The CHOP INTEND is a clinician-rated questionnaire developed to assess motor skill in neuromuscular disease (e.g. SMA). The 16 items are scored from 0 to 4. The global score ranges from 0 to 64, a higher score indicating better motor skills. (See: Glanzman AM, Mazzone E, Main M, Pelliccioni M, Wood J, Swoboda KJ, Scott C, Pane M, Messina S, Bertini E, Mercuri E, Finkel RS. The Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND): test development and reliability. Neuromuscul Disord. 2010 Mar;20(3): 155-61). CHOPS INTEND is validated and has been shown reliable in SMA Type I subjects. It was derived in part from TIMP (Test of Infant Motor Performance) and is designed to measure motor function in weak infants with neuromuscular disease. The test includes active (spontaneous, goal-directed) and elicited reflex movements but does not include respiratory or feeding assessments.

[0666]

[0315] In one embodiment, a subject having neuromuscular disease and being administered a therapy or combination therapy described herein exhibits an increase in at least one of the 16 CHOP items of at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold after administration of the therapy or combination therapy.

[0667] CMAP Test

[0668]

[0316] Neuromuscular damage may be assessed using a compound muscle action potential (CMAP) test which provides an electrical stimulation of a nerve and records the compound muscle action potential from surface electrodes overlying a muscle supplied by that nerve. The test may involve stimulation at the wrist, the elbow, and less frequently, the axilla and the brachial plexus

[0669]

[0317] The CMAP measures the summated voltage response from the individual muscle fiber action potentials. Typically, the electrodes are placed over a target muscle of the subject and CMAP is obtained by giving supramaximal stimuli (i.e., a stimulus having strength significantly above that required to activate all the nerve or muscle fibers in contact with the electrode), which are repeated every 30-60 seconds for a period of 2-3 minutes until stable baseline amplitude is obtained. Then the subject contracts the target muscles for 2-5 minutes, with brief (3-4 seconds) rest every 15 seconds to prevent muscle ischemia. CMAP is recorded every minute when muscle is exercised and every 1 -2 minutes after exercise for a period of 30 minutes or until no further decrease in the amplitude of CMAP is observed. CMAP amplitude is typically measured in millivolts (mV). Percentage of amplitude decrease is calculated by subtracting the smallest amplitude after exercise from the greatest amplitude after exercise and dividing it by the greatest amplitude after exercise. In CMAP tests done on a group of people without muscle disease the CMAP amplitude decrease varied from 5.4% to 28.8% (mean 15%). A decrease of more than 40% in the amplitude of CMAP is considered diagnostic of muscle disease.

[0670]

[0318] In some embodiments, the decrease in amplitude of CMAP in a subject is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, or more. In some embodiments, the amplitude of the negative peak of CMAP in a subject with a muscle disease (e.g., MG) is substantially lower compared to the corresponding amplitude of the negative peak of CMAP in a subject without muscle disease

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[0673] (control subject). In one embodiment, the amplitude of the negative peak of CMAP in a subject with a muscle disease is at least 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, or more, lower than the corresponding amplitude of the negative peak of CMAP in a control subject. CMAP test can be used to determine the effectiveness of therapy by comparing CMAP decrements pre and post treatment, as described herein.

[0674]

[0319] In one embodiment, a subject having a neuromuscular disease / disorder and being administered a therapy or combination therapy described herein exhibits an increase in CMAP of at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold after administration of the therapy or combination therapy.

[0675] MUNE Test

[0676]

[0320] Motor unit number estimation (MUNE) is a test that can be used to determine the approximate number of motor neurons in a muscle or group of muscles. MUNE test provides a calculated value that represents the estimated number of motor neurons or axons (motor control input) supplying the muscle or group of muscles being tested. In addition, MUNE provides a means of measuring motor unit size and enables tracking of loss of motor neurons. MUNE test is typically used most often in neuromuscular disorders such as amyotrophic lateral sclerosis and spinal muscular atrophy.

[0677]

[0321] Typically, in a MUNE test, bipolar electrodes at the skin surface stimulate the nerve strongly enough to activate all of the motor axons within it, resulting in full depolarization and contraction of the muscle, which corresponds with activation of all of its motor units ( / '.e., motor neurons or axons) and component muscle fibers at the site of placement of electrodes. The electrical impulse generated by this muscle activity is recorded by electrodes placed over the muscle on the skin surface. In a healthy muscle, all the motor units and all their muscle fibers are activated simultaneously during this test, generating the compound motor action potential (CMAP), which is the maximum motor response. The amplitude of the CMAP corresponds to the total number of motor units and muscle fibers activated. The amplitude of the third response at each site is summed, then divided by 9 to yield the average single motor unit action potential (SMUP) amplitude. This amplitude is divided into the maximum compound motor unit action potential (CMAP) amplitude to yield the MUNE.

[0678]

[0322] Average MUNE for normal, healthy subjects is 225 (±87), and, for example, was 41.9 (±39) among subjects with a muscle disease (e.g., ALS or SMA) at baseline. Subjects having muscle conditions or disorders exhibit clear decrements over time, with an average rate of decline as high as approximately 9% per month. In one embodiment, the average rate of monthly decline in MUNE values in a subject with a muscle disease or disorder is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, or more, relative to the corresponding MUNE values in a control subject without muscle disease. It is possible that a subject with a muscle disease can have normal CMAP amplitude measurements but a MUNE value less than 50% of the control subject. In one embodiment, the subject with normal CMAP amplitude values has a MUNE value that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, or more, lower relative to the corresponding MUNE values in a control subject without muscle disease.

[0679]

[0323] In one embodiment, a subject having a neuromuscular disease / disorder and being administered a therapy or combination therapy described herein exhibits an increase in a MUNE value of at least 2-

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[0682] fold, at least 3-fold, at least 4-fold, or at least 5-fold after administration of the therapy or combination therapy.

[0683] Effect on Mass and / or Function of Muscle in the Human Subject

[0684]

[0324] In some embodiments, administration of an effective amount of a MuSK agonist, e.g., an antibody, or antigen-binding fragment thereof, described herein to a subject can cause an increase in muscle mass. Preferably, such an increase in muscle mass is clinically meaningful to benefit or otherwise improve the health status of the subject. For example, clinically meaningful changes in muscle mass may improve the patient’s mobility, self-care, metabolism, etc. In some embodiments, the increase in muscle mass is an increase in lean muscle or lean muscles. In some embodiments, such increase in muscle mass is a systemic effect such that muscles in the whole body or substantially whole body show the measurable effect. In other embodiments, effects are localized to certain group / type of muscles. In some embodiments, administration of an effective amount of a MuSK agonist, e.g., an antibody, or antigen-binding fragment thereof, described herein to a subject can cause an increase in the mass of the Gastrocnemius muscle. In some embodiments, administration of an effective amount of a MuSK agonist, e.g., an antibody, or antigen-binding fragment thereof, described herein to a subject can cause an increase in the mass of the Tibialis anterior muscle.

[0685]

[0325] In some embodiments, the mass of the muscle tissue, e.g., lean muscle tissue, is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, the mass of the muscle tissue, e.g., lean muscle tissue, is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%. Such increase in muscle mass may be deduced or measured by any suitable known methods, including measurement of cross-sectional area via MRI (e.g., forearm cross section), circumference, diaphragm width (e.g., via ultrasound), etc. In some embodiments, the body weight of the subject is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%.

[0686]

[0326] In some embodiments, administration of an effective amount of an antibody or antigen-binding fragments thereof described herein to a subject can cause an enhancement in muscle function. Muscle function may be assessed by a variety of measures, including, without limitation: force generation, grip strength (e.g., maximum grip strength), endurance, muscle oxidative capacity, dynamic grip endurance, etc. In some embodiments, serum creatinine levels are used as a validated biomarker indicative of muscle mass, albeit with limited sensitivity. In some embodiments, the muscle function of the human subject is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In some embodiments, the muscle force of the human subject is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%

[0687]

[0327] In some embodiments, administration of the MuSK agonists of the disclosure increases locomotor function in the human subject. In some embodiments, the locomotor function of the human subject is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, the locomotor function

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[0690] of the human subject is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0691]

[0328] In another embodiment, administration of the MuSK agonists of the disclosure increases the muscle strength in the human subject. In some embodiments, the muscle strength of the human subject is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, the muscle strength of the human subject is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0692]

[0329] In some embodiments, administration of the MuSK agonists of the disclosure can cause clinically meaningful changes in muscle function which corresponds to enhanced functionality of the patient. In some embodiments, enhanced functionality includes improvement in the patient’s mobility, self-care, metabolism, etc.

[0693] Effect on Life Quality of the Human Subject

[0694]

[0330] Assessment of the quality of life in patients with severe or chronic conditions, such as MG or SMA patients, may involve integrated approaches to evaluate various aspects of physical, mental, social and other parameters. Generally, a greater degree of quality of life is associated with factors such as: accessibility to assistive technology; community reintegration; functionality with lower limb and walking and / or wheeled mobility; mental health; severity in neurological impairment and autonomic dysfunction; pain management; functional independence and self-care; upper limb strength; and spasticity control. Administration of a MuSK agonist described herein increases the quality of life of the human subject to achieve a clinically meaningful improvement as measured by a standardized quality-of-life test / system.

[0695]

[0331] A number of suitable tests for assessing the quality of life in patients are known in the art, including, without limitation: Spinal Cord Independence Measure (SCIM); Functional Independence Measure (FIM); Incontinence Quality of Life Questionnaire (l-QOL); Life Satisfaction Questionnaire (LISAT-9, LISAT-11 ); Quality of Life Index (QLI); Quality of Life Profile for Adults with Physical Disabilities (QOLP-PD); Quality of Well Being (QWB) and Quality of Well Being- Self-Administered (QWB-SA); Qualiveen; Satisfaction with Life Scale (SWLS, Deiner Scale); Short Form 36 (SF-36); Sickness Impact Profile 68 (SIP 68); and World Health Organization Quality of Life- BREF (WHOQOL-BREF).

[0696]

[0332] In some embodiments, quality of life is assessed in accordance with the SF-36 Quality of Life Scoring System, which is a validated scoring system, in which an 8 point change is considered clinically meaningful. In some embodiments, administration of an effective amount of a MuSK agonist of the disclosure results in a clinically meaningful improvement in a standardized quality-of-life test score.

[0697]

[0333] As used the herein, the term “clinically meaningful improvement” refers to a significant improvement over a standard level. In some embodiments, a patient’s SF-36 Quality of Life scores are increased by at least 8 points, following treatment with an effective amount of an antibody or antigen-binding fragments thereof described herein, as compared to the patient’s score prior to the treatment. In some embodiments, patients achieve higher scores as assessed by the SF-36 Quality of Life Test, for example, at least 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 points

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[0700] increase in the scores from the SF-36 Quality of Life Scoring System. In other embodiments, the scores from the SF-36 Quality of Life Scoring System is increased by at least about 8-10, 10-15, 15-20, 20-30, 30-40, 40-50, 8-20, 8-30, 8-40, or 8-50.

[0701]

[0334] In some embodiments, one or more of Quality of Life measures are employed to assess patients’ quality of life before and after treatment with an activator of MuSK signaling disclosed herein. Advantages of this test include: I) it is easy to administer; ii) it assesses both physical function and mental health; and, iii) it is highly validated for a number of clinical indications.

[0702] Effect on Preventing Muscle Loss or Atrophy

[0703]

[0335] Administration of an effective amount of the MuSK agonist antibody prevents, delays, or alleviates muscle loss or atrophy in the human subject at risk of developing muscle loss and / or atrophy. In some embodiments, muscle loss or atrophy is decreased or prevented by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, muscle loss or atrophy is decreased or prevented by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100% as compared to control group that does not receive a MuSK agonist antibody.

[0704]

[0336] Administration of an effective amount of the MuSK agonist antibody can result in preventing further deterioration of affected muscle, or delaying or slowing of the rate of disease progression in these patients. In some embodiments, administration of an effective amount of the MuSK agonist antibody can delay the muscle loss or atrophy in the human subject at risk of developing muscle loss and / or atrophy, as compared to control group that does not receive MuSK agonist. In some embodiments, muscle loss or atrophy is delayed for at least 1 month, 2 months, 3 months, 6 months, 8 months, 12 months, 2 years, 3 years 5 years or 10 years in the human subject receiving MuSK agonist, as compared to control group that does not receive MuSK agonist.

[0705]

[0337] Preventing further deterioration of affected muscle refers to maintaining disease status which includes for example, maintaining motor functional test scores over longer periods of time as compared to control, slower rate of disease progression, as measured / monitored by motor function test; fewer hospitalizations, fewer injuries (e.g., bone fractures), longer time before requiring ventilator, longer time before becoming wheelchair-bound, etc.

[0706]

[0338] Prevention of muscle loss or atrophy by the use of a MuSK agonist described herein can be readily monitored or assessed by any suitable methods to evaluate motor function involving affected muscles.

[0707] Expression of Biomarkers

[0708]

[0339] Changes in the level of certain biomarkers (e.g., plasma biomarkers) of a neuromuscular disease / disorder may be measured to monitor progression of the pathology, as well as the patients’ responsiveness to treatment. Suitable methods and assays to measure changes in the level of suitable biomarkers from patient samples are known in the art. The detection methods of the invention can thus be used to detect mRNA, protein, cDNA, or genomic DNA, for example, in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of a marker protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations

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[0711] and immunofluorescence. In vitro techniques for detection of genomic DNA include Southern hybridizations. In vivo techniques for detection of mRNA include polymerase chain reaction (PCR), Northern hybridizations and in situ hybridizations. Furthermore, in vivo techniques for detection of a marker protein include introducing into a subject a labeled antibody directed against the protein or fragment thereof. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0712]

[0340] More specifically, monitoring the influence of agents (e.g., MuSK agonist) on the level of expression of a marker can be applied not only in basic drug screening, but also in clinical trials, as well as for evaluating disease maintenance and progression, and patients’ responsiveness to a particular therapy. For example, the effectiveness of an agent to affect marker expression can be monitored in biological samples collected from subjects receiving treatment for a neuromuscular disease / disorder, e.g., prior to, during and / or following treatment and measuring levels of the markers and / or changes in the levels of marker expression over time. In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent(s); (ii) detecting the level of expression of one or more selected markers of the invention in the pre-administration sample; (ill) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression of the marker(s) in the post-administration samples; (v) comparing the level of expression of the marker(s) in the pre-administration sample with the level of expression of the marker(s) in the post-administration sample or samples; and (vi) assessing effectiveness of the therapeutic regimen, and if necessary altering or adjusting treatment accordingly. For example, increased expression of the marker gene(s) during the course of treatment may indicate ineffective dosage and the desirability of increasing the dosage. Conversely, decreased expression of the marker gene(s) may indicate efficacious treatment and no need to change dosage. One or more biomarkers present in serum samples are preferred for ease of sample collection, although in certain cases muscle biomarkers may be used, for example, collected by tissue biopsy.

[0713]

[0341] In some embodiments, plasma protein markers of neuromuscular diseases / disorders are selected from the list below:

[0714] CILP2 (Cartilage intermediate layer protein 2); TNXB (Tenascin XB); CLEC3B (Ctype lectin domain family 3, member B (tetranectin)); TNXB (Tenascin XB); ADAMTSL4 (ADAMTSlike 4); THBS4 (Thrombospondin 4); COMP (Cartilage oligomeric matrix protein); CRTAC1 (Cartilage acidic protein 1); F13B (Coagulation factor XIII, B polypeptide); PEPD (Peptidase D); LUM (Lumican); CD93 (Complement component 1, q subcomponent, receptor 1); Mixed complement C2 / B; ARCS (Amyloid P component, serum); VTN (Vitronectin); DPP4 (Dipeptidylpeptidase 4 (CD26, adenosine deaminase complexing protein 2)); CRP (C-reactive protein, pentraxinrelated); HBB (Hemoglobin beta); GSN (Gelsolin); NCAM1 (Neural cell adhesion molecule 1); CFI I factor (complement); APOA4 (Apolipoprotein AIV); VTN (Vitronectin); F13A1 (Coagulation factor XIII, A1 polypeptide); INHBC (Inhibin, beta C); RPS27A (Ubiquitin and ribosomal protein S27a precursor); CDH13 (Cadherin 13, Hcadherin (heart)); mixed Complement C2 / B; C2 Complement component 2; CP (Ceruloplasmin (ferroxidase)); HBA (Hemoglobin subunit alpha); QSOX1 (Quiescin Q6); LRG1 (Leucine-rich alpha2-glycoprotein 1); C9 (Complement component 9); SERPINA10 (Serpin peptidase inhibitor, clade A(alpha1 antiproteinase, antitrypsin), member 10); ALP (Alkaline phosphatase,

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[0717] liver / bone / kidney); mixed fc-gamma receptor lll-A / B; PROC (Protein C (inactivator of coagulation factors Va and Villa)); VCAM1 (Vascular cell adhesion molecule 1); GAPDH (Glyceraldehyde-3-phosphate dehydrogenase); OMD (Osteomodulin); IGKVD41 (Immunoglobulin kappa variable 41); IGFBP6 (Insulinlike growth factor binding protein 6); PTPRG (Protein tyrosine phosphatase, receptor type, G); S100A9 (S100 calcium binding protein A9 (calgranulin B)); VNN1 (Vanin 1); SERPIND (Serpin peptidase inhibitor, clade D (heparin cofactor), member 1); CA1 (Carbonic anhydrase I); CTSD (Cathepsin D (lysosomal aspartyl peptidase)); HP (Haptoglobin); SELENBP1 (Selenium binding protein 1); ORM2 (Orosomucoid 2); PRDX2 (Peroxiredoxin 2); AOC3 (Amine oxidase, copper containing 3 (vascular adhesion protein 1)); COL6A3 (Collagen, type VI, alpha 3); PZP (Pregnancyzone protein); COL6A1 (Collagen, type VI, alpha 1); PARK7 (Parkinson disease (autosomal recessive, early onset) 7); THBS1 (Thrombospondin 1); CAT (Catalase); LCP1 (Lymphocyte cytosolic protein 1 (Lplastin)); AFM (Afamin); HPR (Haptoglobinrelated protein); SELL1 (Selectin L (lymphocyte adhesion molecule 1)); ENG (Endoglin); PFN1 (Profilin 1); PI16 (Peptidase inhibitor 16); SERPINA6 (Serpin peptidase inhibitor, clade A (alphal antiproteinase, antitrypsin), member 6); F9 (Coagulation factor IX); PROCR (Protein C receptor, endothelial); ORM1 (Orosomucoid 1); NEO1 (Neogenin homolog 1); MMRN2 (Multimerin 2); LGB (Beta-lactoglobulin); CNTN4 (Contactin 4); SHBG (Sex hormonebinding globulin); CA2 (Carbonic anhydrase II); IGFBP5 (Insulinlike growth factor binding protein 5); PLTP (Phospholipid transfer protein); FGA (Fibrinogen alpha chain); TPM4 (Tropomyosin 4); MB (Myoglobin); SPP1 (Osteopontin); AXL (AXL receptor tyrosine kinase); APSC (Amyloid P component, serum); CRP (C-reactive protein, pentraxinrelated); CCL22 (Chemokine (C-C motif) ligand 22 (macrophage derived chemokine)); THBD (Thrombomodulin); CALCA (Calcitonin); LEP (Leptin); NPPB (Brain natriuretic peptide b); MMP2 (Matrix Metalloproteinase 2); CK (Creatine kinase muscle / bone); ACE (Angiotensin converting enzyme); FAPB3 (Fatty acid binding protein (heart)); CD40 (CD40 Ligand); MIF (Macrophage Migration Inhibitory Factor); ANGPT2 (Angiopoietin 2); AHSG (Alpha-2-HS-glycoprotein (fetuin A)); CFH (Complement factor H); IL8 (Interleukin 8); C3 (Complement component 3); PPY (Pancreatic polypeptide); VEGFA (Vascular endothelial growth factor); TF (Transferrin); PGF (Placental growth factor); EGF (Epidermal growth factor); GSTA1 (Glutathione S transferase alpha); SOD1 (Superoxide dismutase 1); VCAM1 (Vascular cell adhesion molecule 1); PAH (Plasminogen activator inhibitor 1); CSF1 (Macrophage colony stimulating factor 1); S100A12 (S100 Protein A12); VTN (Vitronectin); FASLG (Fas ligand); A1 M (Alpha-1 -microglobulin); AST (Astartate transaminase); ACCT (Alpha-1 -antichymotrypsin); CCL3 (Chemokine (C-C motif) ligand 3 (Macrophage Inflammatory Protein 1 beta)); SORT1 (Sortilin); TBG (Thyroxine binding globulin); APOA1 (Apolipoprotein A1); MPO Myeloperoxidase); B2M (Beta 2 microglobulin); EPO (Erythropoietin); MMP10 (Matrix Metalloproteinase 10); PROS1 (Vitamin K Dependent Protein S); MMP7 (Matrix Metalloproteinase 7); AGER (Advanced glycosylation end products receptor); IL18 (Interleukin 18); CCL11 (Chemokine C-C motif ligand 11); IGA (Immunoglobulin A); C peptide (Proinsulin C Peptide); A2M (Alpha-2-macroglobulin); PDGF BB (Platelet Derived Growth Factor); CCL16 (Chemokine C-C motif ligand 16); IL1A (Interleukin 1 alpha); APOA4 (Apolipoprotein A4); MMP9 (Matrix metalloproteinase 9); SPP1 (Osteopontin); CLEC3B (Ctype lectin domain family 3, member B (tetranectin)); IGFBP6 (Insulin-like growth factor binding protein 6); FABP4 (Fatty acid binding protein (adipocyte)); CHI3L1 (Chitinase 3-like 1 (YKL-40)); LEP (Leptin); CTSD (Cathepsin D);

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[0720] MST1 (Macrophage stimulating 1 (hepatocyte growth factor-like)); MIF (Macrophage migration inhibitory factor); S100A4 (S100 calcium binding protein A4); GLO1 (Glyoxalase 1 (lactoylglutathione lyase)); ENG (Endoglin); FTL1 (Fms-related tyrosine kinase 1 (vascular endothelial growth factor receptor)); ERBB2 (Human epidermal growth factor receptor 2 (HER2)); NDKB (Nucleoside phosphatase kinase isoform B); PRDX-4 (Peroxiredoxin 4); PLAUR (Plasminogen activator, urokinase receptor); IL6R (Interleukin 6 receptor); CCL24 (Chemokine (C-C motif) ligand 24 (eotaxin 2)); GSN (Gelsolin); PSAT1 (Phosphoserine aminotransferase 1); MYOG (Myogenin); PAX7 (Paired Box 7) and, TGFpl (Transforming growth factor beta 1).

[0721]

[0342] In some embodiments, muscle biopsy is collected from a patient and is used to examine the morphology of muscle and or NMJ. Muscle biopsy may aid the diagnosis of a particular disease (e.g., muscle disorders, neuromuscular disorders, etc.). For example, in ALS, certain synaptic proteins, such as rapsyn and LRP4, are progressively lost, and therefore, one or more synaptic proteins may serve as biomarkers to assess the structural integrity of NMJs. Moreover, structural defects observed in the architecture of NMJs may inform diagnosis and / or prognosis of a disease. Profound changes to the NMJ architecture are observed in muscle biopsies (intercostal, biceps, laryngeal) from ALS patients. These include, for example, evidence of denervation, fragmented and smaller endplates, flattened synaptic clefts, reduction in the AChR expression. Intracellular recordings showed decreased MEPP amplitude, reduced quantal content and stores (indicative of lesser efficient neurotransmission).

[0722]

[0343] Additionally, or alternatively, any suitable physiological measurements known in the art may be carried out to assess muscle function, including, electrical impedance myography (EIM), quantitative muscle magnetic resonance imaging (qMRI), dual energy X-ray absorptiometry (DEXA), etc.

[0723] Screening methods for MuSK agonist antibodies

[0724]

[0344] To identify or screen for an anti-MuSK antibody capable of potently activating MuSK, methods described herein can be employed. Applicant of the present disclosure successfully identified multiple antibodies that specifically bind each of the known domains within the ECD of MuSK, namely, the Ig1 domain, the Ig2 domain, the Ig3 domain, and the Fz-like domain (plus C-term sequences) of human MuSK. Upon evaluating in vitro potency (e.g., agonist activity), in vivo efficacy and safety, it was determined that a class of antibodies that bind the Fz-like domain in the presence of extra C-terminal amino acids referred herein to as “tail" (e.g., Truncated Tail), exhibited advantageous properties. For this novel class of MuSK agonist antibodies, antigen binding is lost when the extra tail sequences are deleted, indicating that the tail region is required for antibody-antigen interactions. The extracellular portion of the human MuSK protein contains a 45 amino acid-tail between the C-term end of the Fz-like domain and the transmembrane segment. Of this 45 amino acid stretch, the first 7 amino acid segment (“Truncated Tail”) added to the Fz-like domain is sufficient to provide a particularly suitable antigenic target, which is superior to the Fz-like domain alone.

[0725]

[0345] Based on this recognition, the present disclosure provides improved methods for screening or identifying MuSK-activating antibodies or antigen-binding fragments thereof.

[0726]

[0346] According to the invention, the novel antigen that comprises at least a portion of the tail segment of MuSK ECD is used to generate or screen for antibodies or antigen-binding fragments that

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[0729] specifically bind the antigen. In preferred embodiments, the portion of the tail segment is or comprises a 7 amino acid stretch which is referred to as Truncated Tail (set forth as SEQ ID NO: 9). In some embodiments, the MuSK antigen further comprises at least a portion of the Fz-like domain attached to the N-terminus of the tail sequence. Preferably, a full-length Fz-like domain (as set forth as SEQ ID NO: 8) forms a part of the antigen. Most preferably, the antigen does not include the other domains of the MuSK ECD, namely, the Ig1 , Ig2 and Ig3 domains. In preferred embodiments, the antigen is a full-length human Fz-domain sequence attached to the C-terminus Truncated Tail (Fz-like + 7AA; SEQ ID NO: 7). It is contemplated herein that the inclusion of this tail sequence as part of an antigen, as opposed to the Fz-like domain alone, enables identification of antibodies capable of binding MuSK in a unique orientation that enhances stable antibody-antigen interactions and potency.

[0730]

[0347] Suitable in vitro binding assays can be employed to screen for antibodies or antigen-binding fragments capable of binding the antigen comprising Truncated Tail (such as Fz-like domain attached to Truncated Tail). Commonly used techniques include BLI-based assays (such as Octet) and SPR-based assays (such as Biacore). Preferably, one or more antibodies with sufficiently high affinities (such as KD of 5 nM or less, e.g., 1 nM or less) are selected. Further preferably, for purposes of identifying a therapeutic candidate, antibodies with cross-species reactivity (e.g., human MuSK and at least one additional species such as murine MuSK) are desirable. Such species cross-reactivity allows a seamless transition from preclinical to clinical studies of the MuSK agonist. Preferably, such antibody cross-reacts with both human MuSK and mouse MuSK. Most preferably, such antibody cross-reacts with human MuSK, mouse MuSK, and cynomolgus macaques (cyno) MuSK.

[0731]

[0348] To identify agonist antibodies, antibodies or antigen-binding fragments with desired binding activities are evaluated for potency (functional activity), i.e. , the ability to activate MuSK. Suitable in vitro potency assays can be employed, which measures the ability of test antibodies to induce MuSK activation. Such assays may include, for example, phosphorylation assays to measure an increased level of MuSK phosphorylation in the presence of a test antibody; dimerization assays to measure an increased degree of MuSK dimerization, and acetylcholine receptor (AChR) clustering assays. In some embodiments, an AChR clustering assay is performed using cultured muscle cells, e.g., myoblasts and myotubes. Myoblasts may be induced to differentiate into myotubes by, for example, growing cells under a low-serum condition. In some embodiments, the cultured muscle cells are of murine origin. In some embodiments, cells from an animal model known to manifest neuromuscular junction defects can be used in the assay, where the ability of test antibodies to induce AChR clustering can be examined. For example, cells derived from agrin-deficient mice may be used, where exogenously added recombinant agrin may serve as a positive control. In some embodiments, the cultured muscle cells are of human origin, such as primary human cells. In preferred embodiments, antibodies or antigen-binding fragments with sufficiently high potency (e.g., EC50 of single nM or sub-nM range) are selected for further evaluation.

[0732]

[0349] Antibodies or antigen-binding fragments shown to have desirable binding properties and potency may be tested in vivo. Typically, in vivo studies are conducted in well-characterized preclinical animal species such as mice, rats, and cynos, but any suitable animal models can be employed. Preferably, a suitable preclinical model is selected on the basis of previously characterized defects in NMJs. Non-limiting examples of preclinical models that manifest a defect in NMJs include mice carrying a mutation in agrin (e.g., agrin-deficient mice), mice carrying a mutation in Irp4, and mice

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[0735] carrying a mutation in musk. Examples of preclinical models that manifest NMJ defects include one or more mutations in Agrin, such as AgrnNMF380.

[0736]

[0350] In some embodiments, a preclinical model that manifests NMJ disruptions comprises one or more mutations in TDP-43. Non-limiting examples of TDP-43 mutant models include PRP-TDP43A315Tmodel (described in www.jax.org / strain / 010700) and hTDP-43ANLS model (described in www. jax.org / strain / 014650, and www.biospective.com / resources / tdp-43-deltaNLS-mice-for-als-drug-development).

[0737]

[0351] In some embodiments, a preclinical model of ALS is a SOD1-G93A model.

[0738]

[0352] Using such an animal model, test MuSK antibodies or antigen-binding fragments can be tested in vivo for the ability to improve the mutation-related defects, as compared to control (e.g., IgG control). In some embodiments, effects of such antibody on the NMJ are evaluated, e.g., ability to alleviate or rescue the defect in NMJs of the animal. In some embodiments, histology assessments are carried out, including for example immunohistochemistry of select muscle tissues, preferably well characterized muscle groups such as gastrocnemius (gastroc) or a tibialis anterior (TA), as well as diaphragm, where the architecture of NMJs can be readily examined. In some embodiments, changes in body weight of test animals can be monitored to indicate overall health or deterioration, Animals treated with effective MuSK agonists may retain or gain body weight in mutant mice, indicative of efficacy. In some embodiments, specific muscle tissues (muscle groups) can be isolated at study end and weighed. Enhanced muscle weight (e.g., larger muscles) may indicate healthier muscle, while atrophied muscle weighs less. Preferably, well characterized muscle groups such as gastrocnemius (gastroc), tibialis anterior (TA), and / or diaphragm, are examined. In preferred embodiments, effects of test antibodies on muscle function or motor function of the animals are evaluated. Examples of muscle function assessment include, without limitation, muscle force measurement (e.g., maximum force generated at given stimulation frequencies, electrophysiological response, etc.), such measurements may be performed using isolated muscle tissues (ex vivo) or in whole animal (in vivo). Examples of motor function assessment include, without limitation, grip strength tests, locomotive assessment, etc. In some embodiments, effects of test MuSK antibodies or antigen-binding fragments are reflected in statistical changes in the survival rate of the test animals. Based on one or more of efficacy assessments outlined above, suitable MuSK agonist antibody or antibodies can be selected.

[0739]

[0353] Beyond efficacy and safety, the process of identifying a preferred MuSK antibody or fragments thereof, can include protein stability assessment. In some embodiments, protein stability is thermal stability (i.e., thermostability) of the antibody or a fragment(s) thereof. In some embodiments, a differential scanning fluorimetry (DSF) assay is performed to measure Tm of test antibodies. In some embodiments, fragments of test antibodies are used for the assay, such as fragments containing a CH3 domain of the antibodies. In some embodiments, the fragment is a Fab-CH3 fragment of the antibody. Preferable, the Tm of a test antibody fragment is above 68°C. More preferably, the Tm of a test antibody fragment is above 70°C, such as between 70-74°C. More preferably, the Tm is 75°C or higher.

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[0742] Therapeutic use of MuSK agonist antibodies

[0743]

[0354] Each motor unit is comprised of a motor neuron and the skeletal muscle fibers innervated by the neuron's axon terminals. In certain neuromuscular disorders, such as SMA, the underlining defect is in the motor neuron, where mutations in neuronal protein(s) can cause synaptic dysregulation, eventually resulting in denervation of the target muscle, even though the muscle itself is inherently relatively normal. In other neuromuscular disorders, such as muscular dystrophies, genetic defects that drive the pathology exist in the target muscle. Yet in certain neuromuscular disorders, dysregulation of synaptic transmission leads to structural and functional impairments of the neuromuscular junction. Thus, therapies that correct underlining genetic defects, therapies that enhance muscle mass and function, and therapies that stabilize the NMJ are contemplated herein to address neuromuscular impairments in diseases that manifest motor neuron and / or muscle-associated abnormalities. The invention therefore takes into consideration that the motor neuron-NMJ-target muscle axis works as a unit and that both anterograde and retrograde signaling can impact the process. With this backdrop, therapeutic use of MuSK antibodies either alone or in combination with other therapies that address one or more of the components of the motor unit are encompassed herein

[0744]

[0355] In one aspect, the invention provides a MuSK agonist for use in the treatment of a neuromuscular disease / disorder in a subject. In one embodiment, the invention provides a MuSK agonist for use in the treatment of a muscle condition in a subject. Such conditions that may be treated with MuSK antibodies such as those disclosed herein are provided below. Subjects who are diagnosed with or otherwise suffer from one or more of these conditions may benefit from a treatment that includes an anti-MuSK antibody such as those disclosed herein.

[0745]

[0356] Non-limiting examples of neuromuscular diseases that primarily affect muscle are listed below.

[0746] Dystrophies

[0747]

[0357] Muscular dystrophies include several categories: dystrophinopathies, limb girdle muscular dystrophies, congenital muscular dystrophies, distal muscular dystrophies, myofibrillar myopathy, as well as other types of muscular dystrophies. Each of these categories is discussed further below. a) Dystrophinopathies

[0748]

[0358] Dystrophinopathies include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, and DMD-associated dilated cardiomyopathy.

[0749] b) Limb girdle muscular dystrophies (LGMD)

[0750]

[0359] LGMDs as defined by the European Neuromuscular Centre in 2018, are named by the following system: LGMD, recessive or dominant inheritance (R or D), order of discovery (number), affected protein. Examples of LGMSs include the following: LGMD D1 DNAJB6-related; LGMD D2 TNP03-related; LGMD D3 HNRNPDL-related; LGMD D4 calpain3-related; LGMD D5 collagen 6-related; LGMD R1 calpain3-related (Calpainopathy); LGMD R2 dysferlin-related; LGMD R3 a-sarcoglycan-related; LGMD R4 p-sarcoglycan-related; LGMD R5 y-sarcoglycan-related; LGMD R65-sarcoglycan-related; LGMD R7 telethonin-related; LGMD R8 TRIM 32-related; LGMD R9 FKRP-related; LGMD R10 titin-related; LGMD R11 POMT1-related; LGMD R12 anoctamin5-related; LGMD R13 Fukutin-related; LGMD R14 POMT2-related; LGMD R15 POMGnTI -related; LGMD R16 a-dystroglycan-related; LGMD R17 plectin-related; LGMD R18 TRAPPC11 -related; LGMD R19

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[0753] GMPPB-related; LGMD R20 ISPD-related; LGMD R21 POGLUT1 -related; LGMD R22 collagen 6-related; LGMD R23 laminin a2-related; and, LGMD R24 POMGNT2-related.

[0754] c) Congenital muscular dystrophies

[0755]

[0360] Congenital muscular dystrophies include the following: LAMA2-related (merosin deficient) congenital muscular dystrophy (Emery-Dreifuss muscular dystrophy); Collagen Vl-related muscular dystrophy (Bethlem myopathy, Ullrich congenital muscular dystrophy); a-Dystroglycanopathies (Walker-Warburg syndrome, muscle-eye-brain disease); Laminopathies; Fukuyama CMD; and, Rigid spine syndrome.

[0756] d) Distal muscular dystrophy,

[0757]

[0361] Distal muscular dystrophy, also known as distal myopathy, refers to any muscle disease that preferentially affects the hands and / or feet. Examples include: Late adult-onset type 1; Late adultonset type 2a; Late adult-onset type 2b; Early adult-onset type 1 ; Early adult-onset type 2; and, Early adult-onset type 3.

[0758] e) Myofibrillar myopathies

[0759]

[0362] Myofibrillar myopathies are diseases that share histological similarities in affected muscle and include: Desminopathy; Myotilinopathy; Zaspopathy; Filaminopathy; and, Bag3opathy (BAG3-related myofibrillar myopathy).

[0760] f) Others

[0761]

[0363] Other muscular dystrophies include but are not limited to: Myotonic dystrophy;

[0762] Facioscapulohumeral muscular dystrophy (FSHD); Oculopharyngeal muscular dystrophy (OPMD); and, Emery-Dreifuss muscular dystrophy (EDMD).

[0763] Congenital myopathies

[0764]

[0364] Examples of congenital myopathies include: Nemaline myopathy; Central core myopathy; Centronuclear myopathy; Congenital fiber type disproportion; Multi / minicore myopathy; and, Cylindrical spirals myopathy.

[0765] Myopathies associated with metabolic diseases

[0766]

[0365] Mutations causing defects in metabolism can cause muscle damage due to inadequate energy for muscles or accumulation of waste products. Several categories of such conditions are as follows. a) Mitochondrial myopathies are diseases caused by mutations related to mitochondria, and thus are generally inherited from the mother with variable expressivity due to heteroplasmy. These include: Kearns-Sayre syndrome; Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS); Myoclonic epilepsy with ragged red fibers (MERRF); Cytochrome c oxidase (COX) deficiency; Mitochondrial complex I deficiency; Mitochondrial complex II deficiency; Mitochondrial complex III deficiency (cytochrome b deficiency); and, mtDNA deletion.

[0767] b) Myopathies associated with glycogen storage diseases (GSD) are a group of diseases caused by mutations related to glycogen metabolism and include: GSD type II (Pompe disease); GSD type V (McArdle disease); GSD type VII (Tarui disease); GSD type XI (Lactate dehydrogenase deficiency); GSD type X (Phosphoglycerate mutase deficiency); and, Phosphoglycerate kinase deficiency. c) Myopathies associated with Fat oxidation defect include: ; Carnitine palmitoyltransferase I deficiency; Carnitine palmitoyltransferase II (CPTII or CPT2) deficiency; Medium-chain acyl-coenzyme

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[0770] A dehydrogenase deficiency; Long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency; and. Very long-chain acyl-coenzyme A dehydrogenase deficiency.

[0771] d) Other metabolic myopathies include: Myoadenylate deaminase (MADA) deficiency.

[0772] Inflammatory myopathies

[0773]

[0366] Myopathies associated with inflammation include: Inclusion body myositis; Dermatomyositis; Polymyositis; and, Statin-associated autoimmune myopathy.

[0774] Other types of myopathies

[0775]

[0367] Other diseases of muscle that do not squarely fall into the above categories include rippling muscle disease and myopathies associated with drugs / medications.

[0776]

[0368] Certain medications have been reported to cause myopathies, e.g., muscle weakness.

[0777] Examples of drug-induced myopathies are provided below (reviewed in Miernik et al. 2024, “Drug-induced myopathies: A comprehensive review and update” Biomedicines, 12(5): 987).

[0778] a) Statins / hypolipemic drugs

[0779]

[0369] The medications most associated with muscle side effects are p-Hydroxy p-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins). These drugs have a strong potential to reduce the risk of cardiovascular disease, the most common cause of death in developed societies.

[0780] Unfortunately, neuromuscular symptoms are the most common cause of withdrawal of these medications. A significant fraction of patients treated with statins report muscle-related side effects. A higher incidence of myotoxicity is seen with the use of lipophilic statins compared to hydrophilic ones and with higher doses of the drug. According to the FDA (Federal Drug Administration) Adverse Effects Reporting System (AERS), the risk of serious adverse effects such as rhabdomyolysis is several times greater with the use of lovastatin, simvastatin, and atorvastatin compared to pravastatin, rosuvastatin, or fluvastatin. Concomitant use of fibrates increases the risk by 10 times (Tomaszewski M., Stepien K.M., Tomaszewska J., Czuczwar S.J. Statin-induced myopathies. Pharmacol. Rep. 2011;63:859-866. doi: 10.1016 / s1734-1140(11 )70601 -6). Statin-related myotoxicity includes necrotizing myositis, which is associated with the presence of anti-HMG-CoA reductase (HMGCR) antibodies in the body. The main risk factors for muscle-related side effects are elderly age, female gender, Asian origin, concomitant use of drugs that affect statin metabolism (e.g., macrolides, warfarin, cyclosporin, azole antifungals, diltiazem, or fibrates), increased physical activity, hepatopathy, chronic renal disease, hypothyroidism, vitamin D deficiency, or metabolic syndrome. Statin-associated muscle symptoms may be assessed by known guidelines, such as The Statin-Associated Muscle Symptom Clinical Index (SAMS-CI) and Statin Experience Assessment Questionnaire (SEAQ). A strong association between the presence of the HLADRB1 *11 :01 allele and statin-related immune-mediated necrotizing myopathy (IMNM) has been reported, reaching up to 70%. Therefore, patients with this genetic background are at higher risk of developing statin-induced myopathies.

[0781] b) Beta blockers

[0782]

[0370] Beta blockers which are used as cardiological therapy, are known to cause muscular symptoms, such as muscle cramps, weakness and myalgia. Examples of beta blockers include sotalol, propranolol, labetalol, pindolol, carteolol and amiodarone.

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[0785] c) Colchicine

[0786]

[0371] Myopathy associated with colchicine use is a well-known clinical feature. Its symptoms resemble polymyositis with decreased proximal muscle strength, myalgia, and increased CK activity. Associated neuropathy is often present. There is a higher risk of colchicine-induced myopathy in patients with chronic kidney disease. Histopathologically, a picture of lysosomal, vacuolar myopathy is present, with few necrotic fibers. The only type I muscle fibers are vacuolized. Therefore, patients with conditions that primarily impact type I fibers are at higher risk of colchicine-induced myopathies. d) Steroids

[0787]

[0372] Two types of steroid-induced myopathies have been described. The first is uncommon and typically concerns critically ill patients, and has the presentation of acute quadriplegic myopathy (AQM). It occurs mainly in critically ill patients with status asthmaticus or other disorders requiring intensive care, and often in those treated with nondepolarizing muscle-blocking agents (NMBAs) to enable mechanical ventilation. Although AQM mainly affects patients receiving large doses of intravenous corticosteroids, relatively low doses, and even a single dose, have been implicated in case reports.

[0788]

[0373] The second, more frequent type, is associated with chronic / long-term use of glucocorticosteroids (GCS). Mostly, the onset of myopathy is insidious, and the first manifestations are usually a decrease in muscle strength, and difficulty climbing stairs or rising from a sitting position, but less often myalgia. Symptoms usually initially affect the proximal muscles of the lower limbs symmetrically, and then symptoms from the shoulder girdle may also join. A Cushingoid body appearance is predominantly present. The risk of myopathy is higher with doses higher than 40 mg / day of prednisone equivalent. Type II muscle fiber atrophy is present. These symptoms may be alleviated by discontinuation, dose reduction. In some cases, conversion of GCS to non-fluorinated drugs (e.g., prednisolone, hydrocortisone) rather than fluorinated drugs (e.g., triamcinolone, dexamethasone) may also lessen the symptoms.

[0789] e) Anti-malaria drugs

[0790]

[0374] Use of antimalaria drugs such as chloroquine and hydroxychloroquine is associated with myopathy. According to published reports, e incidence of antimalarial myopathy (defined as the presence of microscopic changes with elevated muscle enzyme activity) was 12.6% among patients treated at their rheumatology unit. Of these, about half presented with decreased muscle strength, mostly of mild to moderate severity.

[0791] f) Immunosuppressants / Cyclosporine A

[0792]

[0375] Cyclosporine is a calcineurin inhibitor used as an immunosuppressant, which is most often prescribed for the prevention of transplant rejection. Some patients treated with cyclosporine A experience myopathy characterized by myalgia, muscle weakness and an increase in creatine kinase activity. These manifestations are thought to be associated with mitochondrial dysfunction. Thus, patients treated with immunosuppressive agents such as cyclosporine are at risk of developing drug-induced myopathy.

[0793] g) Reverse transcriptase inhibitors / Zidovudine

[0794]

[0376] Reverse transcriptase inhibitors such as zidovudine are often used to treat retroviral infection such as HIV. Long-term therapy with zidovudine has been reported to lead to mitochondrial myopathy, which more severely affects type I fibers.

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[0797] h) Chelating agents / D-penicillamine

[0798]

[0377] Penicillamine is a chelating agent used in the treatment of Wilson's disease. It is also used to reduce cystine excretion in cystinuria and to treat patients with severe, active rheumatoid arthritis unresponsive to conventional therapy. Penicillamine is used as a form of immunosuppression to treat rheumatoid arthritis. Side effects include increased risk of developing an inflammatory myopathy resembling polymyositis or dermatomyositis. A possible complication includes swallowing difficulty. I) Checkpoint inhibitors

[0799]

[0378] Although immune checkpoint inhibitors are effective in treating a variety of cancer types, they trigger immune stimulation which can cause unwanted inflammation. A common side effect of checkpoint inhibitors is muscle pain, muscle weakness, myasthenia gravis, myalgia and myositis. Myocarditis is a frequent complication of checkpoint inhibitor-associated myositis. The positivity rate of autoantibodies to the acetylcholine receptor (AChR) is estimated to be about 50% in patients with immune checkpoint inhibitor-induced myasthenia gravis.

[0800] j) GLP-1 receptor agonists

[0801]

[0379] GLP-1 receptor agonists are effective weight loss drugs, but a substantial portion of the weight loss is due to muscle loss. Thus, patients receiving GLP-1 RAtherapy are at higher risk of muscle wasting. More recently, similar detrimental effects are implicated in the homeostasis of bone, e.g., reduced bone mineral density, frequent fractures, etc. Thus, patients on incretin-based therapy, such as GLP-1 RA, GIP and / or glucagon therapies, are more prone to losing muscle and / or bone.

[0802] k) Hormone therapies

[0803]

[0380] Hormone therapies such as androgen deprivation therapy are typically used as part of prostate cancer treatment and are associated with muscle atrophy.

[0804]

[0381] Non-limiting examples of neuromuscular diseases that primarily affect the nerve (e.g., motor neurons) are listed below.

[0805]

[0382] Neuromuscular diseases affecting nerve include, for example: Troyer syndrome; Cramp fasciculation syndrome; Hereditary spastic paraplegia; Spinocerebellar ataxia; and, Spinal and bulbar muscular atrophy.

[0806] Neuronopathies

[0807]

[0383] Neuronopathies represent a category of nerve-affecting neuromuscular diseases that primarily affects the cell body (soma) of a nerve cell (i.e., neuron) in the peripheral nervous system (e.g., motor neurons). Examples include, without limitation: Amyotrophic lateral sclerosis (ALS); Spinal muscular atrophy (SMA); Spinal muscular atrophy with respiratory distress type 1; Atypical motor neuron diseases; and, Dorsal root ganglion disorders.

[0808]

[0384] In SMA, a genetic defect associated with the motor neuron protein SMN underlies the progressive degeneration of motor neurons, leading to muscle atrophies. Currently approved therapies aim to increase the amount of functional SMN protein by upregulating the expression of smn2 or replacing the missing smn1. Clinical data have shown that these so-called SMN-targeted therapies can slow the process of neuronal deg...

Claims

SR64-WO-PCT / 127036-05220CLAIMS1. An antibody or antigen-binding fragment thereof that binds and activates human musclespecific kinase (MuSK), wherein the antibody or antigen-binding fragment specifically binds a MuSK fragment consisting of SEQ ID NO: 7 but does not specifically bind a MuSK fragment consisting of SEQ ID NO: 8.

2. The antibody or antigen-binding fragment of claim 1 , wherein the antibody or antigen-binding fragment binds an epitope that comprises one or more amino acid residues of SEQ ID NO: 9.

3. The antibody or antigen-binding fragment of any one of the preceding claims, which has a bivalent binding affinity (kD) for human MuSK of: a) < 10 nM, as measured by BioLayer Interferometry (BLI)-based in vitro binding assay; and / or, b) < 2.5 nM, as measured by Surface Plasmon Resonance (SPR)-based in vitro binding assay.

4. The antibody or antigen-binding fragment of any one of the preceding claims, which has an ECso of < 2.5 nM, as measured by a C2C12 myotube AChR clustering assay and / or an ECso of < 1.0 nM, as measured by a human myotube AChR clustering assay.

5. The antibody or antigen-binding fragments according to any one of the preceding claims, comprising a set of 6 CDRs selected from Table 6 and / or a set of VH / VL sequences selected from Table 7.

6. The antibody or antigen-binding fragments according to any one of the preceding claims, which cross-competes with any one of the antibodies or antigen-binding fragments of claim 5 for binding to human MuSK, optionally wherein the antibody, or antigen-binding fragment thereof, crosscompetes with an antibody or antigen-binding fragment comprising a VH sequence comprising the amino acid sequence set forth in SEQ ID NO: 210, and a VL sequence comprising the amino acid sequence set forth in SEQ ID NO: 211.

7. The antibody or antigen-binding fragments according to any one of the preceding claims, which contains one or more substitution(s) within at least one of the six CDRs of an antibody selected from Table 6.

8. The antibody of any one of the preceding claims, which is a human or humanized antibody.

9. The antibody of claim 8, which is a human IgG 1 or human lgG4 isotype, wherein optionally the antibody of human lgG4 isotype comprises a Ser-to-Pro (S228P) hinge substitution.

10. A method for identifying a MuSK agonist antibody or antigen-binding fragment thereof, the method comprising the steps of:i) screening for antibodies or antigen-binding fragments for the ability to specifically bind a recombinant antigen comprising a human MuSK Truncated Tail sequence as set forth in SEQ ID NO: 9, wherein optionally the antibody or the antigen-binding fragment binds the recombinant antigen with135MEl\59597320.vlSR64-WO-PCT / 127036-05220a KD of 1 nM or less as measured in an in vitro binding assay, wherein further optionally the in vitro binding assay comprises a BLI-based technique or SPR-based technique;ii) carrying out an AChR clustering assay to select from step (i) one or more antibodies or antigen-binding fragments capable of inducing AChR clustering with an EC50 of 1 nM or less, thereby identifying a MuSK agonist antibody or an antigen-binding fragment thereof.

11. The method of claim 10, wherein the recombinant antigen further comprises a human Fz-like domain sequence of SEQ ID NO: 8 or a portion thereof.

12. The method of claim 10, wherein the recombinant antigen is a fragment of human MuSK as set forth in SEQ ID NO: 173 or a portion thereof, wherein optionally the recombinant antigen is a fragment of human MuSK as set forth in SEQ ID NO: 7.13 The method of any one of claims 10-12, wherein the recombinant antigen does not comprise an Ig1 domain sequence, an Ig2 domain sequence, and an Ig3 domain sequence of human MuSK.

14. The method of any one of claims 10-13, wherein the AChR clustering assay of step (ii) comprises a cell-based in vitro potency assay, wherein optionally the cell-based potency assay comprises cultured myoblasts or myotubes.

15. The method of any one of claims 10-14, further comprising carrying out an in vivo study in a preclinical model that manifests a defect in neuromuscular junctions (NMJs) to evaluate the ability of the antibody, relative to control:a) to alleviate or rescue the defect in NMJs;b) to have enhanced body weight;c) to have greater muscle weight at study end;d) to have enhanced muscle or motor function; and / ore) for improved survival.

16. The method of any one of claims 10-15, further comprising determining thermostability of the antibody or a fragment thereof in a differential scanning fluorimetry (DSF) assay.136MEl\59597320.vl

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