Musk inhibition

Abstract

To provide novel methods for treating conditions, disorders and / or symptoms which are alleviated by the inhibition of neuromuscular transmission in subjects.SOLUTION: The present invention provides binding agents for use in treating the conditions, disorders and / or symptoms which are alleviated by the inhibition of neuromuscular transmission in subjects.SELECTED DRAWING: None

Description

[Technical field]

[0001] Provided herein are novel methods for treating diseases, disorders, and / or conditions in a subject that are alleviated by inhibition of MuSK-mediated neuromuscular transmission. The invention also provides binding agents for use in treating the above diseases, disorders, and / or conditions. [Background technology]

[0002] The neuromuscular junction (NMJ) is where the motor neuron contacts the muscle fiber. At this site, the motor neuron and muscle fiber come together to create a specialized structure that allows two-way communication between the two cells. The motor neuron tells the muscle to contract. Several signaling cascades are involved in controlling how this communication occurs. At the heart of neuromuscular transmission is the release of acetylcholine (ACh) by the motor neuron terminals. ACh diffuses through the synaptic cleft to the muscle fiber. There, ACh binds to ACh receptors (AChRs) on the muscle fiber membrane. This generates an action potential in the muscle membrane and ultimately muscle fiber contraction.

[0003] Sufficient release of ACh and the presence of densely packed AChRs are prerequisites for successful neuromuscular transmission and muscle contraction. The agrin-low density lipoprotein receptor-related protein 4 (Lrp4)-muscle-specific kinase (MuSK) signaling cascade is a key regulator of AChR clustering. This signaling cascade is important for the establishment and maintenance of neuromuscular synapses. MuSK internally transduces extracellular signals to promote AChR clustering. Release of ACh and binding to AChRs mediate muscle contraction.

[0004] Many different substances that affect neuromuscular transmission are known and are commonly used in cosmetic and therapeutic situations. A well-known example is the botulinum neurotoxin, which is currently used for several therapeutic and non-therapeutic applications (e.g., to reduce wrinkles or treat muscle spasms). However, due to the extreme toxicity and inherent immunogenicity of these toxins, their use is typically restricted to administration at extremely low doses to avoid or minimize side effects, such as unwanted paralysis and / or host immune responses (Naumann, M., Boo, LM, Ackerman, AH & Gallagher, CJ J Neural Transm (Vienna) 120, 275-290, (2013)).

[0005] Botulinum neurotoxin type A (BoNT / A) is the active substance in preparations currently used for the treatment of several diseases, disorders, and / or conditions that are alleviated by the inhibition of neuromuscular transmission. Botulinum toxin is a biological preparation derived from the bacterium Clostridium botulinum type A. In most treatments, BoNT / A is part of a complex with other proteins. Therefore, modifications in the composition of these products cannot be excluded (see, for example, Botox® / Vistavel®, Dysport® / Azalure®, and Xeomin® / Vocauture®, Frevert J. Drugs R D. 2010;10(2):67-73).

[0006] Treatment with botulinum toxin is also known to have many side effects, including temporary fatigue, dysphagia, neck weakness, hoarseness, and localized pain. Furthermore, many individuals who initially respond to botulinum toxin treatment subsequently become non-responsive to the treatment. Thus, for many individuals, botulinum injections do not provide a satisfactory long-term treatment of the disease. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 97 / 21811 [Non-patent literature]

[0008] [Non-Patent Document 1] Naumann, M., Boo, LM, Ackerman, AH & Gallagher, CJ J Neural Transm (Vienna) 120, 275-290, (2013) [Non-Patent Document 2] FrevertJ.Drugs R D.2010;10(2):67-73 Summary of the Invention [Problem to be solved by the invention]

[0009] There is a need for better blockers of neuromuscular transmission for cosmetic and / or therapeutic use. [Means for solving the problem]

[0010] The present inventors isolated and characterized several MuSK monoclonal antibodies from patients with autoimmune myasthenia gravis (MG) and tested their functional properties to further understand the pathomechanisms of MuSK MG.

[0011] MG is the most common disorder of the neuromuscular synapse, affecting 10 to 20 per 100,000 people in the United States. MG is a debilitating autoimmune disease in which autoantibodies against NMJ proteins impair neuromuscular transmission and cause fatigable muscle weakness. Depending on the subtype of MG, certain muscle subsets are more susceptible to autoimmune attacks, but all skeletal muscles can be affected. Approximately 80% of patients have autoantibodies against muscle nicotinic AChRs, resulting in AChR MG. In most patients, the first symptom is weakness of the extraocular muscles, and if the disease progresses, ocular or generalized weakness of skeletal muscles also occurs. The pathological mechanism by which AChG autoantibodies cause MG is strongly related to the autoantibody isotypes; immunoglobulin (IgG) 1 and IgG3. IgG1 and IgG3 are proinflammatory antibodies that can activate complement, bind to Fc receptors on immune cells, and cross-link and internalize antigens. Furthermore, these AChR antibodies cross-link AChR and induce conformational changes in AChR, resulting in antigen modulation of the AChR, blocking the binding site for ACh, or altering the affinity for ACh. These effector functions contribute overall to disease in AChR MG by decreasing the availability of functional AChRs, resulting in dysfunction and degradation of the NMJ.

[0012] Approximately 5% of MG patients have autoantibodies against MuSK, resulting in MuSK MG (Hoch et al., 2001). MuSK regulates AChR clustering and sub-synaptic gene expression, and thus MuSK is essential for the formation and maintenance of NMJs (Burden et al., 2018). Eye and respiratory muscles are particularly affected by MuSK autoantibodies, which can lead to respiratory crisis in approximately 40% of these patients (Evoli et al., 2003). In contrast to AChR MG, autoantibodies in MuSK MG are predominantly of the IgG4 isotype (McConville et al., 2004). Epitope mapping using polyclonal serum antibodies showed that disease severity correlated with IgG4 activity against the N-terminal Ig-like 1 domain of MuSK (Huijbers et al., 2016). Furthermore, passive transfer of purified human polyclonal IgG4 from MuSK MG patients induced MG in immunodeficient mice, thus confirming the pathogenic nature of these autoantibodies, but not IgG1-3 (Klooster et al., 2012). In vitro studies showed that plasma-purified MuSK IgG4 autoantibodies blocked MuSK-Lrp4 interaction, thereby inducing AChR declustering, resulting in impaired neuromuscular transmission and MG (Huijbers & Zhang 2013, Koneczny et al., 2013, Otsuka et al., 2015).

[0013] We have generated and characterized recombinant MuSK antibodies at the genetic and functional levels from clonal MuSK-specific memory B cell cultures derived from MuSK MG patients. These include IgG1, IgG3, and IgG4 antibodies using distinct heavy and light chain variable region genes that have undergone high levels of affinity maturation in the complementarity determining regions (CDRs) and matched antigen selection. Binding experiments confirmed their specificity for the Ig-like 1 domain of MuSK and their affinity to the mouse NMJ.

[0014] It is known that IgG4 autoantibodies that bind to the Ig-like 1 domain of MuSK can cause the disease MuSK MG. IgG4 is initially generated in vivo in a bivalent monospecific form (i.e., it has two variable regions specific for the same target antigen). Then, over time, IgG4 undergoes exchange of IgG halves (a process literally and herein called Fab-arm exchange, see below), resulting in a pool of bivalent bispecific IgG4 molecules in vivo (i.e., it has two variable regions specific for different target antigens). Thus, over time in vivo, IgG4 becomes functionally monovalent for the original target antigen (i.e., it still has two variable regions, but only one variable region that specifically binds to the original target (in this case, the Ig-like 1 domain of MuSK)). This is a rapid process that occurs within 24 hours for more than 99% of the IgG4 pool (provided that all prerequisites for Fab arm exchange are met, i.e., the required residues are present in the Fc tail of the IgG4).

[0015] The inventors have surprisingly found that MuSK antibodies only inhibit agrin-induced MuSK phosphorylation (and inhibit MuSK dimerization and activation) when they are functionally monovalent with respect to the Ig-like 1 domain of MuSK (i.e., when they possess only one variable region that specifically binds to the Ig-like 1 domain of MuSK). The data provided herein demonstrate for the first time that IgG4 autoantibodies found in MuSK MG patients are only inhibitory in nature when they become functionally monovalent (and thus in vivo after undergoing Fab arm exchange). Thus, the ability of the patient's IgG4 MuSK antibodies to undergo Fab arm exchange appears to be crucial for disease development, since Fab arm exchange renders endogenous MuSK IgG4 bispecific and functionally monovalent with respect to the Ig-like 1 domain of MuSK. Only in this state does IgG4 act as an inhibitor of MuSK activity.

[0016] The inventors have also surprisingly shown that agrin-independent MuSK phosphorylation is induced and increased when a MuSK antibody is bivalent and monospecific for the Ig-like 1 domain of MuSK (i.e., when the MuSK antibody has two variable regions which both specifically bind to the Ig-like 1 domain of MuSK). Thus, the inventors have shown that the valency of an antibody against the Ig-like 1 domain of MuSK determines whether the antibody is a MuSK antagonist (bivalent bispecific antibody, or monovalent monospecific antibody fragment) or an agonist (bivalent monospecific antibody).

[0017] The invention is completely surprising because the data presented herein show that agonistic, bivalent, monospecific MuSK Ig-1-like domain antibodies can be converted into antagonists against the same target antigen when only one variable region that specifically binds to the Ig-like 1 domain of MuSK is retained.

[0018] In one aspect, a binding agent comprising one or more binding regions, where only one binding region specifically binds to the Ig-like 1 domain of a MuSK protein, is provided for use in treating a disease, disorder, and / or condition in a subject that is alleviated by inhibition of neuromuscular transmission.

[0019] Advantageously, the binding agents described herein do not induce (eg are unable to induce) MuSK dimerization and / or phosphorylation and / or activation.

[0020] Suitably the binding agent is a binding protein.

[0021] Suitably, the region which specifically binds to the Ig-like 1 domain of the MuSK protein is a variable region.

[0022] Suitably the MuSK protein is a human MuSK protein.

[0023] Suitably the binding agent is monovalent.

[0024] Suitably the binding agent is bivalent or trivalent, in which case the bivalent or trivalent binding agent does not induce (eg is unable to induce) MuSK dimerisation or activation.

[0025] Suitably the binding agent is an antibody.

[0026] Suitably the antibody is a monoclonal antibody.

[0027] Suitably the antibody is a human or humanized antibody.

[0028] Suitably the antibody is selected from a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody.

[0029] Suitably, the antibody is an IgG. The IgG may be selected from IgG1, IgG2, or IgG3. Alternatively, the IgG may be an IgG4 variant that has a reduced capacity for, or is unable to exchange Fab arms in vivo.

[0030] Suitably the IgG4 variant comprises an IgG4 constant region which comprises one or more amino acid substitutions which reduce the capacity for, or prevent, Fab arm exchange in vivo.

[0031] Suitably, the IgG4 variant comprises an IgG4 constant region comprising an amino acid substitution at amino acid position 228, and / or an amino acid substitution at amino acid position 409, and / or an amino acid substitution at amino acid position 405 of the heavy chain, numbered according to the EU index.

[0032] Suitably the subject is a human.

[0033] Suitably, the disease, disorder and / or condition is selected from the following; (i) A distorted appearance due to excessive muscle activity, or (ii) Any disease, disorder, or condition caused by excessive muscle activity, including dystonia, facial spasms, strabismus, cerebral palsy, stuttering, chronic tension headaches, spasms of the inferior constrictor of the pharynx, pain, migraine headaches, involuntary spasms, muscle spasticity, strabismus, occupational spasms, anal fissures, teeth grinding, and any combination thereof.

[0034] Suitably, the single binding region that specifically binds to the Ig-like 1 domain of the MuSK protein has a sequence selected from the following: a) a VH CDR1 comprising SEQ ID NO: 10, a VH CDR2 comprising SEQ ID NO: 11, a VH CDR3 comprising SEQ ID NO: 12, a VL CDR1 comprising SEQ ID NO: 14, a VL CDR2 comprising SEQ ID NO: 15, and a VL CDR3 comprising SEQ ID NO: 16, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 9, and a light chain variable domain comprising SEQ ID NO: 13; b) a VH CDR1 comprising SEQ ID NO: 18, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 20, a VL CDR1 comprising SEQ ID NO: 22, a VL CDR2 comprising SEQ ID NO: 23, and a VL CDR3 comprising SEQ ID NO: 24, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 17, and a light chain variable domain comprising SEQ ID NO: 21; c) a VH CDR1 comprising SEQ ID NO: 26, a VH CDR2 comprising SEQ ID NO: 27, and a VH CDR3 comprising SEQ ID NO: 28, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 25; d) a VH CDR1 comprising SEQ ID NO: 30, a VH CDR2 comprising SEQ ID NO: 31, a VH CDR3 comprising SEQ ID NO: 32, a VL CDR1 comprising SEQ ID NO: 34, a VL CDR2 comprising SEQ ID NO: 35, and a VL CDR3 comprising SEQ ID NO: 36, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 29, and a light chain variable domain comprising SEQ ID NO: 33; e) a VH CDR1 comprising SEQ ID NO: 38, a VH CDR2 comprising SEQ ID NO: 39, a VH CDR3 comprising SEQ ID NO: 40, a VL CDR1 comprising SEQ ID NO: 42, a VL CDR2 comprising SEQ ID NO: 43, and a VL CDR3 comprising SEQ ID NO: 44, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 37, and a light chain variable domain comprising SEQ ID NO: 41; f) a VH CDR1 comprising SEQ ID NO: 46, a VH CDR2 comprising SEQ ID NO: 47, a VH CDR3 comprising SEQ ID NO: 48, a VL CDR1 comprising SEQ ID NO: 50, a VL CDR2 comprising SEQ ID NO: 51, and a VL CDR3 comprising SEQ ID NO: 52, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 45, and a light chain variable domain comprising SEQ ID NO: 49; g) a VH CDR1 comprising SEQ ID NO: 54, a VH CDR2 comprising SEQ ID NO: 55, a VH CDR3 comprising SEQ ID NO: 56, a VL CDR1 comprising SEQ ID NO: 58, a VL CDR2 comprising SEQ ID NO: 59, and a VL CDR3 comprising SEQ ID NO: 60, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 53, and a light chain variable domain comprising SEQ ID NO: 57; or h) Any combination of the above.

[0035] Also provided is a method for preventing, controlling, or reducing skin wrinkles in a subject, the method comprising administering to the subject a binding agent comprising one or more binding regions, where only one of the binding regions specifically binds to the Ig-like 1 domain of a MuSK protein.

[0036] Also provided is a method for treating or preventing a disease, disorder, and / or condition that is alleviated by inhibition of neuromuscular transmission, the method comprising administering to a subject a binding agent comprising one or more binding domains, where only one of the binding domains specifically binds to the Ig-like 1 domain of a MuSK protein.

[0037] Suitably, the disease, disorder and / or condition is selected from the following: (i) A distorted appearance due to excessive muscle activity, or (ii) Any disease, disorder, or condition caused by excessive muscle activity, including dystonia, facial spasms, strabismus, cerebral palsy, stuttering, chronic tension headaches, spasms of the hypopharyngeal constrictor muscles of the pharynx, pain, migraine headaches, involuntary spasms, muscle spasticity, strabismus, occupational spasms, anal fissures, teeth grinding, and any combination thereof.

[0038] Advantageously, the binding agents described herein do not induce (eg are unable to induce) MuSK dimerization and / or phosphorylation and / or activation.

[0039] Suitably the binding agent is a binding protein.

[0040] Suitably, the region which specifically binds to the Ig-like 1 domain of the MuSK protein is a variable region.

[0041] Suitably the MuSK protein is a human MuSK protein.

[0042] Suitably the binding agent is monovalent.

[0043] Suitably the binding agent is bivalent or trivalent, in which case the bivalent or trivalent binding agent does not induce (eg is unable to induce) MuSK dimerisation or activation.

[0044] Suitably the binding agent is an antibody.

[0045] Suitably the antibody is a monoclonal antibody.

[0046] Suitably the antibody is a human or humanized antibody.

[0047] Suitably the antibody is selected from a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody.

[0048] Suitably, the antibody is an IgG. The IgG may be selected from IgG1, IgG2, or IgG3. Alternatively, the IgG may be an IgG4 variant that has a reduced capacity for, or is unable to exchange Fab arms in vivo.

[0049] Suitably the IgG4 variant comprises an IgG4 constant region which comprises one or more amino acid substitutions which reduce or prevent the capacity for Fab arm exchange in vivo.

[0050] Suitably, the IgG4 variant comprises an IgG4 constant region comprising an amino acid substitution at amino acid position 228, and / or an amino acid substitution at amino acid position 409, and / or an amino acid substitution at amino acid position 405 of the heavy chain, numbered according to the EU index.

[0051] Suitably the subject is a human.

[0052] Suitably, the single binding region that specifically binds to the Ig-like 1 domain of the MuSK protein has a sequence selected from the following: a) a VH CDR1 comprising SEQ ID NO: 10, a VH CDR2 comprising SEQ ID NO: 11, a VH CDR3 comprising SEQ ID NO: 12, a VL CDR1 comprising SEQ ID NO: 14, a VL CDR2 comprising SEQ ID NO: 15, and a VL CDR3 comprising SEQ ID NO: 16, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 9, and a light chain variable domain comprising SEQ ID NO: 13; b) a VH CDR1 comprising SEQ ID NO: 18, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 20, a VL CDR1 comprising SEQ ID NO: 22, a VL CDR2 comprising SEQ ID NO: 23, and a VL CDR3 comprising SEQ ID NO: 24, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 17, and a light chain variable domain comprising SEQ ID NO: 21; c) a VH CDR1 comprising SEQ ID NO: 26, a VH CDR2 comprising SEQ ID NO: 27, and a VH CDR3 comprising SEQ ID NO: 28, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 25; d) a VH CDR1 comprising SEQ ID NO: 30, a VH CDR2 comprising SEQ ID NO: 31, a VH CDR3 comprising SEQ ID NO: 32, a VL CDR1 comprising SEQ ID NO: 34, a VL CDR2 comprising SEQ ID NO: 35, and a VL CDR3 comprising SEQ ID NO: 36, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 29, and a light chain variable domain comprising SEQ ID NO: 33; e) a VH CDR1 comprising SEQ ID NO: 38, a VH CDR2 comprising SEQ ID NO: 39, a VH CDR3 comprising SEQ ID NO: 40, a VL CDR1 comprising SEQ ID NO: 42, a VL CDR2 comprising SEQ ID NO: 43, and a VL CDR3 comprising SEQ ID NO: 44, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 37, and a light chain variable domain comprising SEQ ID NO: 41; f) a VH CDR1 comprising SEQ ID NO: 46, a VH CDR2 comprising SEQ ID NO: 47, a VH CDR3 comprising SEQ ID NO: 48, a VL CDR1 comprising SEQ ID NO: 50, a VL CDR2 comprising SEQ ID NO: 51, and a VL CDR3 comprising SEQ ID NO: 52, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 45, and a light chain variable domain comprising SEQ ID NO: 49; g) a VH CDR1 comprising SEQ ID NO: 54, a VH CDR2 comprising SEQ ID NO: 55, a VH CDR3 comprising SEQ ID NO: 56, a VL CDR1 comprising SEQ ID NO: 58, a VL CDR2 comprising SEQ ID NO: 59, and a VL CDR3 comprising SEQ ID NO: 60, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 53, and a light chain variable domain comprising SEQ ID NO: 57; or h) Any combination of the above.

[0053] In another embodiment, an antibody is provided comprising one or more binding regions, where only one of the binding regions specifically binds to the Ig-like 1 domain of the MuSK protein, and the only binding region that specifically binds to the Ig-like 1 domain of the MuSK protein has a sequence selected from the following: a) a VH CDR1 comprising SEQ ID NO: 10, a VH CDR2 comprising SEQ ID NO: 11, a VH CDR3 comprising SEQ ID NO: 12, a VL CDR1 comprising SEQ ID NO: 14, a VL CDR2 comprising SEQ ID NO: 15, and a VL CDR3 comprising SEQ ID NO: 16, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 9, and a light chain variable domain comprising SEQ ID NO: 13; b) a VH CDR1 comprising SEQ ID NO: 18, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 20, a VL CDR1 comprising SEQ ID NO: 22, a VL CDR2 comprising SEQ ID NO: 23, and a VL CDR3 comprising SEQ ID NO: 24, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 17, and a light chain variable domain comprising SEQ ID NO: 21; c) a VH CDR1 comprising SEQ ID NO: 26, a VH CDR2 comprising SEQ ID NO: 27, and a VH CDR3 comprising SEQ ID NO: 28, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 25; d) a VH CDR1 comprising SEQ ID NO: 30, a VH CDR2 comprising SEQ ID NO: 31, a VH CDR3 comprising SEQ ID NO: 32, a VL CDR1 comprising SEQ ID NO: 34, a VL CDR2 comprising SEQ ID NO: 35, and a VL CDR3 comprising SEQ ID NO: 36, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 29, and a light chain variable domain comprising SEQ ID NO: 33; e) a VH CDR1 comprising SEQ ID NO: 38, a VH CDR2 comprising SEQ ID NO: 39, a VH CDR3 comprising SEQ ID NO: 40, a VL CDR1 comprising SEQ ID NO: 42, a VL CDR2 comprising SEQ ID NO: 43, and a VL CDR3 comprising SEQ ID NO: 44, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 37, and a light chain variable domain comprising SEQ ID NO: 41; f) a VH CDR1 comprising SEQ ID NO: 46, a VH CDR2 comprising SEQ ID NO: 47, a VH CDR3 comprising SEQ ID NO: 48, a VL CDR1 comprising SEQ ID NO: 50, a VL CDR2 comprising SEQ ID NO: 51, and a VL CDR3 comprising SEQ ID NO: 52, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 45, and a light chain variable domain comprising SEQ ID NO: 49; g) a VH CDR1 comprising SEQ ID NO: 54, a VH CDR2 comprising SEQ ID NO: 55, a VH CDR3 comprising SEQ ID NO: 56, a VL CDR1 comprising SEQ ID NO: 58, a VL CDR2 comprising SEQ ID NO: 59, and a VL CDR3 comprising SEQ ID NO: 60, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 53, and a light chain variable domain comprising SEQ ID NO: 57; or h) Any combination of the above.

[0054] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations thereof mean "including but not limited to" and they are not intended to (and do not) exclude other moieties, additives, ingredients, integers, or steps.

[0055] Throughout the description and claims of this specification, the singular encompasses the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification is to be understood as contemplating the plural as well as the singular, unless the context requires otherwise.

[0056] It is to be understood that any feature, integer, property, compound, chemical moiety, or group described in connection with a particular aspect, embodiment, or example of the invention is applicable to any other aspect, embodiment, or example described herein, unless to the contrary.

[0057] Various aspects of the invention are described in further detail below. [Brief description of the drawings]

[0058] Embodiments of the present invention are further described below with reference to the accompanying drawings in which:

[0059] [Figure 1] Figure 1 shows that patient-derived recombinant MuSK antibody binds to mouse NMJs in whole mount levator auris longus muscle. Scale marker is 25 um. [Figure 2a]Figure 2a shows that patient-derived recombinant MuSK antibodies can activate or inhibit MuSK phosphorylation and AChR clustering depending on the antibody titer. Bivalent monospecific recombinant MuSK antibodies (clone # 11-3F6 and 13-3B5) activated MuSK phosphorylation in the presence and absence of agrin (A). Activation of MuSK phosphorylation was dose-dependent (B). Clone 13-3B5 was slightly more potent compared to 11-3F6. Biotin control antibodies did not affect (agrin-dependent) MuSK phosphorylation. Monovalent Fab fragments generated from these recombinant MuSK monoclonal antibodies inhibited MuSK phosphorylation (C). Agrin-dependent AChR clustering was not affected when exposed to biotin control antibodies or their Fab fragments (D). The bivalent monospecific recombinant IgG4 monoclonal MuSK antibody significantly increased AChR clustering compared to the Fab fragment derived from the same monoclonal antibody, although agrin-dependent AChR clustering remained lower than the anti-biotin control (for IgG4: P=0.0004, for Fab fragment: P=0.0001, corrected by one-way ANOVA Dunnett, 11-3F6 IgG4 vs Fab: P=0.0003, 13-3B5 IgG4 vs Fab: P=0.03, anti-biotin IgG4 vs Fab: P=0.3, unpaired t test). The Fab fragment reduced AChR clustering to the level of purified patient IgG4 and the "agrin-free" condition. Bivalent monospecific antibodies further significantly increased AChR clustering, independent of agrin (11-3F6: P=0.03, 13-3B5: P=0.2 one-way ANOVA Dunnett corrected). Data represent mean±SEM. Scale bar represents 50 μm. [Figure 2b] See Figure 2a. [Diagram 3]Figure 3 shows the MuSK reactivity results for eight B cell clones isolated from MuSK MG patients. MuSK was produced in yeast and E. coli. MuSK-reactive B cell clones isolated from MuSK MG patients and healthy donors were single cell sorted and differentiated into plasma cells. Media from these single cell cultures were tested for MuSK reactivity (produced in E. coli) in ELISA using subclass specific (anti-IgG1 or anti-IgG4 or anti-IgG total) secondary antibodies. In these experiments, eight MuSK-reactive B cell clones were isolated, seven of which produced B cell receptor (BCR) sequences. The level of reactivity to MuSK protein varied between clones. A number of wells with MuSK-non-reactive clones from the healthy donor cohort and from MuSK MG patients were also tested. One clone from a healthy donor appeared to have some reactivity to MuSK in this assay (11-1B3). [Figure 4] Figure 4 shows that the reactivity observed against MuSK clones isolated from MuSK MG patients was specific for MuSK, whereas the reactivity observed against a MuSK clone (11-1B3) isolated from a healthy donor was not. The specificity of MuSK reactivity was also tested for four MuSK MG clones compared to a control antigen (acetylcholine receptor alpha subunit) produced in the same system using yeast-produced MuSK. The "MuSK" positive clone (11-1B3) from a healthy donor showed no reactivity to yeast-produced MuSK, suggesting that this antibody / BCR sequence is not MuSK specific but recognizes a protein associated with E. coli. MuSK clones isolated from MuSK MG patients (11-3F6, 11-7C5, 11-8G4, and 11-3D9) showed clear reactivity to yeast-produced MuSK. [Figure 5a] FIG. 5a shows the sequence alignment using the Clustal W alignment program. [Figure 5b] See Figure 5a. [Figure 6] Anti-MuSK / anti-HIV-b12 exchange heterospecific IgG4 antibodies were generated using methods detailed in Labrijn, 2014, Nature Protocols and Genmab, Labrijn, 2013, PNAS, tailored to IgG4. These antibodies were tested for MuSK binding at the mouse neuromuscular junction (NMJ) in ex vivo levator auricle longus muscles. In parallel, equivalent anti-MuSK homospecific antibodies were also tested. The data confirm that both heterospecific MuSK IgG4 antibodies (13-3B5 / HIV IgG4 and 11-3F6 / HIV IgG4) and homospecific MuSK IgG4 antibodies (13-3B5 IgG4 and 11-3F6 IgG4) bind MuSK at the NMJ. [Figure 7] Heterospecific MuSK IgG4 antibodies cause inhibition of agrin-induced MuSK phosphorylation, whereas allospecific MuSK IgG4 antibodies activate phosphorylation in the absence of agrin. [Figure 8] Figure 8 shows that allospecific and xenospecific musk antibodies have different effects on neuromuscular performance in vivo. Nod / scid mice were injected intraperitoneally (Ip) with 5ug / gbw of recombinant antibodies on days 0, 3, and 7. Both xenospecific (functionally monovalent) clones induce rapid and profound muscle weakness and weight loss. Allospecific 11-3F6 IgG4 did not induce muscle weakness or weight loss. Ip = intraperitoneal, gbw = gram body weight. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] We used the variable domains of the MuSK antibody obtained from a MuSK MG patient to generate recombinant IgG1 and IgG4 antibodies that are bivalent and monospecific for the Ig-like 1 domain of MuSK.

[0061] Surprisingly, patient-derived recombinant MuSK monoclonal antibodies (both IgG1 and IgG4) activated rather than inhibited MuSK phosphorylation (Figure 2A). This effect was seen both in the absence and presence of agrin. Activation of MuSK phosphorylation was concentration-dependent (Figure 2B) and differed slightly between the two clones. This suggests that patient-derived bivalent monospecific MuSK antibodies that bind to the Ig-like 1 domain promote MuSK dimerization and activation in vitro.

[0062] However, both recombinant monoclonal IgG1 and IgG4 are involved in bivalent monospecific antibody-antigen interactions. To investigate the functional effects of bispecificity and functional monovalency of Fab-arm-exchanged IgG4 MuSK antibodies in patients, we generated monovalent Fab fragments from these recombinant antibodies by papain digestion. In vitro, these Fab fragments inhibited agrin-dependent MuSK phosphorylation (Figure 2C) and AChR clustering (Figure 2D), similar to anti-MuSK IgG4 from patient serum. In contrast (and consistent with activating MuSK phosphorylation in vitro), bivalent monospecific monoclonal MuSK antibodies activated agrin-dependent AChR clustering compared to Fab fragments derived from the same monoclonal antibodies. Moreover, AChR clustering could be partially induced using bivalent monospecific antibodies independently of agrin (Figure 2D). Thus, monovalent MuSK binding blocks the AChR clustering pathway, whereas bivalent monospecific MuSK antibodies can stimulate MuSK and promote or induce AChR clustering in this tissue culture model.

[0063] AChR clustering at the NMJ is crucial for successful neuromuscular transmission and muscle contraction (Burden et al., 2018). Lower levels of AChR clustering are tolerated in patients until a critical threshold is reached. For example, for MuSK MG, there is a dose-dependent decrease in AChR clustering in mice passively transferred with MuSK antibodies (Klooster et al., 2012). Thus, an increase in AChR clustering leads to improved neuromuscular transmission. Small increases in neuromuscular transmission (e.g., as exemplified by acetylcholinesterase therapy, which is the first-line symptomatic treatment for AChR MG patients) can be therapeutically effective in patients. Advantageously, the bivalent monospecific MuSK antibodies described herein can thus be used as therapeutic agents to improve AChR clustering and synaptic stability at the NMJ.

[0064] The present invention has been illustrated using antibodies. However, the general concept also applies to other binding agents that have binding regions specific for MuSK Ig-like 1 domain. By way of example, the data provided herein demonstrates that bivalent monospecific MuSK Ig-like 1 binding agents (i.e., binding agents that have two binding regions, both of which are specific for MuSK Ig-like 1 domain) can act as MuSK agonists (by inducing MuSK dimerization and / or phosphorylation and / or activation), while bivalent bispecific MuSK Ig-like 1 binding agents (i.e., binding agents that have two binding regions, only one of which is specific for MuSK Ig-like 1 domain) can act as MuSK antagonists (by preventing MuSK dimerization and / or phosphorylation and / or activation).

[0065] The invention described herein is based on the discovery that a binding agent comprising one or more binding domains, where only one binding domain specifically binds to the Ig-like 1 domain of the MuSK protein, can be used as a MuSK antagonist at the NMJ by inhibiting MuSK phosphorylation. Reduction of MuSK phosphorylation impairs neuromuscular transmission. Thus, the binding agent described herein can be used to induce temporary or long-term focal muscle weakness (paresis) in a subject. Advantageously, the binding agent described herein can thus be used as an alternative to botulinum toxin treatment.

[0066] The binding agents described herein have a number of advantages over other known neuromuscular transmission antagonists, such as botulinum toxins: · Botulinum toxin is a multiprotein biologic that varies in composition from batch to batch, making its activity difficult to predict or reproduce. In the EU alone, it is estimated that approximately 400,000 mice per year are used to test the efficacy of each new batch of Botox (Taylor et al., 2019, ALTEX 36(1), 81-90). The binding agents described herein can be produced under stringent GCLP requirements, e.g., as monoclonal antibodies, without any (or minimal) batch-to-batch variation. Advantageously, the binding agents described herein would therefore not require the same stringent batch-to-batch testing as botulinum toxin. Botulinum toxins act at the presynaptic neuronal site and can also be transported to the central nervous system by retrograde axonal transport, where they can produce unwanted side effects. The binding agents described herein are targeted to the postsynaptic muscle membrane, are unlikely to be internalized by motor neurons, and should not harm presynaptic neurons. The binding agents described herein may be a beneficial alternative for subjects who have developed (or are at risk of developing) an allergic reaction to botulinum toxin. The binding agents described herein may be a beneficial alternative for subjects who have developed (or are at risk of developing) an immune response to botulinum toxin. The binding agents described herein, e.g., binding proteins such as antibodies, can be developed in an IgG4 background. Such binding agents are not capable of activating complement and therefore do not cause any membrane damage or local inflammation. The size of the binding agents described herein can be varied and modified to have favorable kinetics. For example, scFvs can be used to more efficiently access target sites and multimeric IgM-like molecules can be created that have longer half-lives.

[0067] binding substance Described herein are binding agents that comprise one or more binding regions, where only one of the binding regions specifically binds to the Ig-like 1 domain of a MuSK protein.

[0068] Advantageously, the binding agents inhibit MuSK activity (and inhibit MuSK function) by inhibiting MuSK phosphorylation and / or dimerization. That is, the binding agents described herein do not induce (e.g., are unable to induce) MuSK phosphorylation and / or dimerization. The binding agents can be used to inhibit neuromuscular transmission in a subject.

[0069] The binding agents are therefore beneficial for treating diseases, disorders, and / or conditions in a subject that are alleviated by the inhibition of neuromuscular transmission.

[0070] As defined in more detail below, "inhibit" or "inhibition" refers to a decrease or reduction, and thus includes partial inhibition (e.g., partial but not complete inhibition of MuSK dimerization, and / or phosphorylation, and / or activation in a subject, and partial but not complete inhibition of neuromuscular transmission in a subject).

[0071] In one particular example, the binding agent is a binding protein comprising one or more binding regions, where only one of the binding regions specifically binds to the Ig-like 1 domain of the MuSK protein. In this context, the binding protein may be an antibody, as described in more detail below, where the binding region is a variable region. In this example, the antibody comprises one or more binding regions, where only one of the binding regions specifically binds to the Ig-like 1 domain of the MuSK protein.

[0072] A binding agent (e.g., a binding protein, e.g., an antibody) may be monovalent or multivalent. As used herein, a "monovalent" binding agent has only one binding region (also referred to herein as an antigen-binding site). As used herein, a "multivalent binding agent" refers to a binding agent that has multiple (i.e., more than one) binding regions (i.e., multiple antigen-binding sites).

[0073] Thus, a multivalent binding agent may have two, three, four, five, six or more binding regions / antigen binding sites. By way of example, a "bivalent" binding agent is a multivalent binding protein having two binding regions / antigen binding sites, while a "trivalent" binding protein is a multivalent binding protein having three binding regions / antigen binding sites.

[0074] The terms "binding region", "antigen-binding site" and "epitope-binding site" are herein interchangeable, unless the context dictates otherwise.

[0075] As an example, a binding agent may have only one binding region (i.e., a binding region that specifically binds to the Ig-like 1 domain of a MuSK protein) and no other binding regions. In this context, a binding agent is monovalent.

[0076] For binding agents (e.g., binding proteins, e.g., antibodies) described herein that are multivalent, the binding agent (e.g., binding protein, e.g., antibody) only has one binding region that specifically binds to the Ig-like 1 domain of the MuSK protein. That is, the remaining binding regions of the multivalent binding agent (i.e., the second, third, fourth, fifth, etc. binding regions of the multivalent binding agent) do not specifically bind to the Ig-like 1 domain of the MuSK protein, but specifically bind to a different antigen / epitope. Typically, the remaining binding regions of the multivalent binding agent (i.e., the second, third, fourth, fifth, etc. binding regions of the multivalent binding agent) specifically bind to a different antigen (e.g., do not specifically bind to MuSK). However, for the avoidance of doubt, in another example, provided that the multivalent binding agent does not (e.g., is unable to) induce MuSK phosphorylation and / or dimerization and / or activation, the remaining binding regions of the multivalent binding agent (i.e., the second, third, fourth, fifth, etc. binding regions of the multivalent binding agent) may specifically bind to another region of the MuSK protein (e.g., the Ig-like 2 domain, the Ig-like 3 domain, the Frizzled domain, etc.). Methods for determining whether MuSK activation and / or dimerization and / or phosphorylation occurs are well known in the art.

[0077] As an example, a binding agent may have two binding regions (i.e., one binding region that specifically binds to the Ig-like 1 domain of a MuSK protein, and another binding region that is specific for another antigen / epitope (i.e., the other binding region does not specifically bind to the Ig-like 1 domain of a MuSK protein)). In this context, the binding agent may be bivalent (and bispecific). This type of binding agent may also be referred to as "functionally monovalent" with respect to the Ig-like 1 domain of a MuSK protein (i.e., it is only capable of binding to the Ig-like 1 domain of a MuSK protein via one binding region).

[0078] As a further example, a binding agent may have three binding regions (i.e., one binding region that specifically binds to the Ig-like 1 domain of a MuSK protein, and two other binding regions specific for another (one or two) antigens / epitopes (i.e., neither of the additional two binding regions specifically binds to the Ig-like 1 domain of a MuSK protein)). In this context, the binding agent may be trivalent (and bispecific or trispecific). As mentioned above, this type of binding agent may also be referred to as "functionally monovalent" with respect to the Ig-like 1 domain of a MuSK protein (i.e., it is only capable of binding to the Ig-like 1 domain of a MuSK protein via one binding region).

[0079] In one particular example, the binding agent described herein is an antibody. In this context, the binding region of the antibody may be a variable region.

[0080] The term "variable region" refers to a region of an immunoglobulin, including one or more Ig domains, substantially encoded by any Vκ, Vλ, and / or VH genes that create the kappa, lambda, and heavy chain immunoglobulin loci, respectively. More specifically, the term refers to the domain of an antibody's heavy or light chain that is involved in binding the antibody to an antigen. An immunoglobulin variable region is thus typically composed of two variable regions, i.e., a heavy chain variable region and a light chain variable region. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have a similar structure, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL may be sufficient to confer specificity of antigen binding.

[0081] The term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain according to Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or amino acid residues from the "hypervariable loops" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H3) in the heavy chain variable domain according to Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). (H1), 53-55 (H2), and 96-101 (H3).

[0082] The amino-terminal portion of each chain contains a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition, commonly referred to in the art and herein as an "Fv domain" or "Fv region." In the variable region, three loops come together in each of the heavy and light chain V domains to form the antigen-binding site. Each loop is called a complementarity determining region (hereinafter also referred to as a "CDR") and is where the variation in amino acid sequence is most pronounced. "Variable" refers to the fact that certain portions of the variable regions differ extensively in sequence among antibodies. The variability within the variable regions is not uniformly distributed. In practice, the V regions consist of relatively invariant stretches of 15 to 30 amino acids, called framework regions (FRs), separated by shorter regions of extreme variability, called "hypervariable regions," each 9 to 15 amino acids long or longer. Each VH and VL is composed of three hypervariable regions ("complementarity determining regions", "CDRs") and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1-CDR1-FR2-CDR2-FR3- CDR3-FR4.

[0083] The antibodies of the present invention may comprise one or more binding regions (i.e., variable regions), with only one binding region (i.e., variable region) specifically binding to the Ig-like 1 domain of the MuSK protein. In the case of antibodies, the terms variable region and binding region are used interchangeably herein. Each variable region may comprise the CDRs of the light chain variable domain (VL) (i.e., VL CDR1, VL CDR2, and VL CDR3) and / or the CDRs of the heavy chain variable domain (VH) (i.e., VH CDR1, VH CDR2, and VH CDR3). As described elsewhere herein, the CDRs from one VL or VH are sufficient to confer antigen-binding specificity (i.e., specific binding to the Ig-like 1 domain of MuSK). In other cases, antigen-binding specificity (i.e., specific binding to the Ig-like 1 domain of MuSK) may be obtained by the presence of CDR1, 2, and 3 from both the VL and the VH.

[0084] Specific examples of CDR combinations that confer antigen binding specificity to the Ig-like 1 domain of MuSK are provided below. CDR sequences were identified using the IMGT / V-QUEST program version: 3.4.17 (19 February 2019) - IMGT / V-QUEST Reference Directory Release: 201910-2 (5 March 2019) (http: / / imgt.org / IMGT_vquest / vquest) and selected for Homo sapiens sequences. These CDR combinations form the binding region (i.e., variable region) of an antibody (including a Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent (di-)scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody) that specifically binds to the Ig-like 1 domain of MuSK.

[0085] 1) CDR1 comprising GFNFSTYT (SEQ ID NO: 10), CDR2 comprising ISSRSAYK (SEQ ID NO: 11), and CDR3 comprising ARDFFQLGPPRFDS (SEQ ID NO: 12). These CDRs may optionally be present in a VH, for example a VH comprising the sequence of SEQ ID NO: 9.

[0086] 2) CDR1 comprising QRISSF (SEQ ID NO: 14), CDR2 comprising GAS (SEQ ID NO: 15), and CDR3 comprising QQSYSPMYT (SEQ ID NO: 16). These CDRs may optionally be present in the VL, for example, the VL comprising the sequence of SEQ ID NO: 13.

[0087] 3) For example, the CDRs (heavy chain) of SEQ ID NO: 10, 11, and 12 and the CDRs (light chain) of SEQ ID NO: 14, 15, and 16 in VH and VL. For example, antigen specificity can be obtained by combining the variable domains of SEQ ID NO: 9 (heavy chain) and SEQ ID NO: 13 (light chain).

[0088] 4) CDR1 comprising GFTFSSYT (SEQ ID NO: 18), CDR2 comprising IGSNGDYI (SEQ ID NO: 19), and CDR3 comprising ARGQLAVAGTHFDY (SEQ ID NO: 20). These CDRs may optionally be present in a VH, for example a VH comprising the sequence of SEQ ID NO: 17.

[0089] 5) CDR1 comprises QKVNKY (SEQ ID NO: 22), CDR2 comprises AAS (SEQ ID NO: 23), and CDR3 comprises QQSYSPLCT (SEQ ID NO: 24). These CDRs may optionally be present in the VL, for example, the VL comprises the sequence of SEQ ID NO: 21.

[0090] 6) For example, the CDRs (heavy chain) of SEQ ID NO: 18, 19, and 20 and the CDRs (light chain) of SEQ ID NO: 22, 23, and 24 in VH and VL. For example, antigen specificity can be obtained by combining the variable domains of SEQ ID NO: 17 (heavy chain) and SEQ ID NO: 21 (light chain).

[0091] 7) CDR1 comprising GFTFSDFT (SEQ ID NO: 26), CDR2 comprising IGSSGTFI (SEQ ID NO: 27), and CDR3 comprising ARGRIAVAGTHFDL (SEQ ID NO: 28). These CDRs may optionally be present in a VH, for example a VH comprising the sequence of SEQ ID NO: 25.

[0092] 8) CDR1 comprising GYTFTGQY (SEQ ID NO: 30), CDR2 comprising INPSSGVT (SEQ ID NO: 31), and CDR3 comprising ATLSLGVYYVGMVA (SEQ ID NO: 32). These CDRs may optionally be present in the VH, for example, the VH comprising the sequence of SEQ ID NO: 29.

[0093] 9) CDR1 comprising GLAQQH (SEQ ID NO: 34), CDR2 comprising KDI (SEQ ID NO: 35), and CDR3 comprising QSGDRTATSVL (SEQ ID NO: 36). These CDRs may optionally be present in the VL, for example, the VL comprising the sequence of SEQ ID NO: 33.

[0094] 10) For example, the CDRs (heavy chain) of SEQ ID NO: 30, 31, and 32 and the CDRs (light chain) of SEQ ID NO: 34, 35, and 36 in VH and VL. For example, antigen specificity can be obtained by combining the variable domains of SEQ ID NO: 29 (heavy chain) and SEQ ID NO: 33 (light chain).

[0095] 11) CDR1 comprising GFDFSAST (SEQ ID NO: 38), CDR2 comprising VSGDSHHI (SEQ ID NO: 39), and CDR3 comprising ARERLLRLGVGFDS (SEQ ID NO: 40). These CDRs may optionally be present in the VH, for example, the VH comprising the sequence of SEQ ID NO: 37.

[0096] 12) CDR1 comprises QRISGF (SEQ ID NO: 42), CDR2 comprises AAS (SEQ ID NO: 43), and CDR3 comprises QQSYSPLYT (SEQ ID NO: 44). These CDRs may optionally be present in the VL, for example, the VL comprises the sequence of SEQ ID NO: 41.

[0097] 13) For example, the CDRs (heavy chain) of SEQ ID NO: 38, 39, and 40 and the CDRs (light chain) of SEQ ID NO: 42, 43, and 44 in VH and VL. For example, antigen specificity can be obtained by combining the variable domains of SEQ ID NO: 37 (heavy chain) and SEQ ID NO: 41 (light chain).

[0098] 14) The CDRs may be as follows: CDR1 comprising GFTFSSYT (SEQ ID NO: 46), CDR2 comprising ISSGGHYI (SEQ ID NO: 47), and CDR3 comprising ARERLLRLGVGFDF (SEQ ID NO: 48). These CDRs may optionally be present in a VH, for example a VH comprising the sequence of SEQ ID NO: 45.

[0099] 15) CDR1 comprises QSISGY (SEQ ID NO: 50), CDR2 comprises AAS (SEQ ID NO: 51), and CDR3 comprises QQSYSALYT (SEQ ID NO: 52). These CDRs may optionally be present in the VL, for example, the VL comprises the sequence of SEQ ID NO: 49.

[0100] 16) For example, the CDRs (heavy chain) of SEQ ID NO: 46, 47, and 48 and the CDRs (light chain) of SEQ ID NO: 50, 51, and 52 in VH and VL. For example, antigen specificity can be obtained by combining the variable domains of SEQ ID NO: 45 (heavy chain) and SEQ ID NO: 49 (light chain).

[0101] 17) CDR1 comprises GFTFSSYW (SEQ ID NO: 54), CDR2 comprises LNEDGSTT (SEQ ID NO: 55), and CDR3 comprises VSDLSGKDEH (SEQ ID NO: 56). These CDRs may optionally be present in the VH, for example, the VH comprises the sequence of SEQ ID NO: 53.

[0102] 18) CDR1 comprises QSLLHSNGYYW (SEQ ID NO: 58), CDR2 comprises LGF (SEQ ID NO: 59), and CDR3 comprises MQGLQ TPYT (SEQ ID NO: 60). These CDRs may optionally be present in the VL, for example, the VL comprises the sequence of SEQ ID NO: 57.

[0103] 19) For example, the CDRs (heavy chain) of SEQ ID NO: 54, 55, and 56 and the CDRs (light chain) of SEQ ID NO: 58, 59, and 60 in VH and VL. For example, antigen specificity can be obtained by combining the variable domains of SEQ ID NO: 53 (heavy chain) and SEQ ID NO: 57 (light chain).

[0104] The above CDRs may be present in an antibody, such as an IgG. For example, the IgG may be selected from IgG1, IgG2, IgG3, or IgG4.

[0105] For example, the CDR and variable domain sequences provided in options 1, 2, 3, 7, 11, 12, or 13 above may be present in the Fc sequence of an IgG1 antibody. An exemplary sequence may be that of SEQ ID NO: 61 or a variant thereof (see, for example, the Fc region of SEQ ID NO: 65, 66, or 67).

[0106] For example, the CDR and variable domain sequences provided in options 8, 9, and 10 above may be present in the Fc sequence of an IgG4 antibody. An exemplary sequence may be that of SEQ ID NO: 64 or a variant thereof (see, for example, the Fc region of SEQ ID NO: 69).

[0107] For example, the CDR and variable domain sequences provided in options 14, 15, or 16 above may be present in the Fc sequence of an IgG3. An exemplary sequence may be that of SEQ ID NO: 63 or a variant thereof (see, for example, the Fc region of SEQ ID NO: 68).

[0108] As described in detail above, the antibodies described herein (including Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody) comprise one or more binding regions, with only one binding region specifically binding to the Ig-like 1 domain of the MuSK protein.

[0109] In one embodiment, a binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain having an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polypeptide of SEQ ID NO:9; and a light chain variable domain having an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polypeptide of SEQ ID NO:13. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0110] Preferably, the heavy chain variable domain has an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide of SEQ ID NO: 9, and comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 having SEQ ID NO: 10, VH CDR2 having SEQ ID NO: 11, and VH CDR3 having SEQ ID NO: 12. the light chain variable domain has an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide of SEQ ID NO: 13 and comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 14, a VL CDR2 having SEQ ID NO: 15, and a VL CDR3 having SEQ ID NO: 16. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0111] Most preferably, the binding region comprises a heavy chain variable domain of SEQ ID NO: 9 and a light chain variable domain of SEQ ID NO: 13. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, where only one binding region specifically binds (e.g. via a sequence as described above) to the Ig-like 1 domain of a MuSK protein.

[0112] In one embodiment, a binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 9; and a light chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 13. Preferably, a binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: a VH CDR1 having SEQ ID NO: 10, a VH CDR2 having SEQ ID NO: 11, and a VH CDR3 having SEQ ID NO: 12. Preferably, a binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 14, a VL CDR2 having SEQ ID NO: 15, and a VL CDR3 having SEQ ID NO: 16. Preferably, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 with SEQ ID NO: 10, VH CDR2 with SEQ ID NO: 11, and VH CDR3 with SEQ ID NO: 12, and at least one, particularly at least two, more particularly at least three light chain CDRs: VL CDR1 with SEQ ID NO: 14, VL CDR2 with SEQ ID NO: 15, and VL CDR3 with SEQ ID NO: 16. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, only one binding region specifically binds to the Ig-like 1 domain of the MuSK protein (e.g., via the sequences described above).

[0113] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain comprising a polypeptide sequence having at least one, two, three, four or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 9; and a light chain variable domain of SEQ ID NO: 13. Preferably, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: a VH CDR1 having SEQ ID NO: 10, a VH CDR2 having SEQ ID NO: 11, and a VH CDR3 having SEQ ID NO: 12. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0114] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain of SEQ ID NO: 9; and a light chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 13. Preferably, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 14, a VL CDR2 having SEQ ID NO: 15, and a VL CDR3 having SEQ ID NO: 16. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0115] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 having SEQ ID NO: 10, VH CDR2 having SEQ ID NO: 11, and VH CDR3 having SEQ ID NO: 12. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0116] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 14, a VL CDR2 having SEQ ID NO: 15, and a VL CDR3 having SEQ ID NO: 16. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0117] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 with SEQ ID NO: 10, VH CDR2 with SEQ ID NO: 11, and VH CDR3 with SEQ ID NO: 12, and at least one, particularly at least two, more particularly at least three light chain CDRs: VL CDR1 with SEQ ID NO: 14, VL CDR2 with SEQ ID NO: 15, and VL CDR3 with SEQ ID NO: 16. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, and only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0118] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises heavy chain CDRs: VH CDR1 with SEQ ID NO: 10, VH CDR2 with SEQ ID NO: 11, and VH CDR3 with SEQ ID NO: 12, and light chain CDRs: VL CDR1 with SEQ ID NO: 14, VL CDR2 with SEQ ID NO: 15, and VL CDR3 with SEQ ID NO: 16. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0119] In one embodiment, an antibody binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain having an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide of SEQ ID NO: 17; and a light chain variable domain having an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide of SEQ ID NO:21. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0120] Preferably, the heavy chain variable domain has an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide of SEQ ID NO: 17, and has at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 having SEQ ID NO: 18, VH CDR2 having SEQ ID NO: 19, and VH CDR3 having SEQ ID NO: 20. the light chain variable domain has an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide of SEQ ID NO: 21 and comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 22, a VL CDR2 having SEQ ID NO: 23, and a VL CDR3 having SEQ ID NO: 24. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0121] Most preferably, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises a heavy chain variable domain of SEQ ID NO: 17 and a light chain variable domain of SEQ ID NO: 21. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0122] In one embodiment, a binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 17; and a light chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 21. Preferably, a binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: a VH CDR1 having SEQ ID NO: 18, a VH CDR2 having SEQ ID NO: 19, and a VH CDR3 having SEQ ID NO: 20. Preferably, a binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 22, a VL CDR2 having SEQ ID NO: 23, and a VL CDR3 having SEQ ID NO: 24. Preferably, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 with SEQ ID NO: 18, VH CDR2 with SEQ ID NO: 19, and VH CDR3 with SEQ ID NO: 20, and at least one, particularly at least two, more particularly at least three light chain CDRs: VL CDR1 with SEQ ID NO: 22, VL CDR2 with SEQ ID NO: 23, and VL CDR3 with SEQ ID NO: 24. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, only one binding region specifically binds to the Ig-like 1 domain of the MuSK protein (e.g., via the sequences described above).

[0123] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 17; and a light chain variable domain of SEQ ID NO: 21. Preferably, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: a VH CDR1 having SEQ ID NO: 18, a VH CDR2 having SEQ ID NO: 19, and a VH CDR3 having SEQ ID NO: 20. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0124] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain of SEQ ID NO: 17; and a light chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 21. Preferably, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 22, a VL CDR2 having SEQ ID NO: 23, and a VL CDR3 having SEQ ID NO: 24. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0125] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 having SEQ ID NO: 18, VH CDR2 having SEQ ID NO: 19, and VH CDR3 having SEQ ID NO: 20. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0126] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 22, a VL CDR2 having SEQ ID NO: 23, and a VL CDR3 having SEQ ID NO: 24. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0127] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 with SEQ ID NO: 18, VH CDR2 with SEQ ID NO: 19, and VH CDR3 with SEQ ID NO: 20, and at least one, particularly at least two, more particularly at least three light chain CDRs: VL CDR1 with SEQ ID NO: 22, VL CDR2 with SEQ ID NO: 23, and VL CDR3 with SEQ ID NO: 24. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, and only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0128] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises heavy chain CDRs: VH CDR1 with SEQ ID NO: 18, VH CDR2 with SEQ ID NO: 19, and VH CDR3 with SEQ ID NO: 20, and light chain CDRs: VL CDR1 with SEQ ID NO: 22, VL CDR2 with SEQ ID NO: 23, and VL CDR3 with SEQ ID NO: 24. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0129] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises a heavy chain variable domain having an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide of SEQ ID NO: 25. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds to the Ig-like 1 domain of the MuSK protein (e.g., via a sequence as described above).

[0130] Preferably, the heavy chain variable domain has an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide of SEQ ID NO: 25 and comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: a VH CDR1 having SEQ ID NO: 26, a VH CDR2 having SEQ ID NO: 27, and a VH CDR3 having SEQ ID NO: 28. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0131] Most preferably, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises a heavy chain variable domain of SEQ ID NO: 25. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. An antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0132] In one embodiment, the binding region specifically binds to the Ig-like 1 domain of the MuSK protein comprises a heavy chain variable domain comprising a polypeptide sequence having at least one, two, three, four or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 25. Preferably, the binding region specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 having SEQ ID NO: 26, VH CDR2 having SEQ ID NO: 27, and VH CDR3 having SEQ ID NO: 28. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds to the Ig-like 1 domain of the MuSK protein (e.g., via a sequence as described above).

[0133] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 having SEQ ID NO: 26, VH CDR2 having SEQ ID NO: 27, and VH CDR3 having SEQ ID NO: 28. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0134] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises the heavy chain CDRs: VH CDR1 having SEQ ID NO: 26, VH CDR2 having SEQ ID NO: 27, and VH CDR3 having SEQ ID NO: 28. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0135] In one embodiment, a binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain having an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polypeptide of SEQ ID NO:29; and a light chain variable domain having an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polypeptide of SEQ ID NO:33. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0136] Preferably, the heavy chain variable domain has an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide of SEQ ID NO:29, and has at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 having SEQ ID NO:30, VH CDR2 having SEQ ID NO:31, and VH CDR3 having SEQ ID NO:32. the light chain variable domain has an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide of SEQ ID NO: 33 and comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 34, a VL CDR2 having SEQ ID NO: 35, and a VL CDR3 having SEQ ID NO: 36. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0137] Most preferably, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises a heavy chain variable domain of SEQ ID NO: 29 and a light chain variable domain of SEQ ID NO: 33. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0138] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 29; and a light chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 33. Preferably, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: a VH CDR1 having SEQ ID NO: 30, a VH CDR2 having SEQ ID NO: 31, and a VH CDR3 having SEQ ID NO: 32. Preferably, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 34, a VL CDR2 having SEQ ID NO: 35, and a VL CDR3 having SEQ ID NO: 36. Preferably, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 with SEQ ID NO: 30, VH CDR2 with SEQ ID NO: 31, and VH CDR3 with SEQ ID NO: 32, and at least one, particularly at least two, more particularly at least three light chain CDRs: VL CDR1 with SEQ ID NO: 34, VL CDR2 with SEQ ID NO: 35, and VL CDR3 with SEQ ID NO: 36. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, only one binding region specifically binds (e.g., via the sequences described above) to the Ig-like 1 domain of the MuSK protein.

[0139] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain comprising a polypeptide sequence having at least one, two, three, four or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 29; and a light chain variable domain of SEQ ID NO: 33. Preferably, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: a VH CDR1 having SEQ ID NO: 30, a VH CDR2 having SEQ ID NO: 31, and a VH CDR3 having SEQ ID NO: 32. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0140] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain of SEQ ID NO: 29; and a light chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 33. Preferably, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 34, a VL CDR2 having SEQ ID NO: 35, and a VL CDR3 having SEQ ID NO: 36. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0141] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 having SEQ ID NO: 30, VH CDR2 having SEQ ID NO: 31, and VH CDR3 having SEQ ID NO: 32. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0142] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 34, a VL CDR2 having SEQ ID NO: 35, and a VL CDR3 having SEQ ID NO: 36. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0143] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 with SEQ ID NO: 30, VH CDR2 with SEQ ID NO: 31, and VH CDR3 with SEQ ID NO: 32, and at least one, particularly at least two, more particularly at least three light chain CDRs: VL CDR1 with SEQ ID NO: 34, VL CDR2 with SEQ ID NO: 35, and VL CDR3 with SEQ ID NO: 36. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, and only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0144] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises heavy chain CDRs: VH CDR1 with SEQ ID NO: 30, VH CDR2 with SEQ ID NO: 31, and VH CDR3 with SEQ ID NO: 32, and light chain CDRs: VL CDR1 with SEQ ID NO: 34, VL CDR2 with SEQ ID NO: 35, and VL CDR3 with SEQ ID NO: 36. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0145] In one embodiment, a binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain having an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide of SEQ ID NO:37; and a light chain variable domain having an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide of SEQ ID NO:41. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0146] Preferably, the heavy chain variable domain has an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide of SEQ ID NO: 37, and has at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 having SEQ ID NO: 38, VH CDR2 having SEQ ID NO: 39, and VH CDR3 having SEQ ID NO: 40. the light chain variable domain has an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identical to the polypeptide of SEQ ID NO: 41 and comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 42, a VL CDR2 having SEQ ID NO: 43, and a VL CDR3 having SEQ ID NO: 44. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0147] Most preferably, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises a heavy chain variable domain of SEQ ID NO: 37 and a light chain variable domain of SEQ ID NO: 41. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0148] In one embodiment, a binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 37; and a light chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 41. Preferably, a binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: a VH CDR1 having SEQ ID NO: 38, a VH CDR2 having SEQ ID NO: 39, and a VH CDR3 having SEQ ID NO: 40. Preferably, a binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 42, a VL CDR2 having SEQ ID NO: 43, and a VL CDR3 having SEQ ID NO: 44. Preferably, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 with SEQ ID NO: 38, VH CDR2 with SEQ ID NO: 39, and VH CDR3 with SEQ ID NO: 40, and at least one, particularly at least two, more particularly at least three light chain CDRs: VL CDR1 with SEQ ID NO: 42, VL CDR2 with SEQ ID NO: 43, and VL CDR3 with SEQ ID NO: 44. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, and only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0149] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain comprising a polypeptide sequence having at least one, two, three, four or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 37; and a light chain variable domain of SEQ ID NO: 41. Preferably, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: a VH CDR1 having SEQ ID NO: 38, a VH CDR2 having SEQ ID NO: 39, and a VH CDR3 having SEQ ID NO: 40. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0150] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain of SEQ ID NO: 37; and a light chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 41. Preferably, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 42, a VL CDR2 having SEQ ID NO: 43, and a VL CDR3 having SEQ ID NO: 44. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0151] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 having SEQ ID NO: 38, VH CDR2 having SEQ ID NO: 39, and VH CDR3 having SEQ ID NO: 40. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0152] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 42, a VL CDR2 having SEQ ID NO: 43, and a VL CDR3 having SEQ ID NO: 44. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0153] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 with SEQ ID NO: 38, VH CDR2 with SEQ ID NO: 39, and VH CDR3 with SEQ ID NO: 40, and at least one, particularly at least two, more particularly at least three light chain CDRs: VL CDR1 with SEQ ID NO: 42, VL CDR2 with SEQ ID NO: 43, and VL CDR3 with SEQ ID NO: 44. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, and only one binding region specifically binds to the Ig-like 1 domain of the MuSK protein (e.g., via a sequence as described above).

[0154] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises heavy chain CDRs: VH CDR1 with SEQ ID NO: 38, VH CDR2 with SEQ ID NO: 39, and VH CDR3 with SEQ ID NO: 40, and light chain CDRs: VL CDR1 with SEQ ID NO: 42, VL CDR2 with SEQ ID NO: 43, and VL CDR3 with SEQ ID NO: 44. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0155] In one embodiment, a binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain having an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polypeptide of SEQ ID NO:45; and a light chain variable domain having an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polypeptide of SEQ ID NO:49. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0156] Preferably, the heavy chain variable domain has an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide of SEQ ID NO: 45, and has at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 having SEQ ID NO: 46, VH CDR2 having SEQ ID NO: 47, and VH CDR3 having SEQ ID NO: 48. the light chain variable domain has an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide of SEQ ID NO: 49 and comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 50, a VL CDR2 having SEQ ID NO: 51, and a VL CDR3 having SEQ ID NO: 52. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0157] Most preferably, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises a heavy chain variable domain of SEQ ID NO: 45 and a light chain variable domain of SEQ ID NO: 49. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0158] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 45; and a light chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 49. Preferably, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: a VH CDR1 having SEQ ID NO: 46, a VH CDR2 having SEQ ID NO: 47, and a VH CDR3 having SEQ ID NO: 48. Preferably, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 50, a VL CDR2 having SEQ ID NO: 51, and a VL CDR3 having SEQ ID NO: 52. Preferably, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 with SEQ ID NO: 46, VH CDR2 with SEQ ID NO: 47, and VH CDR3 with SEQ ID NO: 48, and at least one, particularly at least two, more particularly at least three light chain CDRs: VL CDR1 with SEQ ID NO: 50, VL CDR2 with SEQ ID NO: 51, and VL CDR3 with SEQ ID NO: 52. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, and only one binding region specifically binds (e.g., via the sequences described above) to the Ig-like 1 domain of the MuSK protein.

[0159] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain comprising a polypeptide sequence having at least one, two, three, four or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 45; and a light chain variable domain of SEQ ID NO: 49. Preferably, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: a VH CDR1 having SEQ ID NO: 46, a VH CDR2 having SEQ ID NO: 47, and a VH CDR3 having SEQ ID NO: 48. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0160] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain of SEQ ID NO: 45; and a light chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 49. Preferably, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 50, a VL CDR2 having SEQ ID NO: 51, and a VL CDR3 having SEQ ID NO: 52. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0161] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 having SEQ ID NO: 46, VH CDR2 having SEQ ID NO: 47, and VH CDR3 having SEQ ID NO: 48. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0162] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 50, a VL CDR2 having SEQ ID NO: 51, and a VL CDR3 having SEQ ID NO: 52. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0163] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 with SEQ ID NO: 46, VH CDR2 with SEQ ID NO: 47, and VH CDR3 with SEQ ID NO: 48, and at least one, particularly at least two, more particularly at least three light chain CDRs: VL CDR1 with SEQ ID NO: 50, VL CDR2 with SEQ ID NO: 51, and VL CDR3 with SEQ ID NO: 52. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, and only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0164] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises heavy chain CDRs: VH CDR1 with SEQ ID NO: 46, VH CDR2 with SEQ ID NO: 47, and VH CDR3 with SEQ ID NO: 48, and light chain CDRs: VL CDR1 with SEQ ID NO: 50, VL CDR2 with SEQ ID NO: 51, and VL CDR3 with SEQ ID NO: 52. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0165] In one embodiment, a binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain having an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide of SEQ ID NO:53; and a light chain variable domain having an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide of SEQ ID NO:57. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0166] Preferably, the heavy chain variable domain has an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide of SEQ ID NO: 53, and comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 having SEQ ID NO: 54, VH CDR2 having SEQ ID NO: 55, and VH CDR3 having SEQ ID NO: 56. the light chain variable domain has an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identical to the polypeptide of SEQ ID NO: 57 and comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 58, a VL CDR2 having SEQ ID NO: 59, and a VL CDR3 having SEQ ID NO: 60. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0167] Most preferably, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises a heavy chain variable domain of SEQ ID NO: 53 and a light chain variable domain of SEQ ID NO: 57. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0168] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 53; and a light chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 57. Preferably, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: a VH CDR1 having SEQ ID NO: 54, a VH CDR2 having SEQ ID NO: 55, and a VH CDR3 having SEQ ID NO: 56. Preferably, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 58, a VL CDR2 having SEQ ID NO: 59, and a VL CDR3 having SEQ ID NO: 60. Preferably, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 with SEQ ID NO: 54, VH CDR2 with SEQ ID NO: 55, and VH CDR3 with SEQ ID NO: 56, and at least one, particularly at least two, more particularly at least three light chain CDRs: VL CDR1 with SEQ ID NO: 58, VL CDR2 with SEQ ID NO: 59, and VL CDR3 with SEQ ID NO: 60. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, and only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0169] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain comprising a polypeptide sequence having at least one, two, three, four or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 53; and a light chain variable domain of SEQ ID NO: 57. Preferably, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: a VH CDR1 having SEQ ID NO: 54, a VH CDR2 having SEQ ID NO: 55, and a VH CDR3 having SEQ ID NO: 56. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0170] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises a heavy chain variable domain of SEQ ID NO: 53; and a light chain variable domain comprising a polypeptide sequence having at least one, two, three, four, or five conservative substitutions compared to the polypeptide sequence of SEQ ID NO: 57. Preferably, the binding region that specifically binds to the Ig-like 1 domain of a MuSK protein comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 58, a VL CDR2 having SEQ ID NO: 59, and a VL CDR3 having SEQ ID NO: 60. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein (eg, via a sequence as described above).

[0171] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 having SEQ ID NO: 54, VH CDR2 having SEQ ID NO: 55, and VH CDR3 having SEQ ID NO: 56. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0172] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three light chain CDRs: a VL CDR1 having SEQ ID NO: 58, a VL CDR2 having SEQ ID NO: 59, and a VL CDR3 having SEQ ID NO: 60. Preferably, the antibody is selected from an antibody, a Fab, a bispecific Fab2, a trispecific Fab3, a scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody. An antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0173] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises at least one, particularly at least two, more particularly at least three heavy chain CDRs: VH CDR1 with SEQ ID NO: 54, VH CDR2 with SEQ ID NO: 55, and VH CDR3 with SEQ ID NO: 56, and at least one, particularly at least two, more particularly at least three light chain CDRs: VL CDR1 with SEQ ID NO: 58, VL CDR2 with SEQ ID NO: 59, and VL CDR3 with SEQ ID NO: 60. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, and only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0174] In one embodiment, the binding region that specifically binds to the Ig-like 1 domain of the MuSK protein comprises heavy chain CDRs: VH CDR1 with SEQ ID NO: 54, VH CDR2 with SEQ ID NO: 55, and VH CDR3 with SEQ ID NO: 56, and light chain CDRs: VL CDR1 with SEQ ID NO: 58, VL CDR2 with SEQ ID NO: 59, and VL CDR3 with SEQ ID NO: 60. Preferably, the antibody is selected from an antibody, Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent scFv, bispecific scFv-Fc, bispecific diabody, trispecific triabody, single domain antibody, or bispecific minibody. The antibody comprises one or more binding regions, where only one binding region specifically binds (e.g., via a sequence as described above) to the Ig-like 1 domain of the MuSK protein.

[0175] As used herein, the terms "homology" and "identity" are used interchangeably. Calculations of sequence homology or sequence identity between sequences are performed as follows.

[0176] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence. Next, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is the same as amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, and takes into account the number of gaps, and the length of each gap, that need to be introduced for optimal alignment of the two sequences.

[0177] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the algorithm (Needleman et al. (1970) J. Mol. Biol. 48:444-453) built into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a BLOSUM 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com) using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the set of parameters to be used if the practitioner is unsure what parameters to use to determine whether a molecule is within the sequence identity or homology limits of the invention) is the BLOSUM 62 scoring matrix, with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0178] Alternatively, the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. (1989) CABIOS 4:11-17, as incorporated into the ALIGN program (version 2.0), using a weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0179] As used herein, the term "conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0180] In the context of binding, the terms "specific" and "specifically" are used interchangeably herein to indicate that other biomolecules do not significantly bind to a binding region (e.g., CDRs, variable regions, etc.) that specifically binds to a biomolecule of interest (wherein the biomolecule of interest is the Ig-like 1 domain of a MuSK protein). In some instances, the level of binding to a biomolecule other than the Ig-like 1 domain of a MuSK protein results in negligible (e.g., indeterminable) binding affinity by ELISA or affinity determination methods.

[0181] "Negligible binding" means binding that is at least about 85%, particularly at least about 90%, more particularly at least about 95%, even more particularly at least about 98%, but particularly at least about 99%, and up to 100% lower than binding to the Ig-like 1 domain of the MuSK protein.

[0182] The binding affinity of the binding region with the Ig-like 1 domain of the MuSK protein can be determined using standard binding assays, for example, surface plasmon resonance technology (BIAcore®, GE-Healthcare Uppsala, Sweden). As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows the analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further explanation, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0183] For example, in the context of binding of an antibody to a given antigen / epitope, "specific binding" is defined as a binding intensity greater than or equal to about 10, as determined by surface plasmon resonance (SPR) technology, e.g., in a BIAcore 3000 instrument, using the antibody as the test substance. -7 M or less, e.g., about 10 -8 M or less, e.g., about 10 -9 M or less, about 10 -10 M or less, or about 10 -11It means binding with an affinity corresponding to a KD (equilibrium dissociation constant) of M or lower (low KD refers to high affinity). This term also means that the antibody binds to a given antigen / epitope with an affinity corresponding to a KD that is at least 10 times lower, such as at least 100 times lower, such as at least 1000 times lower, such as at least 10000 times lower, such as at least 100000 times lower, such as at least 100000 times lower than the affinity for binding to a non-specific antigen other than the given antigen or a closely related antigen (e.g. bovine serum albumin, casein). The amount of lower affinity depends on the KD of the antibody, such that if the KD of the antibody is very low (i.e. the antibody is very specific and binds very well), the amount by which the affinity for the antigen is lower than the affinity for a non-specific antigen can be at least 10000 times. As used herein, the term "KD" refers to the dissociation rate constant of a particular antibody-antigen interaction.

[0184] Corresponding affinity values ​​for the "specific binding" of other binding agents described herein are well known in the art.

[0185] The binding region described herein is specific for (i.e., specifically binds to) an epitope within the Ig-like 1 domain of the MuSK protein. As used herein, the term "epitope" refers to a site on a target molecule (e.g., an antigen, e.g., a protein, e.g., the Ig-like 1 domain of the MuSK protein) to which a binding agent (e.g., a binding protein, e.g., an antibody or antibody fragment) binds. An epitope is a group of molecules, e.g., amino acids or sugar side chains, that usually have specific structural characteristics as well as specific charge characteristics. An antigen can have more than one epitope. Epitopes can be formed from both contiguous or adjacent noncontiguous residues (e.g., amino acid residues) of a target molecule. Epitopes formed from contiguous residues (e.g., amino acid residues) are typically also referred to as linear epitopes. An epitope typically includes at least 5 residues and up to about 12 residues, most often between 6 and 10 residues (e.g., amino acid residues). Epitopes may be conformational (i.e., non-linear).

[0186] In one example, the MuSK protein is a human MuSK protein. Human MuSK protein has been well characterized. MuSK protein has been sequenced and recently characterized by Valenzuela et al. (see WO 97 / 21811). MuSK protein is a receptor tyrosine kinase (RTK) located on the cell surface of muscle cells at the NMJ and has the sequence shown in SEQ ID NO: 1. Human MuSK protein can be identified by UniprotKB identifier: O15146. Human MuSK gene information can be found in Ensembl ref: ENSG00000030304. The gene has seven transcripts, as outlined below.

[0187] [Table 1]

[0188] The extracellular region of MuSK contains three Ig-like domains and a frizzled domain. The first N-terminal Ig-like domain in MuSK (also known herein as the Ig-like 1 domain) is required for MuSK to bind to Lrp4 (Zhang et al., 2011). Mutation of one residue, I96, on the solvent-exposed surface of the first Ig-like 1 domain prevents MuSK from binding to Lrp4 and responding to agrin (Stiegler et al., 2006; Zhang et al., 2011). The hydrophobic surface opposite the first Ig-like 1 domain mediates MuSK homodimerization and is essential for MuSK transphosphorylation. MuSK is expressed by muscle but not by motor neurons, but it is essential for presynaptic differentiation as well as postsynaptic differentiation (Burden et al., 2013). MuSK controls presynaptic differentiation by clustering Lrp4 in muscle and functions bidirectionally by acting not only as a receptor for agrin and a ligand for MuSK but also as a direct retrograde signal for presynaptic differentiation. In addition to its role during synapse formation, MuSK is also required for maintaining mature synapses, as inhibition of MuSK in mature muscle results in severe defects in presynaptic and postsynaptic differentiation (Hesser et al., 2006).

[0189] As described herein, one binding region of the binding agent is the Ig-like 1 domain of the human MuSK protein (herein, the immunoglobulin-like 1 domain of MuSK; MuSK Ig-like The Ig-like 1 domain of the human MuSK protein is specific for (i.e., specifically binds to) an epitope within the Ig-like 1 domain; also referred to as the first Ig-like 1 domain of MuSK. The Ig-like 1 domain of the human MuSK protein is set forth in SEQ ID NO:2:

[0190] [ka]

[0191] It has the sequence shown in

[0192] As used herein, a "monospecific" binding agent thus refers to a binding agent that contains only one antigen binding site, or that contains multiple (e.g., 2, 3, 4, 5, etc.) antigen binding sites (e.g., variable regions) that are each identical (or at least that each bind to the same epitope in the target antigen). In the context of a binding agent that acts as a MuSK antagonist, as described herein, a binding agent (e.g., a binding protein, e.g., an antibody) can be monospecific, for example, if it has only one binding region that specifically binds to the Ig-like 1 domain of a MuSK protein.

[0193] With respect to the binding proteins described herein that have multiple binding regions, the binding agent is necessarily "multispecific" (e.g., bispecific or trispecific, etc.). This is because a binding agent described herein may only have one binding region that is specific for the Ig-like 1 domain of the MuSK protein. A "bispecific" binding agent thus comprises two distinct antigen binding sites (thus the bispecific binding protein binds to two different antigens / epitopes).

[0194] The binding agent can be any suitable binding agent known in the art. Non-limiting examples of binding agents described herein include, but are not limited to, aptamers (e.g., nucleic acid aptamers, peptide aptamers, aptabodies, affimers), binding proteins (e.g., antibodies, antibody mimetics, camelid antibodies, duobodies, etc.), and small molecules. Other suitable binding agents are also well known and can be readily ascertained by the skilled artisan using routine experimental procedures.

[0195] As used herein, "aptamer" refers to nucleic acid aptamers and / or peptide aptamers. Examples of aptamers include affimers (evolution of peptide aptamers) and aptabodies (formed by hybridization of two DNA aptamers), which are well known and can be readily ascertained by those skilled in the art using routine experimental procedures.

[0196] In one example, the binding agent is a binding protein. Examples of suitable binding agents include antibodies and antibody mimetics. Examples of antibodies are provided elsewhere herein. Examples of suitable antibody mimetics include affibody molecules (including affimab), affilins, peptide aptamers (including affimers), affitins, alphabodies, anticalins, avimers, DARPins, Fynomers, Kunitz domain peptides, monobodies, nanoCLAMPs, and the like, which are well known and can be readily ascertained by those skilled in the art using routine experimental procedures.

[0197] As described elsewhere herein and in the examples, the binding agent is an antibody. As used herein, the term "antibody" or "antibodies" refers to a molecule or active fragment of a molecule that binds to a known antigen, and in particular to immunoglobulin molecules and immunologically active sites of immunoglobulins, i.e., molecules that contain a binding site that specifically binds to an antigen. As described herein, immunoglobulins can be of any class (IgG, IgM, IgD, IgE, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules and can be based on heavy chain sequences from any species. For example, the species can be, but is not limited to, dog, cat, horse, cow, pig, guinea pig, mouse, rat, etc. The species can be a primate (e.g., a non-human primate). In a preferred example, the species is human.

[0198] The term "antibody" or "antibodies" includes monoclonal, polyclonal, chimeric, single chain, bispecific, human, and humanized antibodies, as well as active fragments thereof. Examples of active fragments of molecules that bind to known antigens and are useful include Fab, bispecific Fab2, trispecific Fab3, scFv, bispecific bivalent (di-)scFv, bispecific scFv-Fc, bispecific diabodies, duobodies, trispecific triabodies, single domain antibodies, and bispecific minibodies, including epitope-binding fragments of any of the above antibodies and fragments, as well as the products of a Fab immunoglobulin expression library.

[0199] In certain instances, the antibody may be a monoclonal antibody. As used herein, the term "monoclonal antibody" refers to an antibody that can be produced in large quantities in a manufacturing facility from a single clone and recognizes only one antigen. Monoclonal antibodies can be produced by any suitable technique known in the art (e.g., by production in HEK cells or insect cells, or by producing B-cell hybridomas).

[0200] As used herein, the term "chimeric antibody" refers to a monoclonal antibody that contains a variable region, i.e., a binding region, derived from one source or species (i.e., non-human primate, human, dog, cat, horse, cow, pig, guinea pig, mouse, rat, etc.), typically prepared by recombinant DNA technology, and at least one portion of the constant region derived from a different source or species. An example is a chimeric antibody that contains a mouse variable region and a human constant region. Such chimeric antibodies are the product of expressed immunoglobulin genes that contain a DNA segment encoding a mouse immunoglobulin variable region and a DNA segment encoding a human immunoglobulin constant region. Other forms of "chimeric antibodies" encompassed by the present disclosure are those in which the class or subclass has been modified or changed from that of the original antibody, for example, changing them so that they are defective in complement and Fc receptor binding. Methods for producing chimeric antibodies include conventional recombinant DNA and gene transfection techniques that are now well known in the art. See, e.g., Morrison, SL, et al., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; US Pat. No. 5,202,238 and US Pat. No. 5,204,244.

[0201] In certain instances, the antibodies described herein are complement and Fc receptor binding deficient.

[0202] In certain instances, the antibody may be a human antibody or a humanized antibody.

[0203] As used herein, the term "humanized antibody" or "humanized form of antibody" refers to an antibody in which the framework or "complementarity determining regions" (CDRs) have been modified to include CDRs of an immunoglobulin of different specificity compared to the framework or "complementarity determining regions" (CDRs) of the parent immunoglobulin. In some instances, the CDRs of VH and VL are grafted into the framework regions of a human antibody to prepare a "humanized antibody". See, for example, Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, MS, et al., Nature 314 (1985) 268-270. The heavy chain variable framework region and the light chain variable framework region can be derived from the same or different human antibody sequences. Both the heavy and light chains may be required for effective antigen (MuSK) binding. The human antibody sequence can be that of a natural human antibody. Human heavy and light chain variable framework regions are described, for example, in Lefranc, M.-P., Current Protocols in Immunology (2000) - Appendix 1P A.1P.1-A.1P.37 and are available, for example, via IMGT (the international ImMunoGeneTics information System®) (http: / / imgt.cines.fr) or via http: / / vbase.mrc-cpe.cam.ac.uk. Optionally, the framework regions can be modified by further mutations. Exemplary CDRs correspond to the CDRs exhibiting sequences recognizing the antigens mentioned above for the chimeric antibodies. In some examples, such humanized versions are chimerized with human constant regions.

[0204] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the state of the art (van Dijk, MA, and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germline immunoglobulin gene array into such germline mutant mice results in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Brueggemann, MD, et al., Year Immunol. 7 (1993) 33-40). Human antibodies can also be produced in phage display libraries (Hoogenboom, HR, and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, JD, et al., J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole, A. et al. and Boerner, P. et al. are also available for the preparation of human monoclonal antibodies (Cole, A., et al., Monoclonal Antibodies and Cancer Therapy, Liss, AR (1985) p. 77; and Boerner, P., et al., J. Immunol. 147 (1991) 86-95).

[0205] In particular examples, the antibody may be selected from a Fab, a bispecific Fab2, a trispecific Fab3, an scFv, a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody.

[0206] Other examples include single domain antibodies, which are widely known in the art, such as those found in camelids, including but not limited to llamas and alpacas; and cartilaginous fish, including but not limited to sharks.

[0207] As used herein, "single chain antibody" refers to a single chain Fv molecule (scFv) in which the VH and VL domains are linked by a peptide linker that allows the two domains to join and form an antigen binding site (Bird et al., 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883, or bispecific single chain Fv (WO 03 / 11161)). Exemplary scFv linkers are well known in the art and are generally 10 to 25 amino acids in length and contain glycine and serine.

[0208] Single domain antibody fragments (VHH or nanobodies), such as functional antibodies produced by camelids lacking light chains and with one N-terminal domain fully capable of antigen binding, are also examples of binding agents as described herein. Such fragments can also form bivalent VHHs, or pentabodies (i.e. with five VHH domains).

[0209] As used herein, "bivalent ScFv (di-ScFv)" refers to a dimerized scFv.

[0210] As used herein, a "minibody" is a minimized antibody-like protein comprising an scFv linked to a CH3 domain. See Hu et al., 1996, Cancer Res. 56:3055-3061. In some cases, the scFv may be linked to an Fc region and may include some or the complete hinge region.

[0211] As used herein, "Fab" or "Fab region" refers to a polypeptide comprising the VH, CH1, VH, and CL immunoglobulin domains. Fab refers to this region alone or in the context of a full-length antibody or antibody fragment or fab fusion protein.

[0212] The terms "Fab", "Fab region", "Fab portion" or "Fab fragment" are understood to define a polypeptide comprising the VH, CH1, VL and CL immunoglobulin domains. Fab may refer to this region alone or in the antibody molecules described herein as well as to full-length immunoglobulins or immunoglobulin fragments. Typically, the Fab region comprises the entire light chain of an antibody. The Fab region may be said to define an arm of an immunoglobulin molecule. The Fab region comprises the epitope-binding portion of the Ig. The Fab region of a native immunoglobulin can be obtained as a proteolytic fragment by papain digestion. The "F(ab')2 portion" is a proteolytic fragment of a pepsin-digested immunoglobulin. The "Fab' portion" is the product resulting from reducing the disulfide bonds of the F(ab')2 portion. As used herein, the terms "Fab", "Fab region", "Fab portion" or "Fab fragment" further comprise the hinge region that defines the C-terminus of an antibody arm (see above). This hinge region corresponds to the hinge region found at the C-terminus of the CH1 domain in full-length immunoglobulins, where the arms of an antibody molecule can be defined as Y. The term hinge region is used in the art because immunoglobulins have some flexibility in this region.

[0213] As used herein, "Fc fusion" refers to a protein in which one or more polypeptides are operatively linked to Fc. Fc fusion is herein meant to be synonymous with the terms "immunoadhesin", "Ig fusion", "Ig chimera", and "receptor globulin" (sometimes with a dash) as used in the prior art (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200). Fc fusions combine the Fc region of an immunoglobulin with a fusion partner, which can generally be any protein, polypeptide, or small molecule. The role of the non-Fc portion of an Fc fusion, i.e., the fusion partner, is to mediate target binding and is thus functionally similar to the variable region of an antibody. Virtually any protein or small molecule can be linked to Fc to generate an Fc fusion. Protein fusion partners may include, but are not limited to, the target binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain. Small molecule fusion partners may include any therapeutic agent that directs the Fc fusion to a therapeutic target. Such a target may be any molecule, for example, an extracellular receptor involved in a disease.

[0214] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody, such as, in some cases, the variable domain thereof, or at least the antigen-binding site thereof. Examples of antibody fragments include diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. scFv antibodies are described, for example, in Huston, JS, Methods in Enzymol. 203 (1991) 46-88. Antibody fragments can be derived from the antibodies described herein by a number of techniques known in the art. For example, purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and subjected to HPLC gel filtration. The appropriate fractions containing Fab fragments can then be collected and concentrated, such as by membrane filtration. For further description of general techniques for isolating active fragments of antibodies, see, e.g., Khaw, BA et al. J. Nucl. Med. 23:1011-1019 (1982); Rousseaux et al. Methods Enzymology, 121:663-69, Academic Press, 1986.

[0215] The binding proteins provided herein can be encoded by nucleic acid molecules that are codon optimized to increase protein expression in a suitable host (e.g., HEK cells or insect cells). Codon optimization and protein production techniques are well known in the art and can be readily adapted to the production of the binding proteins described herein.

[0216] In certain instances, the binding proteins described herein may be IgG. The IgG may be selected from IgG1, IgG2, or IgG3. In alternative instances, the binding proteins described herein may be IgG4 variants (described in more detail below).

[0217] In vivo, IgG4 naturally undergoes Fab arm exchange, rendering it functionally bispecific and preventing antigen cross-linking and internalization (van der Neut Kolfschoten et al., 2007). As used herein, the term "Fab arm exchange" or "FAE" refers to the exchange and recombination of a complete heavy-light chain pair (half-molecule or half-IgG) of an antibody with a complete heavy-light chain pair of another antibody. That is, "Fab arm exchange" is used herein (and more broadly literally) to refer to the process that results in the exchange of "half molecules" of human IgG4 (half-IgG exchange - see e.g., Schuurman et al., mAbs 4:6, 636-636 Nov / Dec 2012). Fab arm exchange is also known as IgG4 shuffling, where the increased susceptibility of the original IgG4 hinge disulfide bond to reduction allows the heavy chains to separate and randomly reassociate, generating a mixed population of IgG4 molecules with randomized heavy and light chain pairs (Aalberse et al., 1999. Int Arch Allergy Immunol 118: 187-189; Labrijn, et al, 2009, Nat Biotechnol 27:767-771; Schuurman et al, 2001. Mol Immunol 38: 1-8; van der Neut Kolfschoten et al, 2007. Science 317: 1554-1557). See also Makaraviciute A, Jackson CD, Millner PA, Ramanaviciene A. J Immunol Methods. 2016 Feb;429:50-6. The terms "Fab arm exchange", "half-antibody exchange" and "half-IgG exchange" are therefore used interchangeably herein.

[0218] In the case of the present invention, and in the case of binding proteins with multiple variable domains (e.g., bispecific binding proteins having only one variable region specific for the Ig-like 1 domain of a MuSK protein), it is advantageous to reduce or eliminate the ability of IgG4 to undergo Fab arm exchange (i.e., ensuring that the binding protein retains only one variable region that specifically binds to the Ig-like 1 domain of a MuSK protein and therefore maintains its MuSK antagonist properties), or to use the introduction of a binding region that specifically binds to the Ig-like 1 domain of a MuSK protein into a background of IgG1 (or IgG2 or IgG3) (i.e., only having one binding region that specifically binds to the Ig-like 1 domain of a MuSK protein, as described elsewhere herein), such that the resulting IgG is bispecific.

[0219] Thus, the binding proteins described herein can be IgG4 variants with reduced capacity for Fab arm exchange in vivo (e.g., IgG4 cannot undergo Fab arm exchange). This type of binding protein is particularly useful because IgG4 antibodies cannot bind complement and cannot recruit immune cells, and are therefore considered "anti-inflammatory" antibodies (Lighaam et al., 2016). Thus, the IgG4 variants described herein are less likely to induce an inflammatory response when administered to a subject as described herein (compared to the corresponding IgG1, IgG2, or IgG3).

[0220] IgG4 antibody variants with reduced capacity for in vivo Fab arm exchange can therefore be generated for use in the methods described herein, where the IgG4 variants comprise two variable regions, with only one variable region of the binding protein specifically binding to the Ig-like 1 domain of the MuSK protein.

[0221] An IgG4 variant with a "reduced capacity for Fab arm exchange in vivo" refers to an IgG4 that has a lower rate of Fab arm exchange than a control IgG4 antibody under the same (in vivo) conditions. A "reduced capacity" refers to a reduction in Fab arm exchange (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% reduction in the rate of Fab arm exchange over a period of time (set from the start of the study, e.g., administration of the IgG4 to a subject, such as a mouse or human subject)). Methods for measuring the rate of Fab arm exchange are known in the art.

[0222] The reduction of IgG4 shuffling / Fab arm exchange can be determined by detecting a lower amount of half antibody molecules produced from an IgG4 variant described herein (e.g., comprising one or more amino acid substitutions in the hinge or CH3 region) compared to the amount of half antibody molecules or arm exchanges produced from an IgG4 molecule comprising an IgG4 hinge or CH3 region that does not comprise one or more amino acid substitutions, detecting new bispecific antibodies or arm exchanges. Any assay well known in the art can be used to detect half antibody production or bispecific antibody molecules. For examples of assays to detect the production of bispecific antibodies, see Van der Neut Kolfschoten et al, 2007, Science, 317: 1554-1557.

[0223] As used herein, an IgG4 variant "not capable of undergoing Fab arm exchange" means that the IgG4 variant is not capable of exchanging and reassociating a complete heavy-light chain pair (half molecules) of the antibody with a complete heavy-light chain pair of another antibody in vivo (e.g. when established from the time of administration of the IgG4 to a subject, such as a mouse or human subject, and measured over a period of 24 hours) or in vitro (e.g. when using a reducing agent, such as glutathione). That is, in this context, an IgG4 variant that is bivalent and bispecific (i.e. one arm that specifically binds to the Ig-like 1 domain of the MuSK protein and one arm that binds to an unrelated protein) will not be capable of Fab arm exchange and will therefore remain bispecific in vivo (e.g. when established from the start of the study, e.g. administration of the IgG4 to a subject, such as a mouse or human subject, and measured over a period of time). Methods for determining whether an IgG4 undergoes Fab arm exchange are known in the art and described above.

[0224] In one example, the IgG4 variant comprises an IgG4 constant region that contains one or more amino acid substitutions that reduce the capacity for Fab arm exchange in vivo.

[0225] For example, an IgG4 variant cannot undergo Fab arm exchange because it comprises an IgG4 constant region containing an amino acid substitution at amino acid position 228 of the heavy chain and / or an amino acid substitution at amino acid position 405 and / or an amino acid substitution at amino acid position 409, numbered according to the EU index (see Edelman et al., 1969, Proc. Natl. Acad. Sci. USA, 63(1): 78-85 and Huijbers 2016). One or more of these amino acid substitutions in the hinge or CH3 region of human IgG4 results in a substantial reduction in intrachain disulfide bond formation, leading to a reduction in IgG4 "half antibody" molecules and a reduction in heterogeneity / shuffling of IgG4 molecules (Bloom et al. 1997, Protein Sci, 6:407- 415; Angal et al, 1993, Molecular Immunology, 30(1): 105-108). These hinge or CH3 mutations (or mutants that delete residues required for Fab arm exchange) can reduce IgG4 shuffling and increase the half-life of IgG4 molecules in vivo (Labrijn, et al, 2009, Nat Biotechnol 27:767-771; Stubenrauch, et al, 2010, Drug Metab Dispos 38:84-91).

[0226] The IgG4 variants described herein may therefore comprise one or more amino acid substitutions in the IgG4 hinge or CH3 region, and IgG4 shuffling is reduced compared to an antibody comprising an IgG4 hinge or CH3 region that does not comprise said one or more amino acid substitutions. In a particular example, the IgG4 hinge or CH3 region comprises one amino acid substitution (either at position 228 (e.g., S228P), or at amino acid position 409 (e.g., R409K), or at amino acid position 405 (e.g., F405L) of the heavy chain, numbered according to the EU index). In another particular example, the IgG4 hinge region or CH3 region comprises two amino acid substitutions (positions 228 (e.g., S228P) and 409 (e.g., R409K) of the heavy chain, numbered according to the EU index; or positions 228 (e.g., S228P) and 405 (e.g., F405L); or positions 405 (e.g., F405L) and 409 (e.g., R409K) of the heavy chain). In another particular example, the IgG4 hinge region or CH3 region comprises three amino acid substitutions (positions 228 (e.g., S228P), 405 (e.g., F405L) and 409 (e.g., R409K) of the heavy chain, numbered according to the EU index). In other particular examples, the IgG4 region further comprises other amino acid substitutions, suitable substitutions being well known in the art.

[0227] Treatment of the subject The binding agents (eg, binding proteins, eg, antibodies) described herein are useful for treating diseases, disorders, and / or conditions in a subject that are alleviated by inhibition of neuromuscular transmission.

[0228] The binding agents (eg, binding proteins, eg, antibodies) described herein can also be used to prevent, control, or reduce skin wrinkles in a subject.

[0229] The terms "subject," "patient," and "individual" are used interchangeably herein.

[0230] In certain examples, the subject is a mammal. Thus, the subject refers to, for example, a dog, a cat, a horse, a cow, a pig, a guinea pig, a mouse, a rat, etc. The subject may be a primate (e.g., a non-human primate). In preferred examples, the subject is a human.

[0231] The subject is typically one in need of treatment for a disease, disorder, and / or condition that is alleviated by inhibition of neuromuscular transmission in the subject.

[0232] The binding agents (eg, binding proteins, e.g., antibodies) provided herein can be used for cosmetic (i.e., non-therapeutic) or therapeutic purposes, as outlined below.

[0233] The binding agents (e.g., binding proteins, e.g., antibodies) may be advantageously used to prevent, control, or reduce skin wrinkles in a subject. In one example, such uses are considered "cosmetic" and / or "non-therapeutic."

[0234] Accordingly, provided are methods of preventing, controlling, or reducing skin wrinkles in a subject, comprising administering to the subject a binding agent (e.g., a binding protein, e.g., an antibody) described herein. In one example, such methods are considered "cosmetic" and / or "non-therapeutic."

[0235] As used herein, the terms "cosmetic" and "non-therapeutic" are used interchangeably and are intended to refer to an intervention performed with the intent of treating (e.g., enhancing, preventing, or controlling) a cosmetic, non-pathological condition in a subject. For example, a cosmetic procedure may be used to repair or enhance the appearance of a subject.

[0236] Thus, the binding agents (e.g., binding proteins, e.g., antibodies) described herein can be used to prevent, control, or reduce skin wrinkles in a subject. Suitable methods of administering the binding agents for this purpose are well known in the art and are not limited to injection.

[0237] The method of preventing, controlling, or reducing skin wrinkles may also be considered as a method of increasing relaxation or slackening of skin tissue. The method may include topically administering a binding agent (e.g., a binding protein, e.g., an antibody) as described herein to skin tissue in an effective amount to increase denervation of a muscle or muscle population present in the skin tissue from the subcutaneous tissue, thereby increasing relaxation or slackening of the skin tissue. In some examples, the binding agent (e.g., a binding protein, e.g., an antibody) is administered subcutaneously.

[0238] Preferably, the relaxation or loosening of skin tissue results in a reduction in wrinkles or fine lines in the skin.

[0239] Advantageously, the binding agents (e.g., binding proteins, e.g., antibodies) described herein can also be used for therapeutic purposes. As used herein, the term "therapeutic" is intended to refer to the "treatment" of a subject.

[0240] As used herein, the terms "treat," "treating," and "treatment" include interventions performed with the intent of preventing the progression of a disease, disorder, or condition, or altering the pathology of a disease, disorder, or condition. Thus, "treatment" refers to both therapeutic treatment and prophylactic or preventive measures, where the subject is to prevent or slow (reduce) the targeted disease, disorder, or condition. As used herein, the terms "disease" and "disorder" are used interchangeably.

[0241] As used herein, the term "subject" refers to an individual (e.g., a human or non-human primate) having or at risk of having a particular disease, disorder, or condition. A subject may be a patient, i.e., a subject in need of treatment according to the present invention. A subject may have been treated for a disease, disorder, or condition. Alternatively, a subject has not been treated prior to treatment according to the present invention.

[0242] The binding agents (e.g., binding proteins, e.g., antibodies) provided herein may be used to treat or prevent diseases, disorders, and / or conditions that are alleviated by inhibition of neuromuscular transmission.

[0243] As will be made clear from the text herein, the binding agents (e.g., binding proteins, e.g., antibodies) described herein can be used to inhibit neuromuscular transmission. In this context, "inhibition" of neuromuscular transmission encompasses a decrease or reduction in neuromuscular transmission (e.g., when comparing the level of neuromuscular transmission in the absence of the binding agent to the level of neuromuscular transmission in the presence of the binding agent). For the avoidance of doubt, "inhibition," "inhibit," "reduce," "decreased," "reduced," or "reduction," and equivalent terms, as used herein, generally refer to a reduction by a statistically significant amount. "Inhibition," "inhibit," "decrease," "decreased," "reduced," or "reduction" typically refers to a decrease of at least 10%, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 10 ... By this is meant at least about a 90%, or up to a 100% reduction, and a 100% reduction, or any decrease between 10-100% as compared to a control (i.e., in the absence of binding agent), or at least about 0.5-fold, or at least about 1.0-fold, or at least about 1.2-fold, at least about 1.5-fold, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold reduction, or any reduction between 1.0-fold and 10-fold or more as compared to a control (i.e., in the absence of binding agent).

[0244] Those skilled in the art will be fully aware of diseases or disorders that are alleviated by the inhibition of neuromuscular transmission, since the use of botulinum toxin A has been widely used for medical and cosmetic treatments for many years (see, e.g., Jankovic (2004) Botulinum in clinical practice. J Neurol Neurosurg Psychiatry 75 951-957). In particular, some diseases or conditions that are alleviated by the inhibition of synaptic transmission mediated by botulinum toxin A are selected from the group consisting of dystonia, facial spasms, strabismus, hypertonia due to cerebral palsy, stuttering, chronic tension headaches, spasms of the inferior pharyngeal constrictor muscles of the pharynx, pain, migraine headaches, or cosmetic treatments, such as wrinkles, reduction of brow furrows, etc.

[0245] The binding agents (e.g., binding proteins, e.g., antibodies) described herein can be used to treat or prevent in a subject a disease, disorder, or condition that is alleviated by inhibition of neuromuscular transmission. Methods of treating or preventing a disease, disorder, or condition that is alleviated by inhibition of neuromuscular transmission are provided, comprising administering to a subject a binding agent (e.g., a binding protein, e.g., an antibody) described herein. Thus, methods of in vivo treatment are provided, which may be prophylactic and / or therapeutic.

[0246] As used herein, treating or preventing "diseases, disorders, and / or conditions that are alleviated by inhibition of neuromuscular transmission" is intended to include treating or preventing pain secondary to spasms or involuntary muscle activity, neurological disorders or diseases in a subject, conditions or diseases resulting from involuntary spasms, muscle spasticity, strabismus, occupational spasms, and anal fissures, migraines, teeth grinding, and combinations thereof.

[0247] In one example, the pain is pain associated with a neuromuscular disorder.

[0248] In one example, the condition or disease resulting from involuntary muscle spasms is selected from the group consisting of hemifacial spasm, blepharospasm, laryngeal dysphoria, head dystonia, neck dystonia, limb dystonia, or rectal spasms.

[0249] As used herein, treating or preventing "diseases, disorders, and / or conditions that are alleviated by the inhibition of neuromuscular transmission" is also intended to include correcting disfigurement in a subject caused by excessive muscle activity.

[0250] As used herein, "excessive muscle activity" refers to increased muscle activity compared to normal. Examples of distorted appearances that can be corrected using the binding agents described herein include muscle spasms and tics.

[0251] As used herein, treating or preventing "diseases, disorders, and / or conditions that are alleviated by the inhibition of neuromuscular transmission" (also referred to herein as treating or preventing "diseases, disorders, and / or conditions that result from excessive muscular activity") also intends to treat or prevent spasms or involuntary contractions of a muscle or group of muscles in a subject. Non-limiting examples of spasms or involuntary contractions include blepharospasm, strabismus, spasmodic torticollis, focal dystonia, jaw dystonia, occupational dystonia, corneal ulcer (protective ptosis), spasmodic dysphonia (laryngeal dystonia), or facial dyskinesis, e.g., Meige syndrome, hemifacial spasm, aberrant regeneration of the facial nerve, or apraxia of eyelid opening.

[0252] The present inventors have shown that the binding agents (e.g., binding proteins, e.g., antibodies) described herein can act as antagonists at the NMJ by inhibiting MuSK phosphorylation. Reduction of MuSK phosphorylation impairs AChR clustering and subsequently blocks neuromuscular transmission. The binding agents (e.g., binding proteins, e.g., antibodies) described herein can therefore be used to induce temporary, localized muscle weakness (e.g., paresis) in a subject. Longer periods of localized muscle weakness / paresis can also be induced (e.g., by multiple administrations). Thus, the binding agents can be used as an alternative to administering botulinum neurotoxin.

[0253] Botulinum neurotoxin type A (BoNT type A; or BoNT / A) has proven to be of great medical importance due to its ability to induce very long neuromuscular paralysis after local injection of minute amounts (1 pM concentration) (Pirazzini-M et al., Pharmacol Rev 2017;69:200). Over the past 30 years, BoNT / A and other botulinum neurotoxins have been successfully utilized for medicinal and cosmetic purposes. These toxins can also suppress NMJs and block the release of neurotransmitters from many types of neurons. Practically every part of the human body, including the brain (Botulinum toxin therapy for neuropsychiatric disorders, US 20040180061 A1), can be treated with BoNT / A. An example of a form of BoNT / A that has been successfully used for cosmetic treatments is Botox. Because NMJ paresis is reversible, sustained relaxation of the muscle requires repeated injections every 3 to 4 months. BoNT / A can block innervation of smooth muscle as well as striated muscle. Thus, botulinum-based therapy has now been expanded to include nearly 100 different disorders, from dystonia to gastrointestinal and urinary disorders.

[0254] The binding agents (e.g., binding proteins, e.g., antibodies) described herein may be intended to be administered to a subject in a therapeutically effective amount. A "therapeutically effective amount" refers to an amount of a drug, compound, antibody, or pharmaceutical binding agent that elicits a desired biological or medical response in the subject. Thus, in therapeutic applications described herein, the agent and / or composition thereof is administered to a subject in an amount sufficient to at least partially arrest a condition or disease and its complications. An amount sufficient to accomplish this is defined as a "therapeutically effective amount or dose." Amounts effective for this use depend on the severity of the disease, as well as the weight and general condition of the patient.

[0255] A binding agent (e.g., a binding protein, e.g., an antibody) as described herein can be part of a composition (e.g., a pharmaceutical composition) that includes the binding agent and one or more other components. The composition can be a composition that includes a binding agent as described herein, and a pharma- ceutically acceptable excipient, adjuvant, diluent, and / or carrier. The composition can include pharma- ceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, supplemental immune enhancers, e.g., adjuvants and cytokines, and optionally other therapeutic agents or compounds, as applicable.

[0256] As used herein, "pharmaceutical acceptable" refers to a substance that is biologically or otherwise undesirable, i.e., the substance may be administered to an individual with a selected binding agent contained in the pharmaceutical composition without causing any undesired biological effects and without interacting in a deleterious manner with any other components of the pharmaceutical composition.

[0257] An excipient is a natural or synthetic substance that is included to enhance the formulation or to provide a therapeutic improvement to the active ingredient in the final dosage form, for example, to facilitate drug absorption or solubility, and is combined with the active ingredient (e.g., a binding agent, as provided herein). Excipients may be useful in the manufacturing process and aid in the handling of the active ingredient in question, such as by promoting powder flow or non-adherent properties, as well as promoting in vitro stability, e.g., preventing degradation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. Suitable excipients are therefore easily identified by those of skill in the art. By way of example, suitable pharma-ceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.

[0258] Adjuvants are pharmacological and / or immunological substances that modify the effect of other substances in the formulation. Pharmaceutically acceptable adjuvants are well known in the art. Suitable adjuvants are therefore easily ascertainable by those skilled in the art.

[0259] A diluent is a substance that dilutes. Pharmaceutically acceptable diluents are well known in the art. Suitable diluents are therefore readily ascertainable by those skilled in the art.

[0260] The carrier is non-toxic to the recipient at the dosage and concentration used and is compatible with other ingredients in the formulation. The term "carrier" refers to an organic or inorganic ingredient, natural or synthetic, that is combined with the active ingredient to facilitate application. Pharmaceutically acceptable carriers are well known in the art. Suitable carriers are therefore easily ascertainable by those skilled in the art.

[0261] In general, the binding agent (e.g., a binding protein, e.g., an antibody) is administered in a pharma- ceutical effective amount. The amount of binding agent actually administered is typically determined by a physician in light of the relevant circumstances, including the disease being treated, the route of administration selected, the actual compound being administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like. Pharmaceutical compositions can be administered by various routes, including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and nasal. The compositions are typically present in unit dosage forms to facilitate accurate dosing. The term "unit dosage form" refers to physically discrete units suitable as unitary doses for human subjects and other mammals, each unit containing a calculated, predetermined amount of active agent, together with suitable pharmaceutical excipients, to produce the desired therapeutic effect. Typical unit dosage forms include prefilled, premeasured ampoules or syringes of liquid compositions, or pills, tablets, capsules, and the like, in the case of solid compositions. In such compositions, the furansulfonic acid compound will usually be a minor component (from about 0.1% to about 50% by weight, or preferably from about 1% to about 40% by weight), with the remainder being various vehicles or carriers and processing aids which aid in forming the desired dosage form.

[0262] Liquid forms suitable for oral administration may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors, etc. Solid forms may, for example, include any of the following ingredients, or compounds of a similar nature: binders such as microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose; disintegrating agents such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0263] Injectable compositions are typically based on injectable sterile saline, or phosphate buffered saline, or other injectable carriers known in the art. As mentioned above, the active compound in such compositions is typically a minor component, often 0.05% to 10% by weight, with the remainder being the injectable carrier, etc.

[0264] Transdermal compositions are typically formulated as topical ointments or creams containing the active ingredient in an amount generally ranging from about 0.01% to about 20% by weight, preferably from about 0.1% to about 20% by weight, preferably from about 0.1% to about 10% by weight, and more preferably from about 0.5% to about 15% by weight. When formulated as an ointment, the active ingredient is typically combined with either a paraffinic or a water-miscible ointment base.

[0265] Preferably, the composition is an injectable or a transdermal composition.

[0266] The ingredients described above for the pharmaceutical compositions are merely representative. Other materials and processing techniques are described in Part 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania.

[0267] The compounds described herein can also be administered in long-term release forms or in long-term release drug delivery systems. A description of representative long-term release materials can be found in Remington's Pharmaceutical Sciences.

[0268] The compounds described herein can be administered as the sole active agent or can be administered in combination with other agents, including other compounds that demonstrate the same or similar therapeutic activity and that have been determined to be safe and effective for combined administration.

[0269] method Provided is a method for preventing, controlling, or reducing skin wrinkles in a subject, the method comprising administering to the subject a binding agent (e.g., a binding protein, e.g., an antibody) that comprises one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein.

[0270] The method is considered cosmetic.

[0271] The characteristics of suitable binding agents, targets, pharmaceutical compositions, means for administration, and tests for determining successful administration are described in detail above and apply equally to this embodiment.

[0272] Also provided is a method for treating or preventing a disease, disorder, and / or condition that is alleviated by inhibition of neuromuscular transmission, comprising administering to a subject a binding agent (e.g., a binding protein, e.g., an antibody) comprising one or more binding regions, where only one of the binding regions specifically binds to the Ig-like 1 domain of a MuSK protein.

[0273] The binding agents are intended to be administered to a suitable subject in a therapeutically effective amount.

[0274] A therapeutically effective amount may be sufficient to reduce MuSK activity in a subject, thereby alleviating a disease, disorder, and / or condition associated with increased (increased or abnormal) levels of neuromuscular transmission in the subject.

[0275] The characteristics of suitable binding agents, subjects, diseases, disorders and conditions, pharmaceutical compositions, means for administration, and tests for determining successful treatment are described in detail above and apply equally to this embodiment.

[0276] As used herein, "decreasing" refers to down-regulation.

[0277] As used herein, "elevated" refers to higher than normal, increased, or upregulated.

[0278] As used herein, "MuSK activity" refers to both biological and functional activity of a MuSK protein, polypeptide, or nucleic acid molecule (e.g., in a MuSK-expressing cell or tissue) determined in vivo or in vitro according to standard techniques. MuSK activity includes stimulation of MuSK dimerization, phosphorylation of MuSK, or assembly of AChRs on the cell membrane at the NMJ. MuSK activation can result in induction or maintenance of postsynaptic differentiation.

[0279] Several methods for measuring neuromuscular transmission are known in the art, for example electromyographic or electrophysiological evidence may be used.

[0280] Preparation of medicines In another aspect, the invention provides for the use of a binding agent (e.g., a binding protein, e.g., an antibody) comprising one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein, in the preparation of a medicament for treating a disease, disorder, and / or condition in a subject that is alleviated by inhibition of neuromuscular transmission. The medicament is for administration to a suitable subject in a therapeutically effective amount.

[0281] The characteristics of suitable binding agents, subjects, disorders and conditions, pharmaceutical compositions, means for administration, and tests for determining successful treatment are described in detail above and apply equally to this embodiment.

[0282] The present invention can be better understood with reference to the following non-limiting examples, which are provided as illustrations of the present invention. The following examples are presented to fully illustrate the preferred embodiments of the present invention, but should not be construed as limiting the broad scope of the present invention in any way. EXAMPLES

[0283] The data provided below demonstrate that functionally monovalent MuSK antibodies (isolated from patients) that bind to the Ig-like 1 domain of MuSK act as antagonists. In contrast, the same antibodies in a bivalent, monospecific form act as agonists. Because Fab arm exchange causes serum IgG4, and IgG4 patient MuSK antibodies cause MG in animal models and humans, the monovalency of these antibodies is believed to be a determining factor in the pathophysiology of MuSK MG.

[0284] result Description of included MuSK MG patients For isolation of MuSK-specific B cells, peripheral blood mononuclear cells (PBMCs) were isolated from two MuSK MG patients. The clinical characteristics of these patients are described in Table 2. The patient producing the most MuSK-specific B cells had relapsed after rituximab treatment.

[0285] [Table 2]

[0286] Isolation and genetic characterization of MuSK autoantibodies from MuSK MG patients Antigen-specific single-cell sorting yielded eight MuSK-binding B cells from two MuSK MG patients. The frequency of circulating MuSK clones was approximately 7 per 100 million PBMCs for patient 1 and 2.5 per 100 million PBMCs for patient 2. An overview of the characteristics of MuSK autoantibodies is given in Table 3. For six of the eight MuSK autoantibody-producing clones, we identified heavy chain (V H ) and light chain (V L We were able to isolate the variable region sequences of V H Only the variable regions could be sequenced.

[0287] [Table 3]

[0288] Interestingly, the majority of the isolated antibodies were of the IgG1 isotype. Furthermore, we identified one IgG4 and one IgG3 clone.

[0289] Fab arm exchange is an important feature of IgG4 and may affect the functional properties of MuSK autoantibodies (Koneczny et al., 2017). Previous studies on purified polyclonal fractions suggested that MuSK MG IgG4 has the ability to undergo Fab arm exchange (Koneczny et al., 2017). We investigated the V of isolated MuSK monoclonal antibodies. H were sequenced to confirm that these antibodies indeed possess the serine at position 228 and arginine at position 409 necessary to undergo Fab arm exchange (data not shown).

[0290] N-linked glycosylation of Fabs may be important for antigen binding, for example in rheumatoid arthritis ACPA autoantibodies (van de Bovenkamp et al., 2018). Therefore, we evaluated the presence of NXST (X cannot be P) motifs in monoclonal MuSK autoantibody variable regions. Only one clone contained such a motif, suggesting that Fab glycosylation is not essential for all MuSK autoantibodies.

[0291] Functional characterization of recombinant MuSK monoclonal autoantibodies The epitopes bound by autoantibodies are important determinants of pathological mechanisms in autoimmune diseases. The Ig-like 1 domain of MuSK was previously identified as the major immunogenic region of MuSK in polyclonal IgG4 patient antibody fractions and serum (Huijbers et al., 2016). For five of the patient-derived MuSK antibodies, epitopes could be mapped to the first Ig-like 1 domain of MuSK (Table 3). No monoclonal autoantibodies against other domains have been identified so far. To define the functional properties of MuSK autoantibodies, recombinant antibodies were produced from IgG1 and IgG4 isolated clones. To assess the importance of the autoantibody isotype in MuSK MG, each of these variable regions was cloned in both IgG1 and IgG4 backbones.

[0292] To assess the ability of patient-derived IgG1 and IgG4 recombinant MuSK antibodies to bind MuSK at the postsynaptic membrane of the NMJ, we performed immunostaining of isolated mouse skeletal muscle. Both the IgG1 and IgG4 forms of the recombinant monoclonal antibodies bound clearly to the NMJ (Figure 1, data shown for the IgG4 recombinant antibody).

[0293] Successful neuromuscular transmission depends on properly clustered AChRs and is therefore tightly regulated through the agrin-Lrp4-MuSK signaling cascade (Burden et al., 2018). Agrin is released by motor neuron terminals and binds to Lrp4, which then binds to MuSK and stimulates MuSK dimerization and transphosphorylation. Activation of MuSK phosphorylation stimulates various intracellular signaling cascades, one of which ends with AChR clustering. Purified patient IgG4 MuSK autoantibodies inhibit MuSK-Lrp4 interaction, MuSK phosphorylation, and thereby reduce AChR clustering in C2C12 myotube cultures and mice (Huijbers & Zhang 2013, Koneczny et al., 2013). Interestingly, when the effect of patient-derived MuSK monoclonal antibodies was assessed on MuSK phosphorylation, both recombinant IgG1 and IgG4 (bivalent and monospecific) antibodies strongly activated, rather than inhibited, MuSK phosphorylation (Fig. 2A). This effect was seen both in the absence and presence of agrin. Activation of MuSK phosphorylation was furthermore concentration-dependent (Fig. 2B) and slightly different between the two clones. Monovalent Fab antibody fragments generated by papain digestion from the same antibodies inhibited MuSK phosphorylation (Fig. 2C).

[0294] Both recombinant monoclonal IgG1 and IgG4, however, are involved in bivalent and monospecific antibody-antigen interactions. To investigate the functional effects of the bispecificity and functional monovalency of Fab-arm exchanged IgG4 MuSK antibodies in patients, we generated monovalent Fab fragments from these recombinant antibodies by papain digestion. In vitro, these Fab fragments inhibited agrin-dependent MuSK phosphorylation (Figure 2C) and AChR clustering (Figure 2D), similar to anti-MuSK IgG4 from patient serum.

[0295] In contrast (and consistent with activating MuSK phosphorylation in vitro), bivalent monospecific monoclonal MuSK antibodies activated agrin-dependent AChR clustering compared to Fab fragments. Moreover, AChR clustering could be partially induced using bivalent monospecific antibodies, independent of agrin (Figure 2D). Thus, while monovalent MuSK binding blocks the AChR clustering pathway, bivalent monospecific MuSK antibodies can stimulate MuSK to promote or induce AChR clustering in this tissue culture model.

[0296] These observations suggest that substances with two MuSK Ig-like 1 domain binding sites have the ability to activate MuSK, whereas substances with only one MuSK Ig-like 1 domain binding site (similar to what was seen with patient-purified IgG4 or Fab fragments) inhibit MuSK activation.

[0297] MuSK antibody characterization MuSK antibodies (isolated from myasthenia gravis patients with MuSK serum antibodies) that bind to the Ig-like 1 domain of MuSK were isolated and sequenced as described below. As shown in Figures 3 and 4, a total of eight MuSK Ig-like 1 domain-reactive antibodies (11-3D9, 11-3F6, 11-8G4, 13-3B5, 13-3D10, 13-4D3, 14-2E9, and 11-7C5) were obtained from MuSK MG patients.

[0298] Variable region sequences / binding regions specific for binding to the Ig-like 1 domain of MuSK protein Variable region amino acid sequences were obtained for seven of these clones (11-3D9, 11-3F6, 11-8G4, 13-3B5, 13-3D10, 13-4D3, and 14-2E9). The variable region of an antibody typically consists of a heavy chain variable domain (VH) and a light chain variable domain (VL). Each VH and VL typically has CDR1, CDR2, and CDR3 sequences that together confer specificity for the target antigen (i.e., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 together confer specificity for the target antigen). However, as described herein above, a single VH or VL domain (i.e., VH CDR1, VH CDR2, and VH CDR3; or VL CDR1, VL CDR2, and VL CDR3) may be sufficient to confer antigen-binding specificity.

[0299] For six clones, both the heavy and light chain variable domains were successfully sequenced, and for one clone (11-8G4), only the heavy chain variable domain sequence was successfully obtained. The heavy and light chain variable domain sequences are provided below for each clone. The light chain type is also indicated below: HC = heavy chain; LC = light chain; VH = heavy chain variable domain; VL = light chain variable domain. The underlined amino acid sequences represent the CDRs (N to C terminus of the amino acid sequence: CDR1, CDR2, CDR3).

[0300] Antibody sequences were selected and analyzed with respect to Homo sapiens sequences using the IMGT / V-QUEST program version: 3.4.17 (19 February 2019) - IMGT / V-QUEST Reference Directory Release: 201910-2 (5 March 2019) (http: / / imgt.org / IMGT_vquest / vquest).

[0301] 11-3D9 VH (SEQ ID NO:9):

[0302] [ka]

[0303] That is, the heavy chain variable domain (VH) CDRs may be as follows: CDR1 comprising GFNFSTYT (SEQ ID NO: 10), CDR2 comprising ISSRSAYK (SEQ ID NO: 11), and CDR3 comprising ARDFFQLGPPRFDS (SEQ ID NO: 12). These CDRs may optionally be present in a VH comprising the sequence of SEQ ID NO: 9.

[0304] 11-3D9 VLk (SEQ ID NO: 13):

[0305] [ka]

[0306] That is, the light chain variable domain (VL) CDRs may be as follows: CDR1 comprising QRISSF (SEQ ID NO: 14), CDR2 comprising GAS (SEQ ID NO: 15), and CDR3 comprising QQSYSPMYT (SEQ ID NO: 16). These CDRs may optionally be present in a VL comprising the sequence of SEQ ID NO: 13.

[0307] Antigen specificity can be obtained by combining the CDRs (heavy chain) of SEQ ID NO: 10, 11, and 12 and the CDRs (light chain) of SEQ ID NO: 14, 15, and 16. For example, antigen specificity can be obtained by combining the variable domains of SEQ ID NO: 9 (heavy chain) and SEQ ID NO: 13 (light chain).

[0308] 11-3F6 VH (SEQ ID NO: 17):

[0309] [ka]

[0310] That is, the heavy chain variable domain CDRs may be as follows: CDR1 comprising GFTFSSYT (SEQ ID NO: 18), CDR2 comprising IGSNGDYI (SEQ ID NO: 19), and CDR3 comprising ARGQLAVAGTHFDY (SEQ ID NO: 20). These CDRs may optionally be present in a VH comprising the sequence of SEQ ID NO: 17.

[0311] 11-3F6 VLk (SEQ ID NO:21):

[0312] [ka]

[0313] That is, the light chain variable domain CDRs may be as follows: CDR1 comprising QKVNKY (SEQ ID NO:22), CDR2 comprising AAS (SEQ ID NO:23), and CDR3 comprising QQSYSPLCT (SEQ ID NO:24). These CDRs may optionally be present in a VL comprising the sequence of SEQ ID NO:21.

[0314] Antigen specificity can be obtained by combining the CDRs (heavy chain) of SEQ ID NO: 18, 19, and 20 and the CDRs (light chain) of SEQ ID NO: 22, 23, and 24. For example, antigen specificity can be obtained by combining the variable domains of SEQ ID NO: 17 (heavy chain) and SEQ ID NO: 21 (light chain).

[0315] 11-8G4 VH (SEQ ID NO:25) (VL sequence was not available for this antibody)

[0316] [ka]

[0317] That is, the heavy chain variable domain CDRs may be as follows: CDR1 comprising GFTFSDFT (SEQ ID NO:26), CDR2 comprising IGSSGTFI (SEQ ID NO:27), and CDR3 comprising ARGRIAVAGTHFDL (SEQ ID NO:28). These CDRs may optionally be present in a VH comprising the sequence of SEQ ID NO:25.

[0318] 13-3B5 VH (SEQ ID NO:29):

[0319] [ka]

[0320] That is, the heavy chain variable domain CDRs may be as follows: CDR1 comprising GYTFTGQY (SEQ ID NO: 30), CDR2 comprising INPSSGVT (SEQ ID NO: 31), and CDR3 comprising ATLSLGVYYVGMVA (SEQ ID NO: 32). These CDRs may optionally be present in a VH comprising the sequence of SEQ ID NO: 29.

[0321] 13-3B5 VLl variable region (SEQ ID NO:33)

[0322] [ka]

[0323] That is, the light chain variable domain CDRs may be as follows: CDR1 comprising GLAQQH (SEQ ID NO:34), CDR2 comprising KDI (SEQ ID NO:35), and CDR3 comprising QSGDRTATSVL (SEQ ID NO:36). These CDRs may optionally be present in a VL comprising the sequence of SEQ ID NO:33.

[0324] Antigen specificity can be obtained by combining the CDRs (heavy chain) of SEQ ID NOs: 30, 31, and 32 and the CDRs (light chain) of SEQ ID NOs: 34, 35, and 36. For example, antigen specificity can be obtained by combining the variable domains of SEQ ID NO: 29 (heavy chain) and SEQ ID NO: 33 (light chain).

[0325] 13-3D10 VH (SEQ ID NO:37)

[0326] [ka]

[0327] That is, the heavy chain variable domain CDRs may be as follows: CDR1 comprising GFDFSAST (SEQ ID NO: 38), CDR2 comprising VSGDSHHI (SEQ ID NO: 39), and CDR3 comprising ARERLLRLGVGFDS (SEQ ID NO: 40). These CDRs may optionally be present in a VH comprising the sequence of SEQ ID NO: 37.

[0328] 13-3D10 VLk (SEQ ID NO: 41)

[0329] [ka]

[0330] That is, the light chain variable domain CDRs may be as follows: CDR1 comprising QRISGF (SEQ ID NO: 42), CDR2 comprising AAS (SEQ ID NO: 43), and CDR3 comprising QQSYSPLYT (SEQ ID NO: 44). These CDRs may optionally be present in a VL comprising the sequence of SEQ ID NO: 41.

[0331] Antigen specificity can be obtained by combining the CDRs (heavy chain) of SEQ ID NOs: 38, 39, and 40 and the CDRs (light chain) of SEQ ID NOs: 42, 43, and 44. For example, antigen specificity can be obtained by combining the variable domains of SEQ ID NO: 37 (heavy chain) and SEQ ID NO: 41 (light chain).

[0332] 13-4D3 VH (SEQ ID NO: 45)

[0333] [ka]

[0334] That is, the heavy chain variable domain CDRs may be as follows: CDR1 comprising GFTFSSYT (SEQ ID NO: 46), CDR2 comprising ISSGGHYI (SEQ ID NO: 47), and CDR3 comprising ARERLLRLGVGFDF (SEQ ID NO: 48). These CDRs may optionally be present in a VH comprising the sequence of SEQ ID NO: 45.

[0335] 13-3D4 VLk (SEQ ID NO: 49)

[0336] [ka]

[0337] That is, the light chain variable domain CDRs may be as follows: CDR1 comprising QSISGY (SEQ ID NO:50), CDR2 comprising AAS (SEQ ID NO:51), and CDR3 comprising QQSYSALYT (SEQ ID NO:52). These CDRs may optionally be present in a VL comprising the sequence of SEQ ID NO:49.

[0338] Antigen specificity can be obtained by combining the CDRs (heavy chain) of SEQ ID NOs: 46, 47, and 48 and the CDRs (light chain) of SEQ ID NOs: 50, 51, and 52. For example, antigen specificity can be obtained by combining the variable domains of SEQ ID NO: 45 (heavy chain) and SEQ ID NO: 49 (light chain).

[0339] 14-2E9 VH (SEQ ID NO:53)

[0340] [ka]

[0341] That is, the heavy chain variable domain CDRs may be as follows: CDR1 comprising GFTFSSYW (SEQ ID NO:54), CDR2 comprising LNEDGSTT (SEQ ID NO:55), and CDR3 comprising VSDLSGKDEH (SEQ ID NO:56). These CDRs may optionally be present in a VH comprising the sequence of SEQ ID NO:53.

[0342] 14-2E9 LCk (SEQ ID NO:57)

[0343] [ka]

[0344] That is, the light chain variable domain CDRs may be as follows: CDR1 comprising QSLLHSNGYYW (SEQ ID NO:58), CDR2 comprising LGF (SEQ ID NO:59), and CDR3 comprising MQGLQTPYT (SEQ ID NO:60). These CDRs may optionally be present in a VL comprising the sequence of SEQ ID NO:57.

[0345] The CDRs (heavy chain) of SEQ ID NOs: 54, 55 and 56 may be present together with the CDRs (light chain) of SEQ ID NOs: 58, 59 and 60. The variable domain (heavy chain) of SEQ ID NO: 53 and the variable domain (light chain) of SEQ ID NO: 57 may also be combined.

[0346] Antigen specificity can be obtained by combining the CDRs (heavy chain) of SEQ ID NO: 54, 55, and 56 and the CDRs (light chain) of SEQ ID NO: 58, 59, and 60. For example, antigen specificity can be obtained by combining the variable domain (heavy chain) of SEQ ID NO: 53 and the variable domain (light chain) of SEQ ID NO: 57.

[0347] Fc region sequence Sufficient cDNA from 5 clones was retained to determine the Fc sequence of the antibody. For subclass analysis, see below. Reference sequences were obtained from ensemble.org.

[0348] Reference sequence IgG1 (SEQ ID NO:61)

[0349] [ka]

[0350] IgG2 (SEQ ID NO:62)

[0351] [ka]

[0352] IgG3 (SEQ ID NO:63)

[0353] [ka]

[0354] IgG4 (SEQ ID NO:64)

[0355] [ka]

[0356] The cDNA generated from the B cell clones was sent for Sanger sequencing, which provided definitive data on the antibody Fc sequence. The DNA sequences were converted using https: / / web.expasy.org / translate / .

[0357] 11-3D9 clone amino acid sequence (translated from cDNA) - SEQ ID NO:65

[0358] [ka]

[0359] 11-8G4 clone amino acid sequence (translated from cDNA) - SEQ ID NO:66

[0360] [ka]

[0361] 13-3D10 clone amino acid sequence (translated from cDNA) - SEQ ID NO:67

[0362] [ka]

[0363] 13-4D3 clone amino acid sequence (translated from cDNA) - SEQ ID NO:68

[0364] [ka]

[0365] 13-3B5 clone amino acid sequence (translated from cDNA) - SEQ ID NO:69

[0366] [ka]

[0367] Using the multiple sequence alignment program Clustal W (1.83), we conclude that 11-3D9, 11-8G4, 13-3D10 all have a standard IgG1 sequence (see Figure 5). 13-3B5 has a standard IgG4 Fc tail, and 13-4D3 has an IgG3 Fc sequence. All variants of 13-4D3 are known on ensemble.org. Color coding indicates the similarity between the subclass reference sequences matched with the different antibodies. The subclass specificity of 11-3F6 and 14-2E9 was also determined by ELISA using an IgG1 specific antibody (see Figure 3).

[0368] Consideration One of the most striking and surprising findings of this study is the fact that (patient-derived) MuSK antibody titers and, consequently, MuSK autoantibody isotypes are key determinants in their ability to inhibit MuSK function and induce MG. Bivalent monospecific MuSK recombinant antibodies binding to the Ig-like 1 domain of MuSK acted as agonists and activated MuSK phosphorylation (and AChR clustering) in myotube cell cultures independently of agrin. Both the original IgG1 and IgG4 antibodies showed this effect. Conversely, functionally monovalent Fab fragments generated from these antibodies inhibited agrin-dependent MuSK phosphorylation (and AChR clustering) as did purified patient IgG4 and IgGtotal (Huijbers & Zhang et al. 2013, Koneczny et al., 2013). The ability of patient IgG4 to undergo Fab arm exchange is therefore likely to be important for disease development, as Fab arm exchange renders endogenously produced MuSK IgG4 bispecific and monovalent to the MuSK antigen. Previous studies have suggested that patient IgG4 has the ability to undergo Fab arm exchange (Koneczny et al. 2017 J autoimmunity), and the sequences of the MuSK monoclonal antibodies described herein confirm that the residues required for this process are present. The data suggest that bivalent monospecific MuSK antibodies that bind to the MuSK Ig-like 1 domain have the ability to dimerize and activate MuSK, while functionally monovalent MuSK Ig-like 1 domain antibodies have the ability to inhibit MuSK dimerization and therefore MuSK activation.

[0369] In summary, MuSK antibody titer is an important determinant of functional effects: bivalent monospecific antibodies with two MuSK Ig-like 1 domain binding sites stimulate MuSK, whereas monovalent or bispecific antibodies with only one MuSK Ig-like 1 domain binding site block MuSK function.

[0370] In conclusion, the results show that the functional monovalency of MuSK antibodies is important for the induction of myasthenic features.

[0371] Materials and Methods Patient Selection MuSK MG patients were recruited at the MG outpatient clinic of the Leiden University Medical Center and selected based on the presence of a positive MuSK antibody test (RSR ltd) and clinical findings consistent with MG. The study was conducted in accordance with the Declaration of Helsinki, approved by the local medical ethics committee, and all patients signed informed consent.

[0372] Isolation of monoclonal autoantibodies from MuSK MG patients MuSK-binding memory B cells were isolated from cryopreserved PBMCs and selected for CD19+, CD20+, and CD27+ cells (CD19-BV421 HIB19 BD, CD20-AF700 2H7 BD, CD27-APCHy7 M-T271 BD, 0,1% BSA, 2mM EDTA / dPBS, all monoclonal antibodies were mouse anti-human). A dump channel was included to exclude dead and non-B cells (7-AAD,00-6993-50, CD3 / FITC UCHT1 BD and CD14 / FITC M5E2, BD and CD56 / FITC HCD56 Biolegend). Selection for antigen-specific cells was performed by using recombinant MuSK produced in E. coli (Huijbers et al., 2016) labeled with R-PE (AS-72113, Anaspec) and MuSK produced in yeast labeled with DyLight 650 (kindly provided by Kostas Lazardinis and Socrates Tzartos, Dylight650 NHS ester, Thermofisher). Single cells were cultured on irradiated L-CD40L feeder cells (kindly provided by Kees van Kooten) in 96-well plates in RPMI medium supplemented with 1% penicillin, streptomycin, - 50uM beta-mercaptoethanol (M3148, Sigma), 20ug / ml human apotransferrin (T2036, Sigma-Aldrich, human IgG depleted using protein A sepharose [GE Healthcare]), 1ng / ml IL1b (201-LB-005, R&D), 50ng / ml IL-21 (PHC0215, Thermofisher), 0.3ng / ml TNFa (210-TA-005, Thermofisher), 0.5ug / ml R848 (SML0196, Sigma) (Lighaam et al., 2014). On average, 50 million PBMCs were used per patient to isolate between 1 and 6 antigen-specific cells.After 2 weeks, media was tested for MuSK-reactive antibodies using a MuSK ELISA as previously described (Huijbers et al., 2016). Briefly, recombinant MuSK was coated overnight on maxisorp plates (NUNC). The next day, plates were washed with PBS and blocked with 2% casein, 0.01% tween in PBS. To detect MuSK-bound antibodies, culture media was incubated 1:10 in blocking buffer and bound antibodies were detected using alkaline phosphatase-conjugated goat anti-human antibody (rabbit anti-human IgG AP, 309-055-008 Jackson Laboratories) and PNP as substrate. Plates were read at 405 nm on a Biotek plate reader.

[0373] RNA isolation, cDNA generation, and antibody sequence isolation After 2 weeks, plates were spun, supernatants were removed, and antibody analysis was performed as described above. A single well containing MuSK antibody producing cells was lysed with 150 μl Qiazol, and RNA was isolated using standard chloroform extraction and isopropanol / ethanol precipitation. RNA was rehydrated in 8 μl H2O and stored at -80°C until further use. cDNA was directly synthesized (without pre-amplification or purification) using Smartscribe reverse transcriptase (Takara Bio Europe), OligodT40 primers, and template switching oligos with 10 rounds of amplification (primer sequences in Table 4). Full-length V(D)J was obtained by ARTISAN PCR. Koning et al., 2017. HC primers represent the heavy chain.

[0374] [Table 4]

[0375] Table 4: Primer sequences. "dT40" represents 40 T nucleotides followed by VN, where "V" represents either A, C, or G nucleotides, and "N" represents any nucleotide (see https: / / www.mathworks.com / help / bioinfo / ref / baselookup.html). In BC-TSO, "rG" represents riboguanosine, and G+ represents one LNA modified guanosine. In hkappaLC rev primers, "M" represents A or C nucleotides, and in hlambdaLC rev primers, "Y" represents C or T nucleotides.

[0376] Recombinant antibody production, purification, and characterization Heavy and light chain sequences in IgG1 and IgG4 backbone pcDNA3.1 vectors were ordered from Geneart (Thermofisher) and transfected into suspension HEK293-F cells (R790-07, Thermofisher) in FreeStyle™ 293 Expression Medium (12338, Thermofisher) using Fectin (12347-019, Thermofisher). To increase transfection and production efficiency, cells were co-transfected with SV40 large T antigen, hp21, and hp27 (kindly provided by Theo Rispens). After 6 days, the culture medium was harvested, cell debris was removed by centrifugation, and IgG was purified on an Akta pure (GE Healthcare) using a HiTrap™ Protein A affinity column (17-0402-01, GE healthcare). Antibodies were dialyzed into PBS, filter-sterilized, and stored at -20°C. Fab fragments were generated from these recombinant antibodies using papain (20341, ThermoFisher) according to the manufacturer's instructions with the following adjustments: the input concentration of the antibodies was 0.5 mg / mL and the duration of digestion was 1 h for 13-3B5 and 3 h for 11-3F6 and anti-biotin (13-3B5 was lost if digestion took longer than 1 h). Depletion of total IgG and Fc fragments was performed with Protein A agarose beads (11134515001, Roche) for 2 h at room temperature, upside down.

[0377] To ensure that equal amounts of MuSK-specific Fab were used, concentrations were measured relative to the parental antibodies in the MuSK ELISA, substituting the antibody combination described above with mouse anti-human Fab (1:1000, SA1-19255, Pierce) and rabbit anti-mouse alkaline phosphatase antibody (1:750, D0314, Dako). Biotin Fab concentrations were determined by comparing MuSK Fab concentrations on Western blots using the same primary antibody and donkey anti-mouse 800CW (1:10.000, 925-32212, Licor). Bound antibody was detected using Odyssey CLx (Licor).

[0378] To determine the binding properties of the recombinant, culture-produced antibodies, we performed an epitope mapping ELISA as previously described (Huijbers et al., 2016). Truncated forms of MuSK protein were immobilized on maxisorp plates and exposed to either single cell culture-derived antibodies at 1:10 or recombinant antibodies. Recombinant antibodies were also used to immunostain mouse levator auris longus muscle at 1:100 overnight at 4°C. AChRs in synaptic regions were labeled with AlexaFluor488-conjugated alpha bungarotoxin (Life technologies) and bound recombinant antibodies were detected with AlexaFluor594-conjugated goat anti-human IgG (Life technologies). Muscles were imaged on a Leica SP8 confocal laser scanning microscope and analyzed using Las X software.

[0379] To determine the ability of antibodies to inhibit MuSK phosphorylation and AChR clustering, we exposed C2C12 myotube cell cultures (Cell lines service) to antibodies as previously described (Huijbers & Zhang et al 2013 PNAS). Differentiated myotubes were stimulated with 0.1 nM agrin (550-AG-100, R&D systems) in the presence or absence of 100 ng / mL recombinant antibodies or Fab fragments. For MuSK phosphorylation data, immunoprecipitation of MuSK was initiated 30 min after exposure. Myotubes were then lysed and MuSK was precipitated using 5 μL / sample of rabbit anti-MuSK polyclonal serum (ab94276 or ab94277, kindly provided by Steve Burden) during overnight incubation at 4°C (Huijbers & Zhang et al. 2013 PNAS). Bound antigen-antibody complexes were precipitated using extensively washed protein A agarose beads (11134515001, Roche). Samples were then run on SDS-PAGE gels and transferred to PDVF membranes. MuSK and phosphorylated MuSK were detected using goat anti-rat MuSK (1:2000, AF562, R&D systems) and mouse anti-phosphorylated tyrosine clone 4G10 (1:1000, 05-321, Millipore) as primary antibodies, as well as donkey anti-mouse 680RD. (1:10,000, 926-68072, Licor) and donkey anti-goat 800CW (1:10,000, 926-32214, Licor). Bound antibodies were detected using Odyssey CLx (Licor).

[0380] AChR clustering was investigated 16 hours after exposure of myotubes to 100 ng / mL recombinant antibodies or Fab fragments in the absence or presence of 0.1 nM agrin. Cells were then washed three times with differentiation medium (DMEM, 31966 Gibco, 2% heat-inactivated horse serum 26050-088, Gibco, 1% pen / strep, and 1% L-glutamine) and incubated with 0.5 μg / mL AlexaFluor488-conjugated alpha bungarotoxin (B13422, ThermoFisher) in differentiation medium for 30 min at 37°C. After staining, cells were fixed in 4% formalin for 5 min, washed with PBS, and mounted using hardset mounting medium (H-1500, Vector Laboratories). Twenty fields per condition, separated by two coverslips, were randomly selected and imaged with a Leica DM5500 microscope. AChR cluster counts and sizes were analyzed using ImageJ (1.48v).

[0381] statistics All data are expressed as the mean ± standard error of the mean (SEM). The statistical significance of differences between treatment groups was tested using Student's t test, with correction for multiple testing, where appropriate. Differences with a P value <0.05 were considered statistically significant.

[0382] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1 94); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those skilled in the art with a general dictionary of many of the terms used in the present invention. Although any methods and materials similar or equivalent to those described herein will suffice for use in the practice of the present invention, preferred methods and materials are described herein. Thus, the terms defined immediately below are more fully described by reference to the specification as a whole. Also, as used herein, the singular terms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary depending on the context in which they are used by those of skill in the art.

[0383] The reader's attention is directed to all articles and documents related to this application, filed contemporaneously with or prior to this application, and open to public inspection herein, the contents of all such articles and documents are incorporated herein by reference.

[0384] All of the features disclosed in this specification (including the accompanying claims, abstract, and drawings), and / or all of the steps of any method or process disclosed, may be combined in any combination, except where at least some of such features and / or steps are mutually exclusive.

[0385] Each feature disclosed in this specification (including the accompanying claims, abstract, and drawings), unless expressly stated otherwise, may be replaced by alternative features serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0386] The invention is not limited to the details of any of the above described embodiments, and extends to any novel, or any novel combination of features disclosed in this specification (including the accompanying claims, abstract and drawings), or any novel, or any novel combination of method or process steps disclosed.

[0387] array SEQ ID NO:1 MuSK (human isoform 205):

[0388] [ka]

[0389] SEQ ID NO:2 Ig-like 1 domain of human MuSK protein:

[0390] [ka]

[0391] [Table 5]

[0392] For sequences 9 to 69, see the Examples section above. For sequences 70 to 78, see FIG.

[0393] [References] TIFF2025016653000031.tif222170TIFF2025016653000032.tif214170

Claims

1. A binding substance comprising one or more binding regions, wherein only one of the binding regions specifically binds to the Ig-like 1 domain of a MuSK protein, for use in treating a disease, disorder, and / or condition in a subject that is alleviated by inhibition of neuromuscular transmission.

2. The binding agent for use according to claim 1 , wherein the binding agent is a binding protein.

3. 3. The method of claim 2, wherein the region that specifically binds to the Ig-like 1 domain of the MuSK protein is a variable region.

4. 4. A binding substance for use according to any one of claims 1 to 3, wherein the MuSK protein is a human MuSK protein.

5. 5. The binding substance for use according to any one of claims 1 to 4, wherein the binding substance is monovalent.

6. 5. The binding substance for use according to any one of claims 1 to 4, wherein the binding substance is bivalent or trivalent.

7. 7. A binding agent for use according to any one of claims 1 to 6, wherein the binding agent is an antibody.

8. The binding agent for use according to claim 7, wherein the antibody is a monoclonal antibody.

9. 9. The binding agent for use according to claim 7 or 8, wherein the antibody is a human antibody or a humanized antibody.

10. The antibody is a Fab, a bispecific Fab 2 , trispecific Fab 3 10. The binding agent for use according to any one of claims 7 to 9, which is selected from a bispecific bivalent scFv, a bispecific scFv-Fc, a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody.

11. 11. The binding agent for use according to any one of claims 7 to 10, wherein the antibody is an IgG.

12. 12. The binding agent for use according to claim 11, wherein the IgG is selected from IgG1, IgG2, or IgG3.

13. 12. The binding agent for use according to claim 11, wherein the IgG is an IgG4 variant that has a reduced capacity for or is unable to exchange Fab arms in vivo.

14. 14. The binding agent for use according to claim 13, wherein the IgG4 variant comprises an IgG4 constant region that comprises one or more amino acid substitutions that reduce the capacity for or prevent Fab arm exchange in vivo.

15. 15. The binding agent for use according to claim 13 or 14, wherein the IgG4 variant comprises an IgG4 constant region comprising an amino acid substitution at amino acid position 228 and / or an amino acid substitution at amino acid position 409 and / or an amino acid substitution at amino acid position 405 of the heavy chain, numbered according to the EU index.

16. 16. The binding agent for use according to any one of claims 1 to 15, wherein the subject is a human.

17. The disease, disorder, and / or condition is (i) A distorted appearance due to excessive muscle activity, or (ii) Any disease, disorder, or condition caused by excessive muscle activity, including dystonia, facial spasms, strabismus, cerebral palsy, stuttering, chronic tension headaches, spasms of the inferior pharyngeal constrictor muscles of the pharynx, pain, migraine headaches, involuntary spasms, muscle spasticity, strabismus, occupational spasms, anal fissures, teeth grinding, and any combination thereof; 17. The binding substance for use according to any one of claims 1 to 16, selected from:

18. A single binding domain that specifically binds to the Ig-like 1 domain of the MuSK protein, a) a VH CDR1 comprising SEQ ID NO: 10, a VH CDR2 comprising SEQ ID NO: 11, a VH CDR3 comprising SEQ ID NO: 12, a VL CDR1 comprising SEQ ID NO: 14, a VL CDR2 comprising SEQ ID NO: 15, and a VL CDR3 comprising SEQ ID NO: 16, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 9, and a light chain variable domain comprising SEQ ID NO: 13; b) a VH CDR1 comprising SEQ ID NO: 18, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 20, a VL CDR1 comprising SEQ ID NO: 22, a VL CDR2 comprising SEQ ID NO: 23, and a VL CDR3 comprising SEQ ID NO: 24, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 17, and a light chain variable domain comprising SEQ ID NO: 21; c) a VH CDR1 comprising SEQ ID NO: 26, a VH CDR2 comprising SEQ ID NO: 27, and a VH CDR3 comprising SEQ ID NO: 28, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 25; d) a VH CDR1 comprising SEQ ID NO: 30, a VH CDR2 comprising SEQ ID NO: 31, a VH CDR3 comprising SEQ ID NO: 32, a VL CDR1 comprising SEQ ID NO: 34, a VL CDR2 comprising SEQ ID NO: 35, and a VL CDR3 comprising SEQ ID NO: 36, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 29, and a light chain variable domain comprising SEQ ID NO: 33; e) a VH CDR1 comprising SEQ ID NO: 38, a VH CDR2 comprising SEQ ID NO: 39, a VH CDR3 comprising SEQ ID NO: 40, a VL CDR1 comprising SEQ ID NO: 42, a VL CDR2 comprising SEQ ID NO: 43, and a VL CDR3 comprising SEQ ID NO: 44, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 37, and a light chain variable domain comprising SEQ ID NO: 41; f) a VH CDR1 comprising SEQ ID NO: 46, a VH CDR2 comprising SEQ ID NO: 47, a VH CDR3 comprising SEQ ID NO: 48, a VL CDR1 comprising SEQ ID NO: 50, a VL CDR2 comprising SEQ ID NO: 51, and a VL CDR3 comprising SEQ ID NO: 52, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 45, and a light chain variable domain comprising SEQ ID NO: 49; g) a VH CDR1 comprising SEQ ID NO: 54, a VH CDR2 comprising SEQ ID NO: 55, a VH CDR3 comprising SEQ ID NO: 56, a VL CDR1 comprising SEQ ID NO: 58, a VL CDR2 comprising SEQ ID NO: 59, and a VL CDR3 comprising SEQ ID NO: 60, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 53, and a light chain variable domain comprising SEQ ID NO: 57; or h) any combination of the above; 18. A binding agent for use according to any one of claims 1 to 17, having a sequence selected from:

19. A method for preventing, controlling, or reducing skin wrinkles in a subject, comprising administering to the subject a binding substance comprising one or more binding domains, where only one of the binding domains specifically binds to the Ig-like 1 domain of a MuSK protein.

20. A method for treating or preventing a disease, disorder, and / or condition that is alleviated by inhibition of neuromuscular transmission, comprising administering to a subject a binding agent comprising one or more binding domains, wherein only one of the binding domains specifically binds to the Ig-like 1 domain of a MuSK protein.

21. The disease, disorder, and / or condition is (i) A distorted appearance due to excessive muscle activity, or (ii) Any disease, disorder, or condition caused by excessive muscle activity, including dystonia, facial spasms, strabismus, cerebral palsy, stuttering, chronic tension headaches, spasms of the inferior pharyngeal constrictor muscles of the pharynx, pain, migraine headaches, involuntary spasms, muscle spasticity, strabismus, occupational spasms, anal fissures, teeth grinding, and any combination thereof; 21. The method of claim 20, wherein the

22. 22. The method of any one of claims 19 to 21, wherein the binding substance is a binding protein.

23. The method of claim 22, wherein the region that specifically binds to the Ig-like 1 domain of the MuSK protein is a variable region.

24. 24. The method of any one of claims 19 to 23, wherein the MuSK protein is a human MuSK protein.

25. 25. The method of any one of claims 19 to 24, wherein the binding agent is monovalent.

26. 25. The method of any one of claims 19 to 24, wherein the binding agent is bivalent or trivalent.

27. 27. The method of any one of claims 19 to 26, wherein the binding agent is an antibody.

28. 28. The method of claim 27, wherein the antibody is a monoclonal antibody.

29. 29. The method of claim 27 or 28, wherein the antibody is a human antibody or a humanized antibody.

30. The antibody is a Fab, a bispecific Fab 2 , trispecific Fab 3 30. The method of any one of claims 27 to 29, wherein the antibody is selected from a bispecific diabody, a trispecific triabody, a single domain antibody, or a bispecific minibody.

31. 31. The method of any one of claims 27 to 30, wherein the antibody is an IgG.

32. The method of claim 31 , wherein the IgG is selected from IgG1, IgG2, or IgG3.

33. The method of claim 31 , wherein the IgG is an IgG4 variant that has a reduced capacity for or is unable to exchange Fab arms in vivo.

34. The method of claim 33, wherein the IgG4 variant comprises an IgG4 constant region that comprises one or more amino acid substitutions that reduce or prevent the capacity for Fab arm exchange in vivo.

35. 35. The method of claim 33 or 34, wherein the IgG4 variant comprises an IgG4 constant region comprising an amino acid substitution at amino acid position 228 and / or an amino acid substitution at amino acid position 409 and / or an amino acid substitution at amino acid position 405 of the heavy chain, numbered according to the EU index.

36. 36. The method of any one of claims 19 to 35, wherein the subject is a human.

37. The binding agent is an antibody that specifically binds to the Ig-like 1 domain of the MuSK protein, the only binding region being a) a VH CDR1 comprising SEQ ID NO: 10, a VH CDR2 comprising SEQ ID NO: 11, a VH CDR3 comprising SEQ ID NO: 12, a VL CDR1 comprising SEQ ID NO: 14, a VL CDR2 comprising SEQ ID NO: 15, and a VL CDR3 comprising SEQ ID NO: 16, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 9, and a light chain variable domain comprising SEQ ID NO: 13; b) a VH CDR1 comprising SEQ ID NO: 18, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 20, a VL CDR1 comprising SEQ ID NO: 22, a VL CDR2 comprising SEQ ID NO: 23, and a VL CDR3 comprising SEQ ID NO: 24, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 17, and a light chain variable domain comprising SEQ ID NO: 21; c) a VH CDR1 comprising SEQ ID NO: 26, a VH CDR2 comprising SEQ ID NO: 27, and a VH CDR3 comprising SEQ ID NO: 28, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 25; d) a VH CDR1 comprising SEQ ID NO: 30, a VH CDR2 comprising SEQ ID NO: 31, a VH CDR3 comprising SEQ ID NO: 32, a VL CDR1 comprising SEQ ID NO: 34, a VL CDR2 comprising SEQ ID NO: 35, and a VL CDR3 comprising SEQ ID NO: 36, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 29, and a light chain variable domain comprising SEQ ID NO: 33; e) a VH CDR1 comprising SEQ ID NO: 38, a VH CDR2 comprising SEQ ID NO: 39, a VH CDR3 comprising SEQ ID NO: 40, a VL CDR1 comprising SEQ ID NO: 42, a VL CDR2 comprising SEQ ID NO: 43, and a VL CDR3 comprising SEQ ID NO: 44, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 37, and a light chain variable domain comprising SEQ ID NO: 41; f) a VH CDR1 comprising SEQ ID NO: 46, a VH CDR2 comprising SEQ ID NO: 47, a VH CDR3 comprising SEQ ID NO: 48, a VL CDR1 comprising SEQ ID NO: 50, a VL CDR2 comprising SEQ ID NO: 51, and a VL CDR3 comprising SEQ ID NO: 52, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 45, and a light chain variable domain comprising SEQ ID NO: 49; g) a VH CDR1 comprising SEQ ID NO: 54, a VH CDR2 comprising SEQ ID NO: 55, a VH CDR3 comprising SEQ ID NO: 56, a VL CDR1 comprising SEQ ID NO: 58, a VL CDR2 comprising SEQ ID NO: 59, and a VL CDR3 comprising SEQ ID NO: 60, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 53, and a light chain variable domain comprising SEQ ID NO: 57; or h) any combination of the above; 37. The method of any one of claims 19 to 36, having a sequence selected from:

38. An antibody comprising one or more binding regions, only one of which specifically binds to the Ig-like 1 domain of a MuSK protein, wherein the only binding region that specifically binds to the Ig-like 1 domain of a MuSK protein is a) a VH CDR1 comprising SEQ ID NO: 10, a VH CDR2 comprising SEQ ID NO: 11, a VH CDR3 comprising SEQ ID NO: 12, a VL CDR1 comprising SEQ ID NO: 14, a VL CDR2 comprising SEQ ID NO: 15, and a VL CDR3 comprising SEQ ID NO: 16, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 9, and a light chain variable domain comprising SEQ ID NO: 13; b) a VH CDR1 comprising SEQ ID NO: 18, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 20, a VL CDR1 comprising SEQ ID NO: 22, a VL CDR2 comprising SEQ ID NO: 23, and a VL CDR3 comprising SEQ ID NO: 24, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 17, and a light chain variable domain comprising SEQ ID NO: 21; c) a VH CDR1 comprising SEQ ID NO: 26, a VH CDR2 comprising SEQ ID NO: 27, and a VH CDR3 comprising SEQ ID NO: 28, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 25; d) a VH CDR1 comprising SEQ ID NO: 30, a VH CDR2 comprising SEQ ID NO: 31, a VH CDR3 comprising SEQ ID NO: 32, a VL CDR1 comprising SEQ ID NO: 34, a VL CDR2 comprising SEQ ID NO: 35, and a VL CDR3 comprising SEQ ID NO: 36, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 29, and a light chain variable domain comprising SEQ ID NO: 33; e) a VH CDR1 comprising SEQ ID NO: 38, a VH CDR2 comprising SEQ ID NO: 39, a VH CDR3 comprising SEQ ID NO: 40, a VL CDR1 comprising SEQ ID NO: 42, a VL CDR2 comprising SEQ ID NO: 43, and a VL CDR3 comprising SEQ ID NO: 44, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 37, and a light chain variable domain comprising SEQ ID NO: 41; f) a VH CDR1 comprising SEQ ID NO: 46, a VH CDR2 comprising SEQ ID NO: 47, a VH CDR3 comprising SEQ ID NO: 48, a VL CDR1 comprising SEQ ID NO: 50, a VL CDR2 comprising SEQ ID NO: 51, and a VL CDR3 comprising SEQ ID NO: 52, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 45, and a light chain variable domain comprising SEQ ID NO: 49; g) a VH CDR1 comprising SEQ ID NO: 54, a VH CDR2 comprising SEQ ID NO: 55, a VH CDR3 comprising SEQ ID NO: 56, a VL CDR1 comprising SEQ ID NO: 58, a VL CDR2 comprising SEQ ID NO: 59, and a VL CDR3 comprising SEQ ID NO: 60, optionally wherein the binding region comprises a heavy chain variable domain comprising SEQ ID NO: 53, and a light chain variable domain comprising SEQ ID NO: 57; or h) any combination of the above; An antibody having a sequence selected from:

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