HK40112910A - Anti-musk antibodies for use in treating neuromuscular disorders - Google Patents

Description

Anti-MuSK antibodies used to treat neuromuscular disorders

Related applications

This application claims priority to U.S. Provisional Patent Application Serial No. 63/364,685, filed May 13, 2022, and EP Application No. 22154118.8, filed January 28, 2022, the entire disclosure of which is incorporated herein by reference.

References to sequence lists

This application includes a sequence list that was filed electronically in ST.26 format and is incorporated herein by reference in its entirety (the copy of ST.26 was created on January 27, 2023, named “196198_SL.XML” and is 364,704 bytes in size).

Technical Field

This invention relates to anti-MuSK antibodies or antigen-binding fragments thereof for the treatment of neuromuscular disorders, such as ALS (amyotrophic lateral sclerosis), in human subjects. In one embodiment, the antibody or antigen-binding fragment thereof is combined with an anticholinergic compound.

Background Technology

ALS is an adult-onset, noncellular, autonomous neuromuscular/neurodegenerative disease that causes progressive loss of upper and lower motor neurons (MNs), leading to gradual paralysis and death within 2 to 5 years. Neuromuscular junction (NMJ) denervation is a hallmark of ALS[1] and is present in several disease models of ALS[2–6], even before MN death[1,3].

Currently approved ALS treatment (riluzole) benefits only 20% of ALS patients by extending lifespan by approximately three months. Riluzole has very limited effects on muscle function. Furthermore, ALS is considered a genetically heterogeneous disease, potentially representing several subgroups with different underlying pathological states. Due to the different underlying disease mechanisms, there are currently no available treatments, and patient-tailored treatments are unlikely to help all ALS patients.

Therefore, new treatments are still needed for ALS and other ALS-like diseases (“ALS-like diseases”).

Attached Figure Description

Figure 1. Combined treatments improved motor function in SOD1G37R mice. ARGX-119 treatment began at P400 (pre-disease onset or asymptomatic), and dafinarax treatment began at approximately P425 (disease onset) and continued until sacrifice (approximately P520). A) A diagram depicting the rotarod apparatus used to assess motor function, coordination, and balance after rotarod acceleration. B) Waiting time (in seconds) for mice treated with ARGX-119 antibody (large gray circles), ARGX-119 + dafinarax combination treatment (black triangles), dafinarax treatment (small gray circles), and double placebo treatment (gray squares) from approximately 400 to 520 days of age to land on the rotarod. C) Grip dynamometer used to monitor overall strength of the forelimbs and hindlimbs of mice. D) Evolution of grip strength measurements during treatment, from approximately P400 to P520 for each group. E) Evolution of body weight measurements during treatment, from approximately P400 to P520 for each group. *p<0.05, ***p<0.001, ****p<0.0001. One-way ANOVA and multiple t-tests.

Figure 2. Treatments improve the contractile properties of EDL muscles. A) Graph showing the setup of the neural and muscle stimulation electrodes and muscle force sensors used to induce muscle contraction. Examples of raw data show muscle contractions induced by neural or muscle stimulation, used to calculate the contractile capacity ratio. B through C) Peak twitch force of EDL muscles generated by neural stimulation (B) or muscle stimulation (C) at different frequencies (5 Hz to 300 Hz) from mice treated with ARGX-119 antibody (gray circles) and ARGX-119 + dafinarax combination (black triangles) versus mice treated with double placebo (gray squares). D) Bar graph showing the mean ± SEM of the EDL muscle contractile capacity ratio as a percentage, representing the ratio of peak force generated by neural stimulation to muscle stimulation (stimulation frequencies from 5 Hz to 100 Hz). E) Histogram showing the mean ± SEM of EDL muscle weight from mice treated with ARGX-119 antibody, combined treatment, and placebo treatment. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Repeated one-way ANOVA and multiple t-tests were performed.

Figure 3. Combination treatments improve the contractile properties of soleus muscle. A to B) Peak twitching force of soleus (SOL) muscle produced by neural stimulation (A) or muscle stimulation (B) at different frequencies (5 Hz to 300 Hz) in mice treated with ARGX-119 antibody (gray circles) and ARGX-119 + dafinarax (black triangles) versus mice treated with double placebo (gray squares). C) Bar graph showing the mean ± SEM of the ratio of SOL muscle contractile capacity as a percentage, representing the ratio of peak force produced by neural stimulation to that produced by muscle stimulation (stimulation frequencies from 5 Hz to 100 Hz). D) Histogram showing the mean ± SEM of EDL muscle weight from mice treated with ARGX-119 antibody, combined treatment, and placebo. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Repeated one-way ANOVA and multiple t-tests were performed.

Figure 4 shows the combined treatments that preserved muscle fatigue characteristics. A) illustrates the EDL fatigue protocol, which consisted of 18 training sessions of 10 stimuli elicited at 120 Hz for 300 ms (one cycle per second). Nine out of ten sessions used separate neural stimulation, with muscle stimulation superimposed on the neural stimulation every ten sessions. A 30-minute recovery period followed the fatigue protocol. B through C) For neural stimulation (B) and neuromuscular (C) of EDL muscles, peak contractile force during the fatigue protocol and recovery period is expressed as a percentage of the initial baseline force generated before the fatigue protocol. Note that placebo treatment (gray squares) showed greater resistance to fatigue compared to animals treated with dual ARGX-119 + dafinavir (black triangles) and those treated with ARGX-119 antibody (gray circles). This illustrates a significant alteration in the characteristics of normal rapid twitching muscles, which are typically highly fatigue-prone. For neural stimulation (D) and neuromuscular stimulation (E) of SOL muscles, peak contractile force during the fatigue and recovery periods is expressed as a percentage of the initial baseline force generated prior to the fatigue program. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Repeated one-way ANOVA and multiple t-tests.

Detailed Implementation

General definition

The following terms and definitions are provided only to aid in understanding the invention. Unless expressly defined herein, all terms used herein have the same meaning as would be understood by one of ordinary skill in the art. For definitions and terms in this art, practitioners specifically refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999). The definitions provided herein should not be construed as having a scope less than that understood by one of ordinary skill in the art.

Unless otherwise specified, it will be apparent to those skilled in the art that all methods, steps, techniques, and operations, which may be performed in a manner known per se and have been carried out without being specifically described in detail, are applicable. For example, refer again to the standard manual, the general background art mentioned above, and other references cited therein.

Unless the context clearly indicates otherwise, nouns without quantifiers used in this article refer to one or more species.

As used herein, the term “comprising” is synonymous with “including” or “containing” and is inclusive or open-ended, without excluding additional undescribed members, compounds, products, elements, or method steps. The expression “consisting of substantially” as used in the context of a product or composition (“a product consisting substantially of” or “a composition consisting substantially of”) means that additional molecules may be present, but such molecules do not alter the characteristics/activity/function of the product or composition. For example, a composition may consist substantially of an antibody or antibody fragment if it itself would exhibit characteristics/activity/function similar to one of the antibodies or antibody fragments.

The enumeration of numerical ranges by endpoints includes all numbers and fractions included in the corresponding range, as well as the enumerated endpoints.

The term "about/approximately" used herein when referring to measurable values such as parameters, quantities, and temporal durations is intended to cover a specified value or a variation relative to the specified value of +/- 10% or less, preferably +/- 5% or less, more preferably +/- 1% or less, and even more preferably +/- 0.1% or less, within which such variation is suitable for implementation in the disclosed invention. It should be understood that the value referred to by the modifier "about" is itself specifically and preferably disclosed.

The terms ‘symptom’ and ‘disease’ are used interchangeably in this article.

The amino acid residues used in this article will be represented by their full names or according to standard three-letter or one-letter amino acid codes.

The terms “polypeptide” or “protein” as used herein are used interchangeably and refer to a polymeric form of amino acids of any length, which may include coding and non-coding amino acids, and chemically or biochemically modified or derived amino acids, and a polypeptide having a modified peptide backbone. A “peptide” is also a polymer of amino acids, typically up to 50 amino acids in length. Polypeptides or peptides are represented by their amino acid sequences.

The terms “nucleic acid molecule,” “polynucleotide,” “polynucleic acid,” and “nucleic acid” as used herein are used interchangeably and refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides or similar molecules) of any length. Nucleic acid molecules are represented by a nucleic acid sequence, characterized primarily by its base sequence. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules can be linear or circular.

As used herein, the term "homology" refers to at least a secondary structural identity or similarity between two macromolecules, particularly two polypeptides or polynucleotides, derived from the same or different taxa, where such similarity is attributed to a common ancestor. Thus, the term "homologous" refers to such related macromolecules having said secondary and optionally tertiary structural similarity. To compare two or more nucleotide sequences, the "sequence identity (percentage)" between a first nucleotide sequence and a second nucleotide sequence can be calculated using methods known to those skilled in the art, for example by dividing the number of nucleotides in the first nucleotide sequence that are identical to the corresponding nucleotides in the second nucleotide sequence by the total number of nucleotides in the first nucleotide sequence and multiplying by 100%, or by using known computer algorithms for sequence alignment, such as NCBIBlast. In determining the degree of sequence similarity between two amino acid sequences, those skilled in the art may consider so-called "conserved" amino acid substitutions, which can generally be described as amino acid substitutions in which one amino acid residue is replaced by another amino acid residue having a similar chemical structure and having little or no effect on the function, activity, or other biological properties of the polypeptide. Possible conserved amino acid substitutions have been illustrated herein. If an amino acid sequence and a nucleic acid sequence have 100% sequence identity over their entire length, they are said to be "identical".

Throughout this application, whenever a specific amino acid sequence SEQ ID NO (for example, SEQ ID NO:Y) is mentioned, it can be replaced with: a polypeptide containing an amino acid sequence that has at least 80% sequence identity or similarity to the amino acid sequence SEQ ID NO:Y. Throughout this application, the phrase "a sequence that is at least X% identical to another sequence" can be replaced with "a sequence that has at least X% sequence identity to another sequence".

In other preferred embodiments, each amino acid sequence described herein shares at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the given amino acid sequence based on its respective percentage of identity (at least 80%). In one preferred embodiment, sequence identity is determined by comparing the full length of the sequences identified herein. In other preferred embodiments, each amino acid sequence described herein has a similarity of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher to the given amino acid sequence based on its respective percentage of similarity (at least 80%). In one preferred embodiment, sequence similarity is determined by comparing the full length of the sequences identified herein. Unless otherwise stated herein, identity or similarity to a given SEQ ID NO means identity or similarity based on the full length of the sequence (i.e., its full length or as a whole).

"Sequence identity" is defined herein as the relationship between two or more amino acid (peptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by sequence comparison. Identity between two amino acid sequences is preferably determined by assessing their identity within the entire SEQ ID NO or a portion thereof as identified herein. A portion may mean at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the length of the SEQ ID NO.

In this art, "identity" also refers to the degree of sequence correlation between amino acid sequences, as determined, such as by matching between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence of a polypeptide and its conserved amino acid substitutes with the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated using known methods, including but not limited to those described below:

Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: lnfo rmatics and Genome Proj ects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds. Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heine, G., Academic Press, 1987; and SequenceAnalysis Primer , Gribskov, M. and Devereux, J., eds, M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988).

The preferred method for determining identity is designed to give the maximum match between the tested sequences. The methods for determining identity and similarity are compiled into publicly available computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, for example, the GCG package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BestFit, FASTA, BLASTN and BLASTP (Altschul, S.F. et al., J.Mol.Biol. 215: 403-410 (1990)), and EMBOSS Needle (Madeira, F., et al., Nucleic Acids Research 47(W1): W636-W641 (2019)). The BLAST procedure is publicly available from NCBI and other sources (BLAST, Altschul, S., et al., NCBI, IMLM, NIH, Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)). The EMBOSS procedure is publicly available from EMBL-EBI. The well-known Smith-Waterman algorithm can also be used to determine identity. The EMBOSS Needle procedure is the preferred procedure.

Preferred parameters for peptide sequence alignment include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48(3): 443-453 (1970); Comparison matrix: BLOSUM62, from Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA. 89: 10915-10919 (1992); Vacancy opening penalty: 10, and vacancy extension penalty: 0.5. The program available for these parameters is publicly available as the EMBOSS Needle program from EMBL-EBI. The aforementioned parameters are the default parameters for global protein pairing sequence alignment (and there is no penalty for terminal vacancies).

Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: DNAfull; Vacancy opening penalty: 10; Vacancy extension penalty: 0.5. The program that can be used with these parameters is publicly available as the EMBOSS Needle program from EMBL-EBI. The aforementioned parameters are the default parameters for global pairing sequence alignment of nucleotide sequences (and there is no penalty for terminal vacancies).

The terms ‘symptom’ and ‘disease’ are used interchangeably in this article.

This document also provides some implementation schemes, wherein any of the implementation schemes described herein can be combined with any one or more other implementation schemes, provided that such combination is not mutually exclusive.

Anti-MuSK antibodies and their antigen-binding fragments

All antibodies or antigen-binding fragments thereof defined herein are, in their own right, covered by this invention. Antibodies or antigen-binding fragments thereof are also used in the treatment of neuromuscular disorders in human subjects.

This invention relates to molecules based on anti-MuSK antibodies, including anti-MuSK antibodies, their epitope-binding domains, their antigen-binding fragments, and antibody derivatives (for the treatment of neuromuscular diseases or conditions). In one embodiment, the term "antibody-based molecule" may be replaced by the word "antibody," or by the expression "antibody or a functional fragment thereof," or by the expression "antibody or antigen-binding fragment."

The term “anti-MuSK antibody” can be replaced by the term “MuSK antibody”.

Any molecule based on an anti-MuSK antibody, including anti-MuSK antibodies, their epitope-binding domains, their antigen-binding fragments, and antibody derivatives capable of binding to muscle-specific tyrosine protein kinase (MuSK), is covered within the scope of this invention. In one embodiment, such an anti-MuSK antibody is also capable of activating MuSK signaling and/or phosphorylation. This invention provides the insight that such antibody-based molecules can be used to treat conditions in which the subject requires enhanced MuSK signaling or MuSK phosphorylation, such as neuromuscular diseases or conditions. Therefore, in a first aspect, an anti-MuSK antibody or its antigen-binding fragment for treating neuromuscular conditions in human subjects is provided.

MuSK is a receptor tyrosine kinase expressed in skeletal muscle and plays a crucial major role in the formation and maintenance of neuromuscular synapses (Burden et al., “The Role of MuSK in Synapse Formation and Neuromuscular Disease,” Cold Spring Harb. Perspect. Biol. 5:a009167 (2013), which is incorporated herein by reference in its entirety). MuSK is a single-channel, 120 kDa transmembrane protein composed of an extracellular region containing three Ig-like domains and one coiled (Fz)-like domain, and an intracellular region containing a juxtamembrane region, a kinase domain, and a short cytoplasmic tail (Jennings et al., “Muscle-Specific trk-Related Receptor with a Kringle Domain Defines a Distinct Class of Receptor Tyrosine Kinases,” Proc. Natl. Acad. S Ci. USA90:2895-2899 (1993) and Valenzuela et al., “Receptor Tyrosine Kinase Specific for the Skeletal Muscle Lineage: Expression in Embryonic Muscle, at the Neuromuscular Junction, and After Injury,” Neuron 15:573-584 (1995), which are incorporated herein by reference in their entirety. MuSK phosphorylation is stimulated by synaptic proteoglycans, which are signals provided by motor neurons. Once activated, MuSK stimulates the following pathways: (1) aggregation and anchoring of AChR and other muscle proteins essential for synaptic transmission, (2) enhanced transcription of genes encoding synaptic proteins in the muscle “synaptic nucleus,” and (3) promotion of retrograde signaling, which promotes presynaptic differentiation and attachment of motor nerve endings to muscles. In the absence of Musk, neuromuscular synapses cannot form (Burden et al., “The Role of Musk in Synapse Formation and Neuromuscular Disease,” Cold Spring Harb. Perspect. Biol. 5:a009167 (2013), which is incorporated herein by reference in its entirety). In addition to its role during synapse formation, MuSK is also required to maintain adult synapses, as inhibition of MuSK expression in adult muscles leads to severe defects in presynaptic and postsynaptic differentiation (Kong et al., “Inhibition of Synapse Assembly in Mammalian Muscle in vivoby RNA Interference,” EMBO Rep 5:183-188 (2004) and Hesser et al., “Synapse Disassembly and Formation of New Synapses in Postnatal Muscle Upon Conditional Inactivation of MuSK,” Mol. Cell. Neurosci. 31:470-480 (2006), which are incorporated herein by reference in their entirety). Consistent with these findings in mice, mutations that impair MuSK kinase activity or inhibit downstream MuSK signaling steps lead to myasthenia gravis (CM), characterized by defects in synaptic structure and function, resulting in muscle weakness and fatigue (Beeson et al., “Dok-7 Mutations Underlie a Neuromuscular Junction Synaptopathy,” Science 313:1975-1978 (2006); Muller et al., “Phenotypical Spectrum of DOK7 Mu…”). "Tations in Congenital Myasthenic Syndromes," Brain 130:1497-1506 (2007); and Selcen et al., "A Compensatory Subpopulation of Motor Neurons in a Mouse Model of Amyotrophic Lateral Sclerosis," J. Comp. Neurol. 490:209-219 (2008), which are incorporated herein by reference in their entirety.

The amino acid sequence of human MuSK has the following amino acid sequence as shown in SEQ ID NO:129.

MRELVNIPLVHI LTLVAFSGTEKLPKAPVITTPLETVDALVEEVATFMCAVESYPQPEISWTRNKILIKLFDTR

YSI RENGQLLTILSVEDSDDGIYCCTANNGVGGAVESCGALQVKMKPKITRPPINVKIIEGLKAVLPCTTMG

NPKPSVSWIKGDSPLRENSRIAVLESGSLRIH NVQKEDAGQYRCVAKNSLGTAYSKVVKLEVVFARILRA

PESH NVTFGSFVTLHCTATGIPVPTITWI ENGNAVSSGSIQESVKDRVIDSRLQLFITKPGLYTCIATN KHGE

KFSTAKAAATISIAEWSKPQKDNKGYCAQYRGEVC NAVLAKDALVFLNTSYADPEEAQELLVHTAWNELK

VVSPVCRPAAEALLCNHIFQECSPGVVPTPIPICREYCLAVKELFCAKEWLVMEEKTHRGLYRSEMHLLS

VPECSKLPSM HWD PTACARLPH LDYN KEN LKTFPPMTSSKPSVDIPN LPSSSSSSFSVSPTYSMTVI ISIM

SSFAIFVLLTITTLYCCRRRKQWKNKKRESAAVTLTTLPSELLLDRLHPN PMYQRMPLLLNPKLLSLEYPR

NNIEYVRDIGEGAFGRVFQARAPGLLPYEPFTMVAVKMLKEEASADMQADFQREAALMAEFDNPNIVKLL

GVCAVGKPMCLLFEYMAYGDLN EFLRSMSPHTVCSLSHSDLSMRAQVSSPGPPPLSCAEQLCIARQVAA

GMAYLSERKFVHRDLATRNCLVGENMVVKIADFGLSRNIYSADYYKANENDAIPIRWMPPESIFYNRYTTE

SDVWAYGVVLWEIFSYGLQPYYGMAHEEVIYYVRDGNILSCPENCPVELYNLMRLCWS

KLPADRPSFTSIHRILERMCERAEGTVSV(SEQ ID NO:129)

According to the present invention, the MuSK antibody-based molecule described herein binds to an epitope within the coiled (Fz)-like domain of the MuSK protein. The Fz-like domain of MuSK has the amino acid sequence shown in SEQ ID NO: 130.

DNKGYCAQYRGEVCNAVLAKDALVFLNTSYADPEEAQELLVHTAWNELKVVSPVCRPAAEALLCNHIFQ

ECSPGVVPTPIPICREYCLAVKELFCAKEWLVMEEKTHRGLYRSEMHLLSVPECSKLPSMHWDPTACARL(SEQ ID NO: 130)

As used herein, the term "epitope" refers to an antigenic determinant capable of binding to an antibody. Epitopes typically contain surface groups of a molecule, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics. The difference between conformational and non-conformational epitopes is that binding to the former (rather than the latter) is lost in the presence of denaturing solvents. Epitopes may contain amino acid residues that directly participate in binding (also known as the immunodominant component of the epitope) and other amino acid residues that do not directly participate in binding, such as amino acid residues that are effectively blocked by a specific antigen-binding peptide (in other words, the amino acid residues are within the "footprint" of the specific antigen-binding peptide). Epitopes typically contain at least three, and more commonly at least five, six, seven, eight, nine, ten, eleven, twelve, thirteen, eleven, eleven, thirteen, twenty, or more spatially conformationally unique amino acids.

In one embodiment, the MuSK antibody or antigen-binding fragment applied according to the invention binds to the MuSK coil (Fz)-like domain. In one embodiment, the MuSK antibody or antigen-binding fragment binds to the epitope in the MuSK Fz-like domain sequence of SEQ ID NO: 130 more frequently, rapidly, persistently, and/or with stronger affinity or affinity, compared to alternative epitopes. In one embodiment, the MuSK antibody-based molecule described herein binds to any 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues of SEQ ID NO: 130 with immunospecific affinity. As used herein, the terms “affinity,” “specific binding,” “binding,” “immunospecific binding,” “binding activity,” or “specific binding activity” refer to the degree to which an antibody or antibody fragment, as defined herein, binds to the epitope within the MuSK Fz-like domain sequence of SEQ ID NO: 130.

In one embodiment, the MuSK antibody-based molecules disclosed herein bind to the MuSK Fz-like domain with an affinity corresponding to a KD of about 10⁻⁷ M or less. For example, the MuSK antibody-based molecules disclosed herein bind to the MuSK Fz-like domain with an affinity corresponding to a _KD of about 10⁻⁸ M, about 10⁻⁹ M, about 10⁻¹⁰ M, about 10⁻¹¹ M, about 10⁻¹² M or less, as determined by, for example, surface plasmon resonance (SPR) technology in a Biacore 3000 instrument (preferably using an antibody as a ligand and MuSK as an analyte). The MuSK antibody-based molecules disclosed herein bind to the MuSK Fz-like domain with an affinity corresponding to the following _KD : _KD is at least 10-fold lower, for example, at least 100-fold lower, for example, at least 1,000-fold lower, for example, at least 10,000-fold lower, for example, at least 100,000-fold lower, for example, at least 100,000-fold lower, for example, at least 100,000-fold lower. The amount of affinity reduction depends on the antibody's KD, so when the antibody's KD is very low (i.e., the antibody is highly specific), then the amount of affinity for the antigen is lower than that for the nonspecific antigen, which can be at least 10,000-fold lower. The term "kd" ( ^seconds⁻¹ or 1/second) as used herein refers to the dissociation rate constant of a specific antibody-antigen interaction. This value is also called the koff value. The term "ka" (M⁻¹×seconds⁻¹ or 1/M) as used herein refers to the binding rate constant of a specific antibody-antigen interaction. As used herein, the term "KD" (M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction and is obtained by dividing kd by ka. The term " _KA " (M⁻¹ or 1/M) refers to the association equilibrium constant of a specific antibody-antigen interaction and is obtained by dividing ka by kd.

In one embodiment, the MuSK-based antibody molecules described herein have a pH-dependent binding affinity for MuSK, which allows antibody recycling to enhance antigen binding. For example, in one embodiment, the association rate constant or dissociation rate constant may differ under acidic versus neutral versus alkaline pH conditions. In one embodiment, the MuSK-based antibody molecules described herein have a higher dissociation rate constant under acidic pH conditions (e.g., <7.0) compared to neutral pH conditions (e.g., pH of about 7.0 to 7.9). In some embodiments, the MuSK-based antibody molecules described herein have a 2- to 3-fold higher dissociation rate constant (i.e., reduced binding affinity) compared to neutral pH (e.g., pH of about 7.4) at acidic pH (e.g., pH of about 5.5). In one embodiment, the MuSK-based antibody molecules bind to the MuSK Fz-like domain with a higher affinity under neutral pH conditions than under acidic pH conditions. In other words, in one embodiment, the molecule based on the MuSK antibody binds to the MuSK Fz-like domain, and its dissociation rate is higher under acidic pH conditions than under neutral pH conditions. Neutral pH conditions can be defined as a pH ranging from 7.0 to 7.9. Acidic pH conditions can be defined as a pH less than 7.0. Higher can mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% higher. Antibodies exhibiting this pH-dependent dissociation characteristic dissociate from the antigen after binding and activation but before lysosomal degradation. Once dissociated, the antibody is transported back into circulation via the nascent Fc receptor and released to bind more antigen.

In some embodiments, the binding of the MuSK antibody of the present invention to their respective epitopes within the Fz-like domain activates MuSK signaling. Specifically, when the MuSK antibody of the present invention binds to their respective epitopes within the MuSK Fz-like domain, this binding induces MuSK phosphorylation and activation. The MuSK antibody of the present invention induces approximately 50% to approximately 100% of MuSK phosphorylation (as measured, for example, in a C2C12 phosphorylation assay) relative to MuSK phosphorylation induced by synaptic glycan activation. In one embodiment, the MuSK antibody of the present invention induces approximately 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (95%) of MuSK phosphorylation (relative to MuSK phosphorylation induced by synaptic glycan activation). In one embodiment, the MuSK antibody-based molecule of the present invention induces approximately 90% to approximately 100% of MuSK phosphorylation upon MuSK binding (relative to MuSK phosphorylation induced by synaptic glycan activation). Phosphorylation of MuSK can be assessed using techniques known to the art, such as Western blot or C2C12 myotube phosphorylation assays. Antibodies that activate MuSK signaling (i.e., induce MuSK dimerization, induce MuSK tyrosine phosphorylation) are agonist antibodies.

In some embodiments, the MuSK antibody of the present invention, i.e., the MuSK antibody binding to the Fz-domain of MuSK, does not interfere with (i.e., does not block, hinder, inhibit, or reduce) natural ligand binding and MuSK stimulation. In some embodiments, the MuSK antibody co-stimulates MuSK activation with its natural ligand (i.e., synaptic proteoglycan) to produce an additive activation effect, such as MuSK phosphorylation. Thus, in some embodiments, the MuSK antibody of the present invention enhances natural MuSK activation, i.e., phosphorylation, induced by natural ligand binding. Such a MuSK antibody is an agonist antibody. In some embodiments, the antibody of the present invention, in combination with a natural ligand, activates MuSK (i.e., phosphorylates MuSK) to >100% of the endogenous activation level, for example, at least 110%, 130%, 150%, or 200% of the endogenous activation level.

Therefore, in one embodiment, the activity of the MuSK-based antibody molecule of the present invention includes: (i) binding to an epitope of human muscle-specific tyrosine protein kinase (MuSK), said epitope being present in the MuSK coil (Fz)-like domain sequence of SEQ ID NO: 130, wherein said antibody-based molecule induces MuSK phosphorylation upon binding to its epitope, and/or (ii) binding to the MuSK Fz-like domain does not block, hinder, or inhibit phosphorylation induced by natural or endogenous MuSK ligands, and may enhance said natural or endogenous MuSK ligand-induced phosphorylation, and (iii) binding to the MuSK Fz-like domain occurs with a higher affinity under neutral pH conditions than under acidic pH conditions. All of these features have been further defined herein.

Antibody-based molecules include, but are not limited to, antibodies, intact antibodies, epitope-binding fragments of intact antibodies, antigen-binding fragments of intact antibodies, and antibody derivatives. Epitope-binding fragments of antibodies can be obtained by actual fragmentation of a parent antibody (e.g., Fab or (Fab)2 fragments). Alternatively, an epitope-binding fragment is an amino acid sequence comprising a portion of the amino acid sequence of such a parent antibody. As used herein, a molecule is referred to as a “derivative” of an antibody (or its related portion) if it is obtained by actual chemical modification of a parent antibody or a portion thereof, or if it comprises an amino acid sequence substantially similar to the amino acid sequence of such a parent antibody or its related portion (e.g., less than 30%, less than 20%, less than 10%, or less than 5% difference from such a parent molecule or its related portion, or a difference of 10 amino acid residues or less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues).

In some embodiments, the antibody-based molecules of the present invention are complete immunoglobulins or molecules in recombinant cells having epitopes encoding such fragments that bind to 333 acids (see, for example, Evans et al. "Rapid Expression of An Anti-Human C5 Chimeric Fab Utilizing A Vector That Replicates In COS And 293 Cells," J. Immunol. Meth. 184:123-38 (1995), which is incorporated herein by reference in its entirety). For example, a chimeric gene encoding a portion of the F(ab')2 fragment may comprise a DNA sequence encoding the CH1 domain and the heavy chain hinge region, followed by translation of a stop codon to produce such a truncated antibody fragment molecule. Suitable fragments capable of binding to the desired epitope can be readily screened for use in the same manner as with complete antibodies.

Antibody derivatives include molecules containing at least one epitope-binding domain of an antibody and are typically formed using recombinant techniques. An exemplary antibody derivative includes a single-chain Fv (scFv). scFv is formed from two domains, VL and VH, of an Fv fragment, which can be encoded by different genes. Using recombination methods, such gene sequences or their encoding cDNAs are linked together by flexible linkers (typically having about 10, 12, 15 or more amino acid residues), which allows them to be made into single protein chains in which VL and VH associate to form monovalent epitope-binding molecules (see, for example, Bird et al. “Single-Chain Antigen-Binding Proteins,” Science 242:423-426 (1988); and Huston et al. “Protein Engineering Of Antibody Binding Sites: Recovery Of Specific Activity In An Anti-Digoxin Single-Chain Fv Analogue Produced In Escherichiacoli,” Proc. Natl. Acad. Sci. (U.S.A.) 85:5879-5883 (1988), which are incorporated herein by reference in their entirety). Alternatively, by using a flexible linker that is not too short (e.g., not less than about 9 residues) to enable the VL and VH of different single polypeptide chains to associate together, a bispecific antibody with binding specificity to two different epitopes can be formed.

In another embodiment, the antibody derivative is a divalent or bivalent single-chain variable fragment modified by linking two scFvs together in tandem (i.e., tandem scFvs) or dimerizing them to form a biantibody (Holliger et al. "'Diabodies': Small Bivalent And Bispecific Antibody Fragments," Proc. Natl. Acad. Sci. (U.S.A.) 90(14), 6444-8 (1993), which is incorporated herein by reference in its entirety). In yet another embodiment, the antibody is a triantibody, i.e., a trivalent single-chain variable fragment modified by linking three scFvs together in tandem or in trimer form to form a triantibody. In yet another embodiment, the antibody is a tetraantibody of four single-chain variable fragments. In another embodiment, the antibody is a “linear antibody” which is an antibody comprising a tandem Fd segment pair (VH-CH1-VH-CH1) forming an antigen-binding region pair (see Zapata et al. Protein Eng. 8(10):1057-1062 (1995), which is incorporated herein by reference in its entirety). In another embodiment, the antibody derivative is a microantibody consisting of a single-chain Fv region (i.e., scFv-CH3) coupled to the CH3 region.

This document also discusses these and other useful antibody fragments and derivatives within the context of this invention. It should also be understood that, unless otherwise stated, the term "antibody-based molecule" also includes antibody-like peptides, such as chimeric and humanized antibodies, antigen-binding fragments, and antibody fragments (epitope-binding, antigen-binding, or functional fragments) that retain the ability to bind specifically to antigens, provided by any known technology, such as enzymatic cleavage, peptide synthesis, and recombinant technologies.

The antibodies generated herein can be of any isotype. As used herein, “isotype” refers to a class of immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by a heavy chain constant region gene. The selection of isotype is typically guided by the desired effector function, such as antibody-dependent cellular cytotoxicity (ADCC) induction. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Particularly useful isotypes of the MuSK antibodies disclosed herein include IgG1 and IgG2.

The human light chain constant region κ or λ can be used. If desired, the class of the MuSK antibody of the present invention can be converted using known methods. For example, an antibody of the present invention that is originally IgM can be converted to an IgG antibody of the present invention. Furthermore, class conversion techniques can be used to convert one IgG subclass to another, for example, from IgG1 to IgG2. Therefore, the effector function of the antibody of the present invention can be altered through isotype conversion to, for example, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies for a variety of therapeutic uses.

In one embodiment, one, two, or more amino acid substitutions are introduced into the Fc region of the IgG constant region to alter the effector function of the antibody-based molecule. For example, one or more amino acid residues selected from amino acid residues 234, 235, 236, 237, 238, 239, 243, 265, 267, 268, 292, 297, 300, 318, 320 , 322, 327, 328, 329, 330, 331, 332, and 396, numbered according to the EU numbering system (https://www.imgt.org/IMGTScientificChart/Hu IGHGnber.html#notes and Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969), can be replaced with different amino acid residues, such that the antibody-based molecule has altered affinity for the effector ligand but retains its antigen-binding ability. In one embodiment, amino acids 234 or 235 have been substituted. In another embodiment, amino acids 234 and 235 have been substituted. In this context, the preferred amino acid sequence of the human IgG constant Fc region comprises SEQ ID NO:266 or SEQ ID NO:267. In this context, for example, amino acids 234 and 235, numbered according to the EU numbering system, correspond to amino acids 7 and 8 in SEQ ID NO:266 and 267 (i.e., the human IgG constant Fc region of the antibody-based molecule disclosed herein), or amino acids 234 and 235, numbered according to the EU numbering system, correspond to amino acids 238 and 239 in SEQ ID NO:268 and 270 (i.e., the human full-length heavy chain of the antibody-based molecule disclosed herein). The positions are generally different because the length of the variable region varies, which introduces a "δ" in the numbering. In the case described above, this δ is 4. Therefore, when identifying the corresponding positions in SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, or SEQ ID NO:270, the same applies to the other amino acid positions identified above according to the EU numbering system (i.e., 236, 237, 238, 239, 243, 265, 267, 268, 292, 297, 300, 318, 320, 322, 327, 328, 329, 330, 331, 332, and 396). In the filed application, the position of the amino acid may be indicated by the EU numbering system or by the actual position in a given Fc region (e.g., SEQ ID NO:266 or SEQ ID NO:267) or the full-length heavy chain (e.g., SEQ ID NO:268 or 270).

Therefore, in one embodiment, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 are substituted into SEQ ID NO:266 or SEQ ID NO:267. In one embodiment, amino acids 1, 2, 3, or 4 are substituted into SEQ ID NO:266 or SEQ ID NO:267. In one embodiment, one or two amino acids are substituted into SEQ ID NO:266 or SEQ ID NO:267. Therefore, in one embodiment, amino acid substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 are introduced into SEQ ID NO:266 or SEQ ID NO:267, and the substitutions are introduced at amino acid residues 234, 235, 236, 237, 239, 243, 267, 292, 297, 300, 318, 320, 322, 328, 330, 332, and 396 selected from the sequence according to the EU numbering system. In one embodiment, one or two amino acid substitutions are introduced into SEQ ID NO:266 or SEQ ID NO:267. In one embodiment, amino acid 234 or 235 of SEQ ID NO:266 or SEQ ID NO:267 according to the EU numbering system has been substituted. In another embodiment, amino acids 234 or 235, which are numbered according to the EU numbering system, in SEQ ID NO: 266 and 267 have been replaced.

The effector ligand with altered affinity can be, for example, an Fc receptor or a C1 component of complement. This method is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260, each incorporated herein by reference in its entirety. In one embodiment, one or more amino acid substitutions may be introduced into the Fc region of the antibody-based molecule described herein to remove potential glycosylation sites on the Fc region, which can reduce Fc receptor binding (see, for example, Shields RLet al., (2001) J Biol Chem 276:6591-604, which is incorporated herein by reference in its entirety). In one embodiment, binding to the same ligand is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or becomes undetectable, compared to binding to an antibody without any amino acid substitutions in its constant Fc region of human IgG.

In the first embodiment, one or more of the following mutations have been introduced into the constant region of the antibody-based molecule described herein (all numbered according to the EU numbering system): N297A substitution; N297Q substitution; L234A substitution; L234D substitution; L234E substitution; L234G substitution; L234H substitution; L234F substitution; L234K substitution; L234Q substitution; L234R substitution; L234S substitution; L234T substitution; L235A substitution; L235D substitution; L235E substitution; L235F substitution; L235G substitution; L235V substitution; L235H substitution; L235I substitution; L235K substitution; L235R substitution; L2 Replace 35S; Replace L235T; Replace L235Q; Replace L237A; Replace S239D; Replace E233P; Replace L234V; C236 is missing; Replace G236E; Replace G236R; Replace G236K; Replace G237A; Replace P238A; Replace F243L; Replace D265A; Replace S267E; Replace H268A; Replace R292P; Replace Y300L; Replace K322A; Replace K322Q; Replace A327Q; Replace L328F; Replace L328R; Replace P329A; Replace P329G; Replace A330L; Replace A330S; Replace P331S; Replace I332E or P396L.

In the second embodiment, one or more of the following mutations have been introduced into the constant region of the antibody-based molecule described herein (all numbered according to the EU numbering system): L234A and/or L235A substitution; L234A and L235A substitution; L234A, L235A and P329G substitution; L234A, L235A and G236K substitution; L234A, L235A and G236E substitution; L234A, L235A and G236R substitution; L234A and G... Replace 236R; Replace L234A, L235S, and G236R; Replace L234A, L235T, and G236R; Replace L234D, L235H, and G236R; Replace L234D, L235K, and G236R; Replace L234D and G236R; Replace L234D, L235Q, and G236R; Replace L234D, L235S, and G236R; Replace L234E, L235D, and G236R; Replace L234E, L235H, and G236R. Replace 6R; Replace L234E, L235I, and G236R; Replace L234G, L235H, and G236R; Replace L234G, L235Q, and G236R; Replace L234G, L235S, and G236R; Replace L234H, L235I, and G236R; Replace L234H, L235S, and G236R; Replace L234K, L235Q, and G236R; Replace L234K, L235R, and G236R; Replace L234K and L235S Replace with G236R; Replace with L234K, L235T and G236R; Replace with L234K, L235V and G236R; Replace with L234Q, L235A and G236R; Replace with L234Q, L235D and G236R; Replace with L234Q, L235H and G236R; Replace with L234Q and G236R; Replace with L234Q, L235Q and G236R; Replace with L234Q, L235R and G236R; Replace with L234Q, L235S and G Replace 236R; Replace L234Q, L235T, and G236R; Replace L234Q, L235V, and G236R; Replace L234R, L235D, and G236R; Replace L234R, L235E, and G236R; Replace L234R, L235H, and G236R; Replace L234R, L235I, and G236R; Replace L234R, L235K, and G236R; Replace L234R and G236R; Replace L234R, L235Q, and G236R. Replace 6R; Replace L234R, L235R, and G236R; Replace L234R, L235T, and G236R; Replace L234S, L235E, and G236R; Replace L234S, L235G, and G236R; Replace L234S, L235H, and G236R; Replace L234S, L235I, and G236R; Replace L234S and G236R; Replace L234S, L235R, and G236R; Replace L234S, L235T, and G236R Replace; L234S, L235V and G236R; L234T, L235A and G236R; L234T, L235D and G236R; L234T, L235H and G236R; L234T, L235I and G236R; L234T, L235K and G236R; L234T, L235Q and G236R; L234T, L235R and G236R; L234T, L235S and G23 Replace 6R; replace L234T, L235T and G236R; replace L234T, L235V and G236R; replace G236R and L328R; replace L234A, L235A, G237A, P238S, H268A, A330S and P331S; replace E233P, L234V, L235A, missing G326, A327G, A330S and P331S; replace L235A and G236R; replace L235S and G236R.

In the third embodiment, one or more of the following mutations have been introduced into the Fc region of the antibody-based molecule described herein, SEQ ID NO:266 or SEQ ID NO:267 (both according to EU numbering system): N297A substitution; N297Q substitution; L234A substitution; L234D substitution; L234E substitution; L234G substitution; L234H substitution; L234F substitution; L234K substitution; L234Q substitution; L234R substitution; L234S substitution; L234T substitution; L235A substitution; L235D substitution; L235E substitution; L235F substitution; L235G substitution; L235V substitution; L235H substitution; L235I substitution; L235K substitution. Replace with: L235R; L235S; L235T; L235Q; L237A; S239D; E233P; L234V; C236 missing; G236E; G236R; G236K; G237A; P238A; F243L; D265A; S267E; H268A; R292P; Y300L; K322A; K322Q; A327Q; L328F; L328R; P329A; P329G; A330L; A330S; P331S; I332E or P396L.

In the fourth embodiment, one or more of the following mutations have been introduced into the Fc region of the antibody-based molecule described herein, SEQ ID NO:266 or SEQ ID NO:267 (both according to the EU numbering system): L234A and/or L235A substitution; L234A and L235A substitution; L234A, L235A and P329G substitution; L234A, L235A and G236K substitution; L234A, L235A and G236E substitution; L23 Replace 4A, L235A, and G236R; replace L234A and G236R; replace L234A, L235S, and G236R; replace L234A, L235T, and G236R; replace L234D, L235H, and G236R; replace L234D, L235K, and G236R; replace L234D and G236R; replace L234D, L235Q, and G236R; replace L234D, L235S, and G236R; replace L234E, L235D, and G2... Replace 36R; Replace L234E, L235H, and G236R; Replace L234E, L235I, and G236R; Replace L234G, L235H, and G236R; Replace L234G, L235Q, and G236R; Replace L234G, L235S, and G236R; Replace L234H, L235I, and G236R; Replace L234H, L235S, and G236R; Replace L234K, L235Q, and G236R; Replace L234K, L235R, and G Replace 236R; Replace L234K, L235S, and G236R; Replace L234K, L235T, and G236R; Replace L234K, L235V, and G236R; Replace L234Q, L235A, and G236R; Replace L234Q, L235D, and G236R; Replace L234Q, L235H, and G236R; Replace L234Q and G236R; Replace L234Q, L235Q, and G236R; Replace L234Q, L235R, and G236R. Replace L234Q, L235S, and G236R; Replace L234Q, L235T, and G236R; Replace L234Q, L235V, and G236R; Replace L234R, L235D, and G236R; Replace L234R, L235E, and G236R; Replace L234R, L235H, and G236R; Replace L234R, L235I, and G236R; Replace L234R, L235K, and G236R; Replace L234R and G236R; L234 Replace R, L235Q, and G236R; Replace L234R, L235R, and G236R; Replace L234R, L235T, and G236R; Replace L234S, L235E, and G236R; Replace L234S, L235G, and G236R; Replace L234S, L235H, and G236R; Replace L234S, L235I, and G236R; Replace L234S and G236R; Replace L234S, L235R, and G236R; Replace L234S, L235... Replace T and G236R; Replace L234S, L235V and G236R; Replace L234T, L235A and G236R; Replace L234T, L235D and G236R; Replace L234T, L235H and G236R; Replace L234T, L235I and G236R; Replace L234T, L235K and G236R; Replace L234T, L235Q and G236R; Replace L234T, L235R and G236R; Replace L234T, L235S Replace with G236R; replace L234T, L235T and G236R; replace L234T, L235V and G236R; replace G236R and L328R; replace L234A, L235A, G237A, P238S, H268A, A330S and P331S; replace E233P, L234V, L235A, missing G326, A327G, A330S and P331S; replace L235A and G236R; replace L235S and G236R.

In one embodiment, one or more of the following mutations are introduced into the Fc region of the antibody-based molecule described herein, SEQ ID NO:266 or SEQ ID NO:267: L234A and/or L235A substitutions (both according to EU numbering system). In another embodiment, the following mutations are introduced into the Fc region of the antibody-based molecule described herein, SEQ ID NO:266 or SEQ ID NO:267: L234A and L235A substitutions according to EU numbering system. This embodiment produces an antibody-based molecule having a heavy chain represented by SEQ ID NO:268 or SEQ ID NO:270.

In the context of this invention, antibodies with altered, weakened, or even eliminated effector functions are attractive.

In one embodiment, an anti-MuSK antibody or its antigen-binding fragment is provided, wherein: - it is an agonist MuSK antibody and/or

- The function of the effector has been reduced or eliminated.

The antibody or its antigen-binding fragment is preferably used to treat neuromuscular diseases in human subjects.

In one embodiment, an anti-MuSK antibody or an antigen-binding fragment thereof is provided, which: - binds to the MuSK coil (Fz)-like domain sequence of SEQ ID NO:129,

- For the agonist MuSK antibody and/or

- The function of the effector has been reduced or eliminated.

The antibody or its antigen-binding fragment is preferably used to treat neuromuscular diseases in human subjects.

As previously described herein, reduced or eliminated effector function can be obtained by introducing mutations into the constant Fc region of human IgG. Preferably, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 amino acids are substituted into the Fc region. More preferably, at least 1, 2, 3, or 4 amino acids are substituted into the Fc region.

The Fc region may contain SEQ ID NO:266 or SEQ ID NO:267 and the substitution is introduced at the amino acid positions of amino acid residues 234, 235, 236, 237, 238, 239, 243, 265, 267, 268, 292, 297, 300, 318, 320, 322, 327, 328, 329, 330, 331, 332 and 396 selected from the sequence according to the EU numbering system.

In one embodiment, the Fc region may contain SEQ ID NO:266 or SEQ ID NO:267 and the substitution is introduced at the amino acid position of amino acid residue 234 or 235 selected from the sequence according to the EU numbering system.

In one embodiment, the Fc region may contain SEQ ID NO:266 or SEQ ID NO:267 and the substitution is introduced at the amino acid positions of amino acid residues 234 and 235 selected from the sequence according to the EU numbering system.

In one embodiment, one or more of the following mutations (all numbered according to the EU numbering system) have been introduced into the constant Fc region of human IgG in the antibody-based molecule described herein, in SEQ ID NO:266 or SEQ ID NO:267: N297A substitution; N297Q substitution; L234A substitution; L234D substitution; L234E substitution; L234G substitution; L234H substitution; L234F substitution; L234K substitution; L234Q substitution; L234R substitution; L234S substitution; L234T substitution; L235A substitution; L235D substitution; L235E substitution; L235F substitution; L235G substitution; L235V substitution; L235H substitution; L235I substitution; L2 Replace 35K; Replace L235R; Replace L235S; Replace L235T; Replace L235Q; Replace L237A; Replace S239D; Replace E233P; Replace L234V; C236 is missing; Replace G236E; Replace G236R; Replace G236K; Replace G237A; Replace P238A; Replace F243L; Replace D265A; Replace S267E; Replace H268A; Replace R292P; Replace Y300L; Replace K322A; Replace K322Q; Replace A327Q; Replace L328F; Replace L328R; Replace P329A; Replace P329G; Replace A330L; Replace A330S; Replace P331S; Replace I332E or P396L.

In one embodiment, each of the combinations of mutations previously described in the fourth embodiment of this application in the constant Fc region of the human IgG of the antibody-based molecule described herein can be prepared.

In a preferred embodiment, an L234A or L235A substitution is introduced into the human IgG constant Fc region of the antibody-based molecule described herein. In a more preferred embodiment, L234A and L235A substitutions are introduced into the human IgG constant Fc region of the antibody-based molecule described herein. This embodiment produces an antibody-based molecule having the heavy chain represented by SEQ ID NO:268 or SEQ ID NO:270.

In one or even a more preferred embodiment, the anti-MuSK antibody or its antigen-binding fragment comprises:

a) A heavy chain variable domain (VH) containing an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and

b) A light chain variable domain (VL) containing an amino acid sequence that has at least 80% identity or similarity to SEQ ID NO:235.

In this context, the identity or similarity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

Preferred anti-MuSK antibodies or their antigen-binding fragments include:

a) Full-length heavy chain, comprising SEQ ID NO: 268 and

b) A full-length light chain containing SEQ ID NO: 269.

Preferred anti-MuSK antibodies or their antigen-binding fragments include:

a) Full-length heavy chain, comprising SEQ ID NO: 270 and

b) A full-length light chain containing SEQ ID NO: 271.

In one embodiment, the antibody-based molecules of the present invention are “humanized,” particularly if they are intended for therapeutic purposes. The term “humanized” refers to a chimeric molecule typically prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species and a residual immunoglobulin structure based on the structure and/or sequence of a human immunoglobulin. The antigen-binding site may comprise a complete non-human antibody variable domain fused to a human constant domain, or a complementarity-determining region (CDR) of such a variable domain grafted onto a suitable human framework region of the human variable domain. The framework residues of such humanized molecules may be wild-type (e.g., fully human), or they may be modified to include one or more amino acid substitutions not present in the human antibody (the sequence of which serves as the basis for humanization). Humanization reduces or eliminates the likelihood that the constant regions of a molecule will act as immunogens in a human individual, but the likelihood of an immune response to exogenous variable regions remains unchanged (LoBuglio, A.F. et al. "Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics and Immune Response," Proc. Natl. Acad. Sci. USA 86: 4220-4224 (1989), which is incorporated herein by reference in its entirety). Another approach focuses not only on providing human-derived constant regions but also on modifying variable regions to reconstruct them as closely as possible to their human form. Both heavy and light chain variable regions contain three complementarity-determining regions (CDRs) that vary in response to the antigen in question and determine binding capacity. The CDRs are flanked by four framework regions (FRs), which are relatively conserved in a given species and are presumed to provide a scaffold for the CDRs. When preparing nonhuman antibodies against a specific antigen, the variable region can be "reconstructed" or "humanized" by grafting a CDR derived from the nonhuman antibody onto the FR present in the human antibody to be modified. Suitable methods for humanizing the nonhuman antibodies described herein are known in the art, see, for example...

Sato, K. et al., Cancer Res 53; 851-856 (1993); Riechmann, L et al., "Reshaping Human Antibodies for Therapy," Nature 332: 323-327 (1988); Verhoeyen, M.et al., "Reshaping Human Antibodies: Grafting An Antilysozyme Activity," Science 239: 1534-1536 (1988); Kettleborough, C.A. et al., "Humanization OfA Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues OnLoop Conformation," Protein Engineering 4: 773-3783 (1991); Maeda, H.et al., "Construction Of Reshaped Human Antibodies With HIV-Neutralizing Activity," Human Antibodies Hybridoma 2: 124-134 (1991); Gorman, S.D. et al., "Re shaping A Therapeutic CD4 Antibody," Proc. Natl. Acad. Sci. USA 88: 4181-4185 (1991); Tempest, P. R. et al., "Reshaping A Human Monoclonal Antibody To Inhi bit Humanized Antibodies For Antiviral Therap y," Proc. Natl. Acad. Sci. USA 88: 2869-2873 (1991); Carter, P. et al., "Humanization OfAnAnti-p185her2Antibody For Human Cancer Therapy," Proc Natl. Acad. Sci.USA 89: 4285-4289 (1992); and Co, M.S. et al., "Chimeric And Humanized Antibodies WithSpecificity For The CD33Antigen," J. Immunol. 148: 1149-1154 (1992)

The entire contents of this document are incorporated herein by reference. In some embodiments, the humanized MuSK antibody of the present invention retains all CDR sequences (e.g., a humanized antibody comprising all six CDRs from an antibody derived from a llama or mouse). In other embodiments, the humanized MuSK antibody of the present invention has one or more CDRs (one, two, three, four, five, or six) altered relative to the original antibody. Methods for humanizing antibodies are well known in the art and suitable for humanizing the antibodies disclosed herein (see, for example, U.S. Patent No. 5,225,539 to Winter; U.S. Patent Nos. 5,530,101 and 5,585,089 to Queen and Selick; U.S. Patent No. 5,859,205 to Robert et al.; U.S. Patent No. 6,407,213 to Carter; and U.S. Patent No. 6,881,557 to Foote, which are incorporated herein by reference in their entirety).

In some antibodies, only a subset of the CDR, namely the set of CDR residues required for binding called "specificity-determining residues" (SDRs), is needed to maintain antibody binding. CDR residues that do not contact the antigen and are not in the SDR can be identified based on previous studies of the Kabat CDR region located outside the Chothia hypervariable ring (see Kabat et al., Sequences of Proteins of Immunological Interest. National Institutes of Health Publication No. 91-3242 (1992); Chothia, C. et al., "Canonical Structures For The Hypervariable Regions of I..."). "Munoglobulins," J. Mol. Biol. 196: 901-917 (1987), which is incorporated herein by reference in its entirety, are identified by molecular modeling and/or primarily empirically or as described in Gonzales, N. R. et al., "SDR Grafting of A Murine Antibody Using Multiple Human Germline Templates To Minimize Its Immunogenicity," Mol. Immunol. 41: 863-872 (2004) (which is incorporated herein by reference in its entirety). In such humanized antibodies, at positions where one or more donor CDR residues are absent or where the entire donor CDR is omitted, the amino acid residue occupying said position can be an amino acid residue occupying the corresponding position in the acceptor antibody sequence (by Kabat number). The number of such substitutions of the acceptor for the donor amino acid in the included CDR reflects a balance of competition considerations. Such substitutions can help reduce the number of non-human amino acids in humanized antibodies, thereby reducing potential immunogenicity. However, substitutions can also cause changes in affinity, and a significant decrease in affinity is preferably avoided. Substitutions can also lead to changes in activity. Such substitutions that result in a significant decrease in activity are also preferably avoided. In this case, the antibody or antibody fragment should still exhibit detectable antibody activity as defined earlier herein, or at least to some extent, antibody activity. The substitution sites within the CDR and the amino acids to be substituted can also be selected empirically.

Phage display technology can alternatively be used to increase (or decrease) the CDR affinity of the antibody-based molecules of the present invention. This technique (referred to as affinity maturation) employs mutagenesis, or “CDR walking,” and reselection using the target antigen or fragments thereof to identify antibodies that have a CDR that binds to the antigen with a higher (or lower) affinity when compared to the initial or parental antibody (see, for example, Glaser et al., “Antibody Engineering By Codon-Based Mutagenesis In A Filamentous Phage Vector System,” J. Immunology 149:3903-3913 (1992), which is incorporated herein by reference in its entirety). A semi-random library is created by mutagenesis of the entire codon, rather than individual nucleotides, resulting in amino acid mutations. The library can consist of a library of variant clones, each variant clone differing from another member of such a library in that it contains a single amino acid change in a single CDR and contains variants that may represent every possible amino acid substitution for each CDR residue. Mutants exhibiting increased (or decreased) binding affinity to antigens can be screened by contacting immobilized mutants with labeled antigens. Any screening method known in the art can be used to identify variant-based antibody-binding molecules with increased or decreased affinity to antigens (e.g., ELISA) (see Wu, H. et al., “Stepwise In Vitro Affinity Maturation Of Vitaxin, An Alphav Beta3-Specific Humanized mAb,” Proc. Natl. Acad. Sci. USA 95:6037-6042 (1998); Yelton et al., “Affinity Maturation Of The BR96 Anti-Carcinoma Antibody By Codon-Based Mutagenesis,” J. Immunology 155:1994 (1995), which are incorporated herein by reference in their entirety). The "CDR step" that randomizes the light chain can be used (see Schier, R. et al., "Isolation of Picomolar Affinity Anti-c-erbB-2 Single-Chain Fv By Molecular Evolution Of The Complementarity Determining Regions In The Center Of The Antibody Binding Site," J. Mol. Biol. 263:551-567 (1996), which is incorporated herein by reference in its entirety).

Methods for affinity maturation of Musk antibody molecules are described herein and disclosed, for example,

Krause, J.C.et al., “An Insertion Mutation That Distorts AntibodyBinding Site Arcnitecture Enhancements Function of a Human Antibody,” MBio.2(1):e00345-10(2011); Kuan, C.T.et al., “Affinity-Matured Anti- Glycoprotein NMBRecombinant Immunotoxins Targeting Malignant Gliomas And Melanomas," Int.J.Cancer 10.1002/ijc.25645(2010); Hackel, B.J.et al., "Stability And CDRComposition Biases Enrich Binder Func tionality Landscapes,” J.Mol.Biol.401(1):84-96(2010);Montgomery, D.L.et al., “Affinity Maturation An dCharacterizationOf A Human Monoclonal Antibody Against HlV-1gp41." MAbs1(5):462-474(2009); Gustch ina, E.et al., “Affinity Maturation By Targeted Diversification Of TheCDR-H2Loop Of A Monoclonal Fab Derived From A SyntheticHuman AntibodyLibrary And Directed Against The Internal Trimeric Coiled -Coil Of Gp41 YieldsA Set Of Fabs With Improved HIV-1Neutralization Potency And Breadth," Virology 393(1):112-119(2009); Finlay, WJ.et al., “Affinity Maturation Of A Humanized RatAntibody For Anti-RA GE Therapy: Comprehensive Mutagenesis Reveals A High Level Of Mutational Plasticity Both Inside And Outside The Complementarity-Determining Regions,” J.Mol.Biol.388(3):541-558(2009); Bostrom, J.et al ., "Improving Antibody Binding Affinity And Specificity For Therapeutic Development," Methods Mol. Biol. 525: 353-376 (2009); Steidl, S. et al., "ln VitroAffinity Maturation Of Human GM-CSF Antibodies By T argeted CDR-Diversification," Mol. Immunol. 46(1): 135-144 (2008); and Barderas, R. et al., "Affinity Maturat ion Of Antibodies Assisted By ln Silico Modeling,” Proc.Natl.Acad.Sci.USA 105(26):9029-9034(2008),

It is incorporated here by reference.

In the context of this application, amino acid alteration (change or modification) can be amino acid substitution, addition, deletion or chemical modification.

In one embodiment, the MuSK antibody-based molecule as described herein comprises any one, any two, any three, any four, any five, or any six amino acid sequences of CDRs as provided in Tables 1 and 2 herein.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) includes a heavy chain variable domain, wherein the heavy chain variable domain comprises:

(i) Complementarity-determining region 1 (CDR-H1) comprising an amino acid sequence of any one of SEQ ID NO: 1 to 16, 135, 136, 147 to 149, or a modified amino acid sequence of any one of SEQ ID NO: 1 to 16, 135, 136, 147 to 149, wherein the modified sequence has 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO: 1 to 16, 135, 136, 147 to 149; (i i) Complementarity-determining region 2 (CDR-H2) comprising an amino acid sequence of any one of SEQ ID NO: 17 to 32, 137, 138, 150 to 155, or a modified amino acid sequence of any one of SEQ ID NO: 17 to 32, 137, 138, 150 to 155, wherein the modified sequence has 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO: 17 to 32, 137, 138, 150 to 155; and

(iii) Complementarity-determining region 3 (CDR-H3) comprising an amino acid sequence of any one of SEQ ID NO: 33 to 48, 139, 140, 156 to 158, 240 to 251, or a modified amino acid sequence of any one of SEQ ID NO: 33 to 48, 139, 140, 156 to 158, or 240 to 251, wherein the modified sequence has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO: 33 to 48, 139, 140, 156 to 158, or 240 to 251.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises: (i) a heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:1 or CDR-H1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:1, CDR-H2 of SEQ ID NO:17 or CDR-H2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:17, and CDR-H3 of SEQ ID NO:33 or CDR-H2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:33. (ii) a heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:2 or CDR-H1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 or CDR-H2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:18, and CDR-H3 of SEQ ID NO:34 or CDR-H3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:34; (iii) a heavy chain variable domain comprising: S (iv) A heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:3 or CDR-H1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:3; CDR-H2 of SEQ ID NO:19 or CDR-H2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:19; and CDR-H3 of SEQ ID NO:35 or CDR-H3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:35; O:4 CDR-H1 having 1, 2, 3, 4 or 5 amino acid changes, CDR-H2 of SEQ ID NO:20 or CDR-H2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:20, and CDR-H3 of SEQ ID NO:36 or CDR-H3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:36; (v) heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:5 or CDR-H1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:5, SEQ ID NO:20, CDR-H2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:20, CDR-H2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:36, CDR-H3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:36, CDR-H2 ... CDR-H2 of SEQ ID NO:21 or CDR-H2 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:21, and CDR-H3 of SEQ ID NO:37 or CDR-H3 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:37; (vi) a heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:6 or CDR-H1 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:6, CDR-H2 of SEQ ID NO:22 or CDR-H2 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:22. CDR-H2 with 1, 2, 3, 4, or 5 amino acid changes, and CDR-H3 of SEQ ID NO:38 or CDR-H3 with 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:38; (vii) heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:7 or CDR-H1 with 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:7, CDR-H2 of SEQ ID NO:23 or CDR-H2 with 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:23, and SEQ ID NO:38. (viii) A heavy chain variable domain comprising: CDR-H1 of SEQ ID NO: 8 or CDR-H1 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO: 8; CDR-H2 of SEQ ID NO: 24 or CDR-H2 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO: 24; and CDR-H3 of SEQ ID NO: 40 or CDR-H3 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO: 39; NO:40 has CDR-H3 with 1, 2, 3, 4 or 5 amino acid changes; (ix) heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:9 or CDR-H1 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:9, CDR-H2 of SEQ ID NO:25 or CDR-H2 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:25, and CDR-H3 of SEQ ID NO:41 or CDR-H3 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:41; (x) A heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:10 or CDR-H1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:10, CDR-H2 of SEQ ID NO:26 or CDR-H2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:26, and CDR-H3 of SEQ ID NO:42 or CDR-H3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:42; (xi) A heavy chain variable domain comprising: SEQ ID NO:11 The CDR-H1 of SEQ ID NO:12 or a CDR-H1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:11, the CDR-H2 of SEQ ID NO:27 or a CDR-H2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:27, and the CDR-H3 of SEQ ID NO:43 or a CDR-H3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:43; (xii) a heavy chain variable domain comprising: the CDR-H1 of SEQ ID NO:12 or a CDR-H1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:12. CDR-H1 with 2, 3, 4, or 5 amino acid changes, CDR-H2 of SEQ ID NO:28 or CDR-H2 with 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:28, and CDR-H3 of SEQ ID NO:44 or CDR-H3 with 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:44; (xiii) a heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:13 or CDR-H1 with 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:13, SEQ I CDR-H2 of SEQ ID NO:29 or CDR-H2 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:29, and CDR-H3 of SEQ ID NO:45 or CDR-H3 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:45; (xiv) heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:14 or CDR-H1 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:14, CDR-H2 of SEQ ID NO:30 or CDR-H2 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:29, and CDR-H2 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:29; O:30 CDR-H2 with 1, 2, 3, 4 or 5 amino acid changes, and CDR-H3 of SEQ ID NO:46 or CDR-H3 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:46; (xv) heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:15 or CDR-H1 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:15, CDR-H2 of SEQ ID NO:31 or CDR-H2 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:31. 2, and CDR-H3 of SEQ ID NO:47 or CDR-H3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:47; (xvi) heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:16 or CDR-H1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:16, CDR-H2 of SEQ ID NO:32 or CDR-H2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:32, and CDR-H3 of SEQ ID NO:48 or CDR-H3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:47. SEQ ID NO:48 CDR-H3 having 1, 2, 3, 4, or 5 amino acid alterations; (xvii) heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:135 or CDR-H1 having 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:135, CDR-H2 of SEQ ID NO:137 or CDR-H2 having 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:137, and CDR-H3 of SEQ ID NO:139 or CDR-H3 having 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:139. (xviii) CDR-H3 with 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:136; and (xviii) heavy chain variable domains comprising: CDR-H1 of SEQ ID NO:136 or CDR-H1 with 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:136; CDR-H2 of SEQ ID NO:138 or CDR-H2 with 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:138; and CDR-H3 of SEQ ID NO:140 or CDR-H3 with 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:140. The sequences of the heavy chain CDRs are provided in Table 1.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises: (ii.a) a heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:2 or CDR-H1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 or CDR-H2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:18, and CDR-H3(X2m1) of SEQ ID NO:240 or CDR-H2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:18, and CDR-H3(X2m1) of SEQ ID NO:240 or CDR-H2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:240. (ii.b) CDR-H3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:240; and (ii.b) a heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:2 or CDR-H1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 or CDR-H2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:18, and CDR-H3 (X2m2) of SEQ ID NO:241 or CDR-H3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:240. NO:241 has CDR-H3 with 1, 2, 3, 4 or 5 amino acid changes; (ii.c) heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:2 or CDR-H1 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 or CDR-H2 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:18, and CDR-H3 (X2m3) of SEQ ID NO:242 or CDR-H3 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:242. 242 CDR-H3 having 1, 2, 3, 4 or 5 amino acid alterations; (ii.d) heavy chain variable domains comprising: CDR-H1 of SEQ ID NO:2 or CDR-H1 having 1, 2, 3, 4 or 5 amino acid alterations relative to SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 or CDR-H2 having 1, 2, 3, 4 or 5 amino acid alterations relative to SEQ ID NO:18, and CDR-H3(X2m4) of SEQ ID NO:243 or CDR-H3 having 1, 2, 3, 4 or 5 amino acid alterations relative to SEQ ID NO:243. (ii.e) CDR-H3 with 2, 3, 4, or 5 amino acid alterations; (ii.e) heavy chain variable domains comprising: CDR-H1 of SEQ ID NO:2 or CDR-H1 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 or CDR-H2 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:18, and CDR-H3(X2m5) of SEQ ID NO:244 or CDR-H3 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:244. (ii.f) CDR-H3 with 4 or 5 amino acid alterations; (ii.f) Heavy chain variable domains comprising: CDR-H1 of SEQ ID NO:2 or CDR-H1 with 1, 2, 3, 4 or 5 amino acid alterations relative to SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 or CDR-H2 with 1, 2, 3, 4 or 5 amino acid alterations relative to SEQ ID NO:18, and CDR-H3(X2m6) of SEQ ID NO:245 or CDR-H3 with 1, 2, 3, 4 or 5 amino acid alterations relative to SEQ ID NO:245. (ii.g) Acid-modified CDR-H3; (ii.g) Heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:2 or CDR-H1 having 1, 2, 3, 4 or 5 amino acid modifications relative to SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 or CDR-H2 having 1, 2, 3, 4 or 5 amino acid modifications relative to SEQ ID NO:18, and CDR-H3(X2m7) of SEQ ID NO:246 or CDR-H3 having 1, 2, 3, 4 or 5 amino acid modifications relative to SEQ ID NO:246. CDR-H3; (ii.h) Heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:2 or CDR-H1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 or CDR-H2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:18, and CDR-H3(X2m8) of SEQ ID NO:247 or CDR-H3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:247.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises: (xvii.a) a heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:135 or CDR-H1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:135, CDR-H2 of SEQ ID NO:137 or CDR-H2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:137, and CDR-H3 (X17m1) of SEQ ID NO:248 or CDR-H3 (X17m1) having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:137, and CDR-H3 (X17m1) of SEQ ID NO:248. O:248 CDR-H3 having 1, 2, 3, 4, or 5 amino acid alterations; (xvii.b) heavy chain variable domains comprising: CDR-H1 of SEQ ID NO:135 or CDR-H1 having 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:135, CDR-H2 of SEQ ID NO:137 or CDR-H2 having 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:137, and CDR-H3 (X17m2) of SEQ ID NO:249 or CDR-H3 having 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:249. CDR-H3 with 1, 2, 3, 4, or 5 amino acid alterations; (xvii.c) heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:135 or CDR-H1 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:135, CDR-H2 of SEQ ID NO:137 or CDR-H2 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:137, and CDR-H3 (X17m3) of SEQ ID NO:250 or CDR-H3 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:250. The modified CDR-H3; (xvii.d) heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:135 or CDR-H1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:135, CDR-H2 of SEQ ID NO:137 or CDR-H2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:137, and CDR-H3 (X17m6) of SEQ ID NO:251 or CDR-H3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:251.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises a heavy chain variable domain, wherein the heavy chain variable domain comprises: (xix) a heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:147 or CDR-H1 having 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:147, CDR-H2 of SEQ ID NO:150 or CDR-H2 having 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:150, and CDR-H3 of SEQ ID NO:156 or CDR-H3 having 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:156; and (xx) a heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:148 or CDR-H1 having 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:148. CDR-H1, CDR-H2 of SEQ ID NO:151 or CDR-H2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:151, and CDR-H3 of SEQ ID NO:157 or CDR-H3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:157; (xxi) heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:149 H1 or CDR-H1 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:149, CDR-H2 of SEQ ID NO:152 or CDR-H2 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:152, and CDR-H3 of SEQ ID NO:158 or CDR-H3 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:158.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises a heavy chain variable domain, wherein the heavy chain variable domain comprises (xxii) a heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:147 or CDR-H1 having 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:147, CDR-H2 of SEQ ID NO:153 or CDR-H2 having 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:153. CDR-H2 with 3, 4, or 5 amino acid alterations, and CDR-H3 (3B2g1m1/3B2g2m1) of SEQ ID NO:156 or CDR-H3 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:156; (xxiii) heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:147 or CDR-H1 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:147, S CDR-H2 of SEQ ID NO:154 or CDR-H2 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:154, and CDR-H3 (3B2g1m2/3B2g2m2) of SEQ ID NO:156 or CDR-H3 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156; (xxi) heavy chain variable domain comprising: CDR-H1 of SEQ ID NO:147 or the same For CDR-H1 of SEQ ID NO:147 with 1, 2, 3, 4 or 5 amino acid changes, CDR-H2 of SEQ ID NO:155 or CDR-H2 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:155, and CDR-H3 (3B2g1m4/3B2g2m4) of SEQ ID NO:156 or CDR-H3 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156, the sequences of the heavy chain CDRs are provided in Table 1.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises: (i) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:1, CDR-H2 of SEQ ID NO:17, and CDR-H3 of SEQ ID NO:33; and (ii) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18, and CDR-H3 of SEQ ID NO:33. (iii) a heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:3, CDR-H2 of SEQ ID NO:19, and CDR-H3 of SEQ ID NO:35; (iv) a heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:4, CDR-H2 of SEQ ID NO:20, and CDR-H3 of SEQ ID NO:36; (v) a heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:34, CDR-H2 of SEQ ID NO:20, and CDR-H3 of SEQ ID NO:36; (vi) A heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:37; (vii) a heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:6, CDR-H2 of SEQ ID NO:22, and CDR-H3 of SEQ ID NO:38; (vii) a heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:7, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:38. (viii) CDR-H2 of SEQ ID NO:23 and CDR-H3 of SEQ ID NO:39; (viii) a heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:8, CDR-H2 of SEQ ID NO:24 and CDR-H3 of SEQ ID NO:40; (ix) a heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:9, CDR-H2 of SEQ ID NO:25 and CDR-H3 of SEQ ID NO:40. (x) A heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:10, CDR-H2 of SEQ ID NO:26, and CDR-H3 of SEQ ID NO:42; (xi) A heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:11, CDR-H2 of SEQ ID NO:27, and CDR-H3 of SEQ ID NO:43; (xii) A heavy chain variable structural domain comprising (xiii) CDR-H1 of SEQ ID NO:12, CDR-H2 of SEQ ID NO:28, and CDR-H3 of SEQ ID NO:44; (xiv) Heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:13, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:45; (xv) CDR-H2 of SEQ ID NO:30 and CDR-H3 of SEQ ID NO:46; (xv) Heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:15, CDR-H2 of SEQ ID NO:31 and CDR-H3 of SEQ ID NO:47; (xvi) Heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:16, CDR-H2 of SEQ ID NO:32 and CDR-H3 of SEQ ID NO:47. CDR-H3 of SEQ ID NO:48; (xvii) heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:135, CDR-H2 of SEQ ID NO:137, and CDR-H3 of SEQ ID NO:139; and (xviii) heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:136, CDR-H2 of SEQ ID NO:138, and CDR-H3 of SEQ ID NO:140. The sequences of the heavy chain CDR sequences are provided in Table 1 below.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises: (ii.a) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18, and CDR-H3(X2m1) of SEQ ID NO:240; and (ii.b) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18, and CDR-H3(X2m1) of SEQ ID NO:240. (ii.c) Heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18, and CDR-H3(X2m2) of SEQ ID NO:241; (ii.d) Heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18, and CDR-H3(X2m3) of SEQ ID NO:242; (ii.e) CDR-H3(X2m4) of SEQ ID NO:243; (ii.e) heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18, and CDR-H3(X2m5) of SEQ ID NO:244; (ii.f) heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18, and CDR-H3(X2m5) of SEQ ID NO:245. R-H3(X2m6); (ii.g) heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 and CDR-H3(X2m7) of SEQ ID NO:246; (ii.h) heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 and CDR-H3(X2m8) of SEQ ID NO:247.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises: (xvii.a) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:135, CDR-H2 of SEQ ID NO:137, and CDR-H3 (X17m1) of SEQ ID NO:248; (xvii.b) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:135, CDR-H2 of SEQ ID NO:137, and CDR-H3 (X17m1) of SEQ ID NO:248. CDR-H3(X17m2) of SEQ ID NO:249; (xvii.c) heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:135, CDR-H2 of SEQ ID NO:137 and CDR-H3(X17m3) of SEQ ID NO:250; (xvii.d) heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:135, CDR-H2 of SEQ ID NO:137 and CDR-H3(X17m6) of SEQ ID NO:251.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises a heavy chain variable domain, wherein the heavy chain variable domain comprises: (xix) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:150, and CDR-H3 of SEQ ID NO:156; (xx) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:148, CDR-H2 of SEQ ID NO:151, and CDR-H3 of SEQ ID NO:157; and (xxi) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:149, CDR-H2 of SEQ ID NO:152, and CDR-H3 of SEQ ID NO:158.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises a heavy chain variable domain, wherein the heavy chain variable domain comprises: (xxii) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:153, and CDR-H3 (3B2g1m1/3B2g2m1) of SEQ ID NO:156; and (xxiii) a heavy chain variable domain comprising (xxiv) CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:154, and CDR-H3 (3B2g1m2/3B2g2m2) of SEQ ID NO:156; and (xxiv) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:155, and CDR-H3 (3B2g1m4/3B2g2m4) of SEQ ID NO:156. The sequences of the heavy chain CDR sequences are provided in Table 1 below.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises a heavy chain variable domain, wherein the heavy chain variable domain comprises CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:153, or a CDR-H2 amino acid sequence having at least 0, 1, 2, 3, 4, or 5 alterations relative to SEQ ID NO:153, and CDR-H3 (3B2g2m1) of SEQ ID NO:156. In one embodiment, the CDR-H2 amino acid sequence has at least 0, 1, 2, 3, 4, or 5 alterations relative to SEQ ID NO:153. According to this embodiment, the CDR-H2 amino acid sequence has at least 0, 1, 2, 3, 4, or 5 alterations relative to SEQ ID NO:153, wherein said alterations are present at residues 1, 2, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or any combination thereof.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) includes a heavy chain variable domain, wherein the heavy chain variable domain comprises:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156.

In one embodiment, the antibody's CDR-H2 contains proline (P) at position 3, tryptophan (W) at position 4, and serine (S) or asparagine (N) at position 5.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) includes a heavy chain variable domain, wherein the heavy chain variable domain comprises:

- Contains the CDR-H1 amino acid sequence consisting of SEQ ID NO:147 or SEQ ID NO:147.

- Contains the CDR-H2 amino acid sequence of SEQ ID NO:153 or composed of SEQ ID NO:153, and

- Contains the CDR-H3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:156 or SEQ ID NO:156.

Table 1 below provides the sequences of heavy chain CDR sequences.

In some embodiments, the MuSK antibody-based molecules disclosed herein also include a light chain variable domain. The light chain variable domain includes:

(i) Complementarity-determining region 1 (CDR-L1) having an amino acid sequence of any one of SEQ ID NO:49 to 64, 141, 142, 159 to 169, or a modified amino acid sequence of any one of SEQ ID NO:49 to 64, 141, 142 or 159 to 169, wherein the modified sequence has at least 80% sequence identity with any one of SEQ ID NO:49 to 64, 141, 142 or 159 to 169;

(ii) Complementarity-determining region 2 (CDR-L2) having an amino acid sequence of any one of SEQ ID NO: 65 to 80, 143, 144, 170 to 179, or a modified amino acid sequence of any one of SEQ ID NO: 65 to 80, 143, 144, or 170 to 179, wherein the modified sequence has at least 80% sequence identity with any one of SEQ ID NO: 65 to 80, 143, 144, or 170 to 179; and

(iii) Complementarity-determining region 3 (CDR-L3) having an amino acid sequence of any one of SEQ ID NO: 81 to 96, 145, 146, 180 to 195, or a modified amino acid sequence of any one of SEQ ID NO: 81 to 96, 145, 146, or 180 to 195, wherein the modified sequence has at least 80% sequence identity with any one of SEQ ID NO: 81 to 96, 145, 146, or 180 to 195.

In some embodiments, the MuSK antibody-based molecules disclosed herein also include a light chain variable domain. The light chain variable domain includes:

(iv) Complementarity-determining region 1 (CDR-L1) having an amino acid sequence of any one of SEQ ID NO:49 to 64, 141, 142, 159 to 169, or a modified amino acid sequence of any one of SEQ ID NO:49 to 64, 141, 142 or 159 to 169, wherein the modified sequence has 1, 2, 3, 4 or 5 amino acid changes relative to any one of SEQ ID NO:49 to 64, 141, 142 or 159 to 169;

(v) Complementarity-determining region 2 (CDR-L2) having an amino acid sequence of any one of SEQ ID NO: 65 to 80, 143, 144, 170 to 179, or a modified amino acid sequence of any one of SEQ ID NO: 65 to 80, 143, 144, or 170 to 179, wherein the modified sequence has 1, 2, 3, 4, or 5 amino acid changes relative to any one of SEQ ID NO: 65 to 80, 143, 144, or 170 to 179; and

(vi) Complementarity-determining region 3 (CDR-L3) having an amino acid sequence of any one of SEQ ID NO: 81 to 96, 145, 146, 180 to 195, or a modified amino acid sequence of any one of SEQ ID NO: 81 to 96, 145, 146, or 180 to 195, wherein the modified sequence has 1, 2, 3, 4, or 5 amino acid changes relative to any one of SEQ ID NO: 81 to 96, 145, 146, or 180 to 195.

In one embodiment, the light chain variable domain of the MuSK antibody-based molecule disclosed herein comprises (i) a light chain variable domain comprising: CDR-L1 of SEQ ID NO:49 or CDR-L1 having 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:49, CDR-L2 of SEQ ID NO:65 or CDR-L2 having 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:65, and CDR-L3 of SEQ ID NO:81 or CDR-L3 having 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:81. CDR-L3; (ii) a light chain variable domain comprising: CDR-L1 of SEQ ID NO:50 or CDR-L1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:50, CDR-L2 of SEQ ID NO:66 or CDR-L2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:66, and CDR-L3 of SEQ ID NO:82 or CDR-L3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:82; (iii) a light chain variable domain comprising: SEQ ID NO:50, CDR-L1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:82; (iv) A light chain variable domain comprising: CDR-L1 of SEQ ID NO:51 or CDR-L1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:51; CDR-L2 of SEQ ID NO:67 or CDR-L2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:67; and CDR-L3 of SEQ ID NO:83 or CDR-L3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:83; O:52 CDR-L1 with 1, 2, 3, 4 or 5 amino acid changes, CDR-L2 of SEQ ID NO:68 or CDR-L2 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:68, and CDR-L3 of SEQ ID NO:84 or CDR-L3 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:84; (v) a light chain variable domain comprising: CDR-L1 of SEQ ID NO:53 or CDR-L1 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:53, (vi) A light chain variable domain comprising: CDR-L2 of SEQ ID NO:69 or CDR-L2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:69, and CDR-L3 of SEQ ID NO:85 or CDR-L3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:85; (ii) A light chain variable domain comprising: CDR-L1 of SEQ ID NO: 70 having 1, 2, 3, 4 or 5 amino acid changes, and CDR-L3 of SEQ ID NO: 86 or having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO: 86; (vii) a light chain variable domain comprising: CDR-L1 of SEQ ID NO: 55 or having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO: 55, CDR-L2 of SEQ ID NO: 71 or having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO: 71. -L2, and CDR-L3 of SEQ ID NO:87 or CDR-L3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:87; (viii) light chain variable domain comprising: CDR-L1 of SEQ ID NO:56 or CDR-L1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:56, CDR-L2 of SEQ ID NO:72 or CDR-L2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:72, and CDR-L3 of SEQ ID NO:88 or (ix) A light chain variable domain comprising: CDR-L1 of SEQ ID NO:57 or CDR-L1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:57; CDR-L2 of SEQ ID NO:73 or CDR-L2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:73; and CDR-L3 of SEQ ID NO:89 or CDR-L3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:89. (x) A light chain variable domain comprising: CDR-L1 of SEQ ID NO:58 or CDR-L1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:58, CDR-L2 of SEQ ID NO:74 or CDR-L2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:74, and CDR-L3 of SEQ ID NO:90 or CDR-L3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:90; (xi) A light chain variable domain comprising: S CDR-L1 of SEQ ID NO:59 or CDR-L1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:59, CDR-L2 of SEQ ID NO:75 or CDR-L2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:75, and CDR-L3 of SEQ ID NO:91 or CDR-L3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:91; (xii) a light chain variable domain comprising: CDR-L1 of SEQ ID NO:60 or CDR-L1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:59; IDNO:60 having a CDR-L1 with 1, 2, 3, 4 or 5 amino acid changes, SEQ ID NO:76 having a CDR-L2 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:76, and SEQ ID NO:92 having a CDR-L3 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:92; (xiii) a light chain variable domain comprising: SEQ ID NO:61 having a CDR-L1 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:61. L1, CDR-L2 of SEQ ID NO:77 or CDR-L2 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:77, and CDR-L3 of SEQ ID NO:93 or CDR-L3 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:93; (xiv) light chain variable domain comprising: CDR-L1 of SEQ ID NO:62 or CDR-L1 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:62, CDR-L2 of SEQ ID NO:78 or CDR-L2 with 1, 2, 3, 4 or 5 amino acid changes relative to SE Q ID NO:78 CDR-L2 with 1, 2, 3, 4 or 5 amino acid changes, and SEQ ID NO:94 CDR-L3 or CDR-L3 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:94; (xv) light chain variable domain comprising: SEQ ID NO:63 CDR-L1 or CDR-L1 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:63, SEQ ID NO:79 CDR-L2 or CDR-L2 with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:79. CDR-L2, and CDR-L3 of SEQ ID NO:95 or CDR-L3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:95; (xvi) light chain variable domain comprising: CDR-L1 of SEQ ID NO:64 or CDR-L1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:64, CDR-L2 of SEQ ID NO:80 or CDR-L2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:80, and CDR-L3 of SEQ ID NO:96. Or a CDR-L3 having 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO: 96; (xvii) a light chain variable domain comprising: CDR-L1 of SEQ ID NO: 141 or a CDR-L1 having 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO: 141, CDR-L2 of SEQ ID NO: 143 or a CDR-L2 having 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO: 143, and CDR-L3 of SEQ ID NO: 145 or a CDR-L3 having 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO: 145. (xviii) CDR-L3 with 1, 2, 3, 4, or 5 amino acid alterations; (xviii) a light chain variable domain comprising: CDR-L1 of SEQ ID NO:142 or CDR-L1 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:142, CDR-L2 of SEQ ID NO:144 or CDR-L2 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:144, and CDR-L3 of SEQ ID NO:146 or CDR-L3 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:146. The sequences of the light chain CDRs are provided in Table 2 below.

In one embodiment, the light chain variable domain of the MuSK antibody-based molecule disclosed herein comprises (xix) light chain variable domains, which include: CDR-L1 of SEQ ID NO:159 or CDR-L1 having 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:159, CDR-L2 of SEQ ID NO:170 or CDR-L2 having 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:170, and CDR-L3 of SEQ ID NO:180 or CDR-L3 having 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:170. ID NO:180 CDR-L3 having 1, 2, 3, 4 or 5 amino acid alterations; (xx) light chain variable domains comprising: CDR-L1 of SEQ ID NO:159 or CDR-L1 having 1, 2, 3, 4 or 5 amino acid alterations relative to SEQ ID NO:159, CDR-L2 of SEQ ID NO:171 or CDR-L2 having 1, 2, 3, 4 or 5 amino acid alterations relative to SEQ ID NO:171, and CDR-L3 of SEQ ID NO:181 or CDR-L3 having 1, 2, 3, 4 or 5 amino acid alterations relative to SEQ ID NO:180. O:181 CDR-L3 having 1, 2, 3, 4, or 5 amino acid modifications; (xxi) light chain variable domain comprising: CDR-L1 of SEQ ID NO:160 or CDR-L1 having 1, 2, 3, 4, or 5 amino acid modifications relative to SEQ ID NO:160, CDR-L2 of SEQ ID NO:172 or CDR-L2 having 1, 2, 3, 4, or 5 amino acid modifications relative to SEQ ID NO:172, and CDR-L3 of SEQ ID NO:182 or CDR-L3 having 1, 2, 3, 4, or 5 amino acid modifications relative to SEQ ID NO:180. 182 CDR-L3 having 1, 2, 3, 4, or 5 amino acid alterations; (xxii) light chain variable domains comprising: CDR-L1 of SEQ ID NO:159 or CDR-L1 having 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:159, CDR-L2 of SEQ ID NO:172 or CDR-L2 having 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:172, and CDR-L3 of SEQ ID NO:183 or CDR-L3 having 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:183. (xxiii) CDR-L3 having 1, 2, 3, 4, or 5 amino acid alterations; (xxiii) a light chain variable domain comprising: CDR-L1 of SEQ ID NO:159 or CDR-L1 having 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:159, CDR-L2 of SEQ ID NO:171 or CDR-L2 having 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:171, and CDR-L3 of SEQ ID NO:184 or CDR-L3 having 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:184. CDR-L3 having 1, 2, 3, 4, or 5 amino acid alterations; (xxiv) light chain variable domains comprising: CDR-L1 of SEQ ID NO:159 or CDR-L1 having 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:159, CDR-L2 of SEQ ID NO:173 or CDR-L2 having 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:173, and CDR-L3 of SEQ ID NO:185 or CDR-L3 having 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:185. CDR-L3 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:159; (xxv) light chain variable domains comprising: CDR-L1 of SEQ ID NO:159 or CDR-L1 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:159, CDR-L2 of SEQ ID NO:173 or CDR-L2 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:173, and CDR-L3 of SEQ ID NO:186 or CDR-L3 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:186. CDR-L3 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:161; (xxvi) light chain variable domains comprising: CDR-L1 of SEQ ID NO:161 or CDR-L1 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:161; CDR-L2 of SEQ ID NO:174 or CDR-L2 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:174; and CDR-L3 of SEQ ID NO:187 or CDR-L3 with 1, 2, 3, 4, or 5 amino acid alterations relative to SEQ ID NO:187. CDR-L3 with 4 or 5 amino acid alterations; (xxvii) light chain variable domains comprising: CDR-L1 of SEQ ID NO:162 or CDR-L1 with 1, 2, 3, 4 or 5 amino acid alterations relative to SEQ ID NO:162, CDR-L2 of SEQ ID NO:174 or CDR-L2 with 1, 2, 3, 4 or 5 amino acid alterations relative to SEQ ID NO:174, and CDR-L3 of SEQ ID NO:188 or CDR-L3 with 1, 2, 3, 4 or 5 amino acid alterations relative to SEQ ID NO:188. CDR-L3 with 4 or 5 amino acid alterations; (xxviii) light chain variable domains comprising: CDR-L1 of SEQ ID NO:163 or CDR-L1 with 1, 2, 3, 4 or 5 amino acid alterations relative to SEQ ID NO:163, CDR-L2 of SEQ ID NO:174 or CDR-L2 with 1, 2, 3, 4 or 5 amino acid alterations relative to SEQ ID NO:174, and CDR-L3 of SEQ ID NO:188 or CDR-L3 with 1, 2, 3, 4 or 5 amino acid alterations relative to SEQ ID NO:188. CDR-L3 with or 5 amino acid alterations; (xxix) light chain variable domains comprising: CDR-L1 of SEQ ID NO:164 or CDR-L1 with 1, 2, 3, 4 or 5 amino acid alterations relative to SEQ ID NO:164, CDR-L2 of SEQ ID NO:174 or CDR-L2 with 1, 2, 3, 4 or 5 amino acid alterations relative to SEQ ID NO:174, and CDR-L3 of SEQ ID NO:189 or CDR-L3 with 1, 2, 3, 4 or 5 amino acid alterations relative to SEQ ID NO:189. CDR-L3 with one amino acid modification; (xxx) light chain variable domains comprising: CDR-L1 of SEQ ID NO:165 or CDR-L1 with one, two, three, four or five amino acid modifications relative to SEQ ID NO:165, CDR-L2 of SEQ ID NO:175 or CDR-L2 with one, two, three, four or five amino acid modifications relative to SEQ ID NO:175, and CDR-L3 of SEQ ID NO:190 or CDR-L3 with one, two, three, four or five amino acid modifications relative to SEQ ID NO:190. The modified CDR-L3; (xxxi) light chain variable domain comprising: CDR-L1 of SEQ ID NO:166 or CDR-L1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:166, CDR-L2 of SEQ ID NO:176 or CDR-L2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:176, and CDR-L3 of SEQ ID NO:191 or CDR-L3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:191. CDR-L3; (xxxi) light chain variable domain comprising: CDR-L1 of SEQ ID NO:167 or CDR-L1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:167, CDR-L2 of SEQ ID NO:177 or CDR-L2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:177, and CDR-L3 of SEQ ID NO:192 or CDR-L3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:192. L3; (xxxii) Light chain variable domain comprising: CDR-L1 of SEQ ID NO:168 or CDR-L1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:168, CDR-L2 of SEQ ID NO:178 or CDR-L2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:178, and CDR-L3 of SEQ ID NO:193 or CDR-L3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:193. 3; (xxxiii) Light chain variable domains comprising: CDR-L1 of SEQ ID NO:169 or CDR-L1 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:169, CDR-L2 of SEQ ID NO:179 or CDR-L2 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:179, and CDR-L3 of SEQ ID NO:194 or CDR-L3 having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:194.

In one embodiment, the light chain variable domain of the MuSK antibody-based molecule disclosed herein comprises: (i) a light chain variable domain comprising CDR-L1 of SEQ ID NO:49, CDR-L2 of SEQ ID NO:65, and CDR-L3 of SEQ ID NO:81; and (ii) a light chain variable domain comprising CDR-L1 of SEQ ID NO:50, CDR-L2 of SEQ ID NO:66, and CDR-L3 of SEQ ID NO:81. (iii) a light chain variable structural domain comprising CDR-L1 of SEQ ID NO:51, CDR-L2 of SEQ ID NO:67, and CDR-L3 of SEQ ID NO:83; (iv) a light chain variable structural domain comprising CDR-L1 of SEQ ID NO:52, CDR-L2 of SEQ ID NO:68, and CDR-L3 of SEQ ID NO:84; (v) a light chain variable structural domain comprising... (vi) A light chain variable structural domain comprising CDR-L1 of SEQ ID NO:53, CDR-L2 of SEQ ID NO:69, and CDR-L3 of SEQ ID NO:85; (vii) a light chain variable structural domain comprising CDR-L1 of SEQ ID NO:54, CDR-L2 of SEQ ID NO:70, and CDR-L3 of SEQ ID NO:86; (viii) a light chain variable structural domain comprising CDR-L1 of SEQ ID NO:71, CDR-L2 of SEQ ID NO:72, and CDR-L3 of SEQ ID NO:88; (ix) a light chain variable structural domain comprising CDR-L1 of SEQ ID NO:57, CDR-L2 of SEQ ID NO:73, and CDR-L3 of SEQ ID NO:88. (x) A light chain variable structural domain comprising CDR-L1 of SEQ ID NO:58, CDR-L2 of SEQ ID NO:74, and CDR-L3 of SEQ ID NO:90; (xi) A light chain variable structural domain comprising CDR-L1 of SEQ ID NO:59, CDR-L2 of SEQ ID NO:75, and CDR-L3 of SEQ ID NO:91; (xii) A light chain variable structural domain, It includes CDR-L1 of SEQ ID NO:60, CDR-L2 of SEQ ID NO:76, and CDR-L3 of SEQ ID NO:92; (xiii) a light chain variable structural domain, which includes CDR-L1 of SEQ ID NO:61, CDR-L2 of SEQ ID NO:77, and CDR-L3 of SEQ ID NO:93; (xiv) a light chain variable structural domain, which includes CDR-L1 of SEQ ID NO:62, (xv) CDR-L2 of SEQ ID NO:78 and CDR-L3 of SEQ ID NO:94; (xvi) Light chain variable structural domains comprising CDR-L1 of SEQ ID NO:63, CDR-L2 of SEQ ID NO:79 and CDR-L3 of SEQ ID NO:95; (xvi) Light chain variable structural domains comprising CDR-L1 of SEQ ID NO:64, CDR-L2 of SEQ ID NO:80 and CDR-L3 of SEQ ID NO:95. CDR-L3 of SEQ ID NO:96; (xvii) light chain variable structural domains comprising CDR-L1 of SEQ ID NO:141, CDR-L2 of SEQ ID NO:143, and CDR-L3 of SEQ ID NO:145; (xviii) light chain variable structural domains comprising CDR-L1 of SEQ ID NO:142, CDR-L2 of SEQ ID NO:144, and CDR-L3 of SEQ ID NO:146. The sequences of the light chain CDRs are provided in Table 2 below.

In one embodiment, the light chain variable domains of the MuSK antibody-based molecule disclosed herein comprise (xix) light chain variable domains comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:170, and CDR-L3 of SEQ ID NO:180; (xx) light chain variable domains comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:171, and CDR-L3 of SEQ ID NO:181; (xxi) light chain variable domains comprising CDR-L1 of SEQ ID NO:160, CDR-L2 of SEQ ID NO:172, and CDR-L3 of SEQ ID NO:182; (xxii) light chain variable domains comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:172, and CDR-L3 of SEQ ID NO:182. 2 and SEQ ID NO:183 CDR-L3; (xxiii) light chain variable structural domains comprising SEQ ID NO:159 CDR-L1, SEQ ID NO:171 CDR-L2 and SEQ ID NO:184 CDR-L3; (xxiv) light chain variable structural domains comprising SEQ ID NO:159 CDR-L1, SEQ ID NO:173 CDR-L2 and S CDR-L3 of SEQ ID NO:185; (xxv) light chain variable structural domains comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:173, and CDR-L3 of SEQ ID NO:186; (xxvi) light chain variable structural domains comprising CDR-L1 of SEQ ID NO:161, CDR-L2 of SEQ ID NO:174, and CDR-L3 of SEQ ID NO:186. CDR-L3 of SEQ ID NO:187; (xxvii) light chain variable structural domains comprising CDR-L1 of SEQ ID NO:162, CDR-L2 of SEQ ID NO:174, and CDR-L3 of SEQ ID NO:188; (xxviii) light chain variable structural domains comprising CDR-L1 of SEQ ID NO:163, CDR-L2 of SEQ ID NO:174, and CDR-L3 of SEQ ID NO:188. :188 CDR-L3; (xxix) light chain variable structural domains comprising SEQ ID NO:164 CDR-L1, SEQ ID NO:174 CDR-L2 and SEQ ID NO:189 CDR-L3; (xxx) light chain variable structural domains comprising SEQ ID NO:165 CDR-L1, SEQ ID NO:175 CDR-L2 and SEQ ID NO:190 C DR-L3; (xxxi) light chain variable structural domains comprising CDR-L1 of SEQ ID NO:166, CDR-L2 of SEQ ID NO:176, and CDR-L3 of SEQ ID NO:191; (xxxi) light chain variable structural domains comprising CDR-L1 of SEQ ID NO:167, CDR-L2 of SEQ ID NO:177, and CDR-L3 of SEQ ID NO:192. 3; (xxxii) a light chain variable structural domain comprising CDR-L1 of SEQ ID NO:168, CDR-L2 of SEQ ID NO:178 and CDR-L3 of SEQ ID NO:193; (xxxiii) a light chain variable structural domain comprising CDR-L1 of SEQ ID NO:169, CDR-L2 of SEQ ID NO:179 and CDR-L3 of SEQ ID NO:194.

In one embodiment, the light chain variable domain of the MuSK antibody-based molecule disclosed herein comprises CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:172, and CDR-L3 of SEQ ID NO:195, or CDR-L3 having 1, 2, 3, 4, or 5 amino acid changes relative to the amino acid sequence of SEQ ID NO:195, wherein said changes are present at residues 1, 2, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or any combination thereof.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) includes a light chain variable domain, wherein the light chain variable domain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one embodiment, the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 have at least 0, 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO: 159, 172, or 195 (respectively).

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) includes a light chain variable domain, wherein the light chain variable domain contains:

- Contains a CDR-L1 amino acid sequence consisting of SEQ ID NO:159 or SEQ ID NO:159.

- Contains the CDR-L2 amino acid sequence consisting of SEQ ID NO:172 or SEQ ID NO:172, and

Contains the CDR-L3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:195 or SEQ ID NO:195.

Table 2 below provides the sequences of light chain CDR sequences.

Suitable amino acid modifications to the heavy chain CDR sequences and/or light chain CDR sequences of the MuSK-based antibody molecules disclosed herein include, for example, conserved substitutions or functionally equivalent amino acid residue substitutions that result in variant CDR sequences having binding characteristics similar to or enhanced than those of the CDR sequences disclosed herein. This invention covers the CDRs of Tables 1 and 2, which contain 1, 2, 3, 4, 5, or more amino acid changes (depending on the length of the CDR) that maintain or enhance MuSK binding of the antibody. Suitable amino acid modifications to the heavy chain CDR sequences of Table 1 and/or the light chain CDR sequences of Tables 1 and 2 include, for example, conserved substitutions or functionally equivalent amino acid residue substitutions that result in variant CDR sequences having binding characteristics similar to or enhanced than those of the CDR sequences in Tables 1 and 2. Conserved substitutions are those occurring within the amino acid families associated with their side chains. Genetically encoded amino acids can be divided into four families: (1) acidic (aspartic acid, glutamic acid); (2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. Alternatively, the amino acid library can be divided into (1) acidic (aspartic acid, glutamic acid); (2) basic (lysine, arginine, histidine); (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), wherein serine and threonine are optionally separately classified as aliphatic-hydroxy; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur-containing (cysteine and methionine) (Stryer (ed.), Biochemistry, 2nd ed., WH Freeman and Co., 1981, which is incorporated herein by reference in its entirety). Non-conservative substitutions can also be made to the heavy chain CDR sequences in Table 1 and the light chain CDR sequences in Table 2. Non-conservative substitutions involve replacing one or more amino acid residues of a CDR with one or more amino acid residues from different classes of amino acids to improve or enhance the binding properties of the CDR. The amino acid sequences of the heavy chain CDR in Table 1 and/or the light chain CDR in Table 2 may also contain one or more internal neutral amino acid insertions or deletions that maintain or enhance MuSK binding.

In one implementation, the MuSK antibody-based molecule contains:

(i) a heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:1, CDR-H2 of SEQ ID NO:17 and CDR-H3 of SEQ ID NO:33, and a light chain variable structural domain comprising CDR-L1 of SEQ ID NO:49, CDR-L2 of SEQ ID NO:65 and CDR-L3 of SEQ ID NO:81;

(ii) Heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 and CDR-H3 of SEQ ID NO:34, and light chain variable structural domains comprising CDR-L1 of SEQ ID NO:50, CDR-L2 of SEQ ID NO:66 and CDR-L3 of SEQ ID NO:82;

(iii) Heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:3, CDR-H2 of SEQ ID NO:19 and CDR-H3 of SEQ ID NO:35, and light chain variable structural domains comprising CDR-L1 of SEQ ID NO:51, CDR-L2 of SEQ ID NO:67 and CDR-L3 of SEQ ID NO:83;

(iv) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:4, CDR-H2 of SEQ ID NO:20 and CDR-H3 of SEQ ID NO:36, and light chain variable domains comprising CDR-L1 of SEQ ID NO:52, CDR-L2 of SEQ ID NO:68 and CDR-L3 of SEQ ID NO:84;

(v) A heavy chain variable domain comprising CDR-H1 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:21 and CDR-H3 of SEQ ID NO:37, and a light chain variable domain comprising CDR-L1 of SEQ ID NO:53, CDR-L2 of SEQ ID NO:69 and CDR-L3 of SEQ ID NO:85;

(vi) A heavy chain variable domain comprising CDR-H1 of SEQ ID NO:6, CDR-H2 of SEQ ID NO:22 and CDR-H3 of SEQ ID NO:38, and a light chain variable domain comprising CDR-L1 of SEQ ID NO:54, CDR-L2 of SEQ ID NO:70 and CDR-L3 of SEQ ID NO:86;

(vii) Heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:7, CDR-H2 of SEQ ID NO:23 and CDR-H3 of SEQ ID NO:39, and light chain variable structural domains comprising CDR-L1 of SEQ ID NO:55, CDR-L2 of SEQ ID NO:71 and CDR-L3 of SEQ ID NO:87;

(viii) A heavy chain variable domain comprising CDR-H1 of SEQ ID NO:8, CDR-H2 of SEQ ID NO:24 and CDR-H3 of SEQ ID NO:40, and a light chain variable domain comprising CDR-L1 of SEQ ID NO:56, CDR-L2 of SEQ ID NO:72 and CDR-L3 of SEQ ID NO:88;

(ix) Heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:9, CDR-H2 of SEQ ID NO:25 and CDR-H3 of SEQ ID NO:41, and light chain variable structural domains comprising CDR-L1 of SEQ ID NO:57, CDR-L2 of SEQ ID NO:73 and CDR-L3 of SEQ ID NO:89;

(x) A heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:10, CDR-H2 of SEQ ID NO:26 and CDR-H3 of SEQ ID NO:42, and a light chain variable structural domain comprising CDR-L1 of SEQ ID NO:58, CDR-L2 of SEQ ID NO:74 and CDR-L3 of SEQ ID NO:90;

(xi) a heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:11, CDR-H2 of SEQ ID NO:27 and CDR-H3 of SEQ ID NO:43, and a light chain variable structural domain comprising CDR-L1 of SEQ ID NO:59, CDR-L2 of SEQ ID NO:75 and CDR-L3 of SEQ ID NO:91;

(xii) Heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:12, CDR-H2 of SEQ ID NO:28 and CDR-H3 of SEQ ID NO:44, and light chain variable structural domains comprising CDR-L1 of SEQ ID NO:60, CDR-L2 of SEQ ID NO:76 and CDR-L3 of SEQ ID NO:92;

(xiii) Heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:13, CDR-H2 of SEQ ID NO:29 and CDR-H3 of SEQ ID NO:45, and light chain variable structural domains comprising CDR-L1 of SEQ ID NO:61, CDR-L2 of SEQ ID NO:77 and CDR-L3 of SEQ ID NO:93;

(xiv) includes heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:14, CDR-H2 of SEQ ID NO:30 and CDR-H3 of SEQ ID NO:46, and light chain variable structural domains comprising CDR-L1 of SEQ ID NO:62, CDR-L2 of SEQ ID NO:78 and CDR-L3 of SEQ ID NO:94;

(xv) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:15, CDR-H2 of SEQ ID NO:31 and CDR-H3 of SEQ ID NO:47, and a light chain variable domain comprising CDR-L1 of SEQ ID NO:63, CDR-L2 of SEQ ID NO:79 and CDR-L3 of SEQ ID NO:95;

(xvi) Heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:16, CDR-H2 of SEQ ID NO:32 and CDR-H3 of SEQ ID NO:48, and light chain variable structural domains comprising CDR-L1 of SEQ ID NO:64, CDR-L2 of SEQ ID NO:80 and CDR-L3 of SEQ ID NO:96;

(xvii) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:135, CDR-H2 of SEQ ID NO:137, and CDR-H3 of SEQ ID NO:139, and light chain variable domains comprising CDR-L1 of SEQ ID NO:141, CDR-L2 of SEQ ID NO:143, and CDR-L3 of SEQ ID NO:145; and

(xviii) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:136, CDR-H2 of SEQ ID NO:138 and CDR-H3 of SEQ ID NO:140, and light chain variable domains comprising CDR-L1 of SEQ ID NO:142, CDR-L2 of SEQ ID NO:144 and CDR-L3 of SEQ ID NO:146.

In one implementation, the MuSK antibody-based molecule contains:

(ii.a) A heavy chain variable domain comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 and CDR-H3 of SEQ ID NO:240, and a light chain variable domain (X2m1) comprising CDR-L1 of SEQ ID NO:50, CDR-L2 of SEQ ID NO:66 and CDR-L3 of SEQ ID NO:82;

(ii.b) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 and CDR-H3 of SEQ ID NO:241, and light chain variable domains (X2m2) comprising CDR-L1 of SEQ ID NO:50, CDR-L2 of SEQ ID NO:66 and CDR-L3 of SEQ ID NO:82;

(ii.c) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 and CDR-H3 of SEQ ID NO:242, and light chain variable domains (X2m3) comprising CDR-L1 of SEQ ID NO:50, CDR-L2 of SEQ ID NO:66 and CDR-L3 of SEQ ID NO:82;

(ii.d) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 and CDR-H3 of SEQ ID NO:243, and light chain variable domains (X2m4) comprising CDR-L1 of SEQ ID NO:50, CDR-L2 of SEQ ID NO:66 and CDR-L3 of SEQ ID NO:82;

(ii.e) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 and CDR-H3 of SEQ ID NO:244, and light chain variable domains (X2m5) comprising CDR-L1 of SEQ ID NO:50, CDR-L2 of SEQ ID NO:66 and CDR-L3 of SEQ ID NO:82;

(ii.f) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 and CDR-H3 of SEQ ID NO:245, and light chain variable domains (X2m6) comprising CDR-L1 of SEQ ID NO:50, CDR-L2 of SEQ ID NO:66 and CDR-L3 of SEQ ID NO:82;

(ii.g) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 and CDR-H3 of SEQ ID NO:246, and light chain variable domains comprising CDR-L1 of SEQ ID NO:50, CDR-L2 of SEQ ID NO:66 and CDR-L3 of SEQ ID NO:82 (X2m7);

(ii.f) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 and CDR-H3 of SEQ ID NO:247, and light chain variable domains (X2m8) comprising CDR-L1 of SEQ ID NO:50, CDR-L2 of SEQ ID NO:66 and CDR-L3 of SEQ ID NO:82.

In one implementation, the MuSK antibody-based molecule contains:

(xvii.a) includes heavy chain variable domains comprising CDR-H1 of SEQ ID NO:135, CDR-H2 of SEQ ID NO:137 and CDR-H3 of SEQ ID NO:248, and light chain variable domains comprising CDR-L1 of SEQ ID NO:141, CDR-L2 of SEQ ID NO:143 and CDR-L3 of SEQ ID NO:145 (X17m1);

(xvii.b) contains heavy chain variable domains of CDR-H1 of SEQ ID NO:135, CDR-H2 of SEQ ID NO:137 and CDR-H3 of SEQ ID NO:249, and light chain variable domains (X17m2) containing CDR-L1 of SEQ ID NO:141, CDR-L2 of SEQ ID NO:143 and CDR-L3 of SEQ ID NO:145;

(xvii.c) contains heavy chain variable domains of CDR-H1 of SEQ ID NO:135, CDR-H2 of SEQ ID NO:137 and CDR-H3 of SEQ ID NO:250, and light chain variable domains (X17m3) containing CDR-L1 of SEQ ID NO:141, CDR-L2 of SEQ ID NO:143 and CDR-L3 of SEQ ID NO:145;

(xvii.d) contains heavy chain variable domains of CDR-H1 of SEQ ID NO:135, CDR-H2 of SEQ ID NO:137 and CDR-H3 of SEQ ID NO:251, and light chain variable domains of CDR-L1 of SEQ ID NO:141, CDR-L2 of SEQ ID NO:143 and CDR-L3 of SEQ ID NO:145 (X17m6).

In one implementation, the MuSK antibody-based molecule contains:

(i) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:150 and CDR-H3 of SEQ ID NO:156, and a light chain variable domain comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:170 and CDR-L3 of SEQ ID NO:180 (14D10);

(ii) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:150 and CDR-H3 of SEQ ID NO:156, and light chain variable domains comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:171 and CDR-L3 of SEQ ID NO:181 (7G4);

(iii) A heavy chain variable domain comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:150 and CDR-H3 of SEQ ID NO:156, and a light chain variable domain comprising CDR-L1 of SEQ ID NO:160, CDR-L2 of SEQ ID NO:172 and CDR-L3 of SEQ ID NO:182 (3C4);

(iv) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:150 and CDR-H3 of SEQ ID NO:156, and light chain variable domains comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:172 and CDR-L3 of SEQ ID NO:183 (3B2);

(v) A heavy chain variable domain comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:150 and CDR-H3 of SEQ ID NO:156, and a light chain variable domain comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:171 and CDR-L3 of SEQ ID NO:184 (3G3);

(vi) A heavy chain variable domain comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:150 and CDR-H3 of SEQ ID NO:156, and a light chain variable domain comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:173 and CDR-L3 of SEQ ID NO:185 (31G2);

(vii) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:150 and CDR-H3 of SEQ ID NO:156, and light chain variable domains comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:173 and CDR-L3 of SEQ ID NO:186 (31B7);

(viii) A heavy chain variable domain comprising CDR-H1 of SEQ ID NO:148, CDR-H2 of SEQ ID NO:151 and CDR-H3 of SEQ ID NO:157, and a light chain variable domain comprising CDR-L1 of SEQ ID NO:161, CDR-L2 of SEQ ID NO:174 and CDR-L3 of SEQ ID NO:187 (17H10);

(ix) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:148, CDR-H2 of SEQ ID NO:151 and CDR-H3 of SEQ ID NO:157, and light chain variable domains comprising CDR-L1 of SEQ ID NO:162, CDR-L2 of SEQ ID NO:174 and CDR-L3 of SEQ ID NO:188 (23B6);

(x) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:148, CDR-H2 of SEQ ID NO:151 and CDR-H3 of SEQ ID NO:157, and light chain variable domains comprising CDR-L1 of SEQ ID NO:163, CDR-L2 of SEQ ID NO:174 and CDR-L3 of SEQ ID NO:188 (30E1);

(xi) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:148, CDR-H2 of SEQ ID NO:151 and CDR-H3 of SEQ ID NO:157, and light chain variable domains comprising CDR-L1 of SEQ ID NO:164, CDR-L2 of SEQ ID NO:174 and CDR-L3 of SEQ ID NO:189 (30A11);

(xii) A heavy chain variable domain comprising CDR-H1 of SEQ ID NO:149, CDR-H2 of SEQ ID NO:152 and CDR-H3 of SEQ ID NO:158, and a light chain variable domain (16F11) comprising CDR-L1 of SEQ ID NO:165, CDR-L2 of SEQ ID NO:175 and CDR-L3 of SEQ ID NO:190;

(xiii) A heavy chain variable domain comprising CDR-H1 of SEQ ID NO:149, CDR-H2 of SEQ ID NO:152 and CDR-H3 of SEQ ID NO:158, and a light chain variable domain comprising CDR-L1 of SEQ ID NO:166, CDR-L2 of SEQ ID NO:176 and CDR-L3 of SEQ ID NO:191 (4C11);

(xiv) includes heavy chain variable domains comprising CDR-H1 of SEQ ID NO:149, CDR-H2 of SEQ ID NO:152 and CDR-H3 of SEQ ID NO:158, and light chain variable domains comprising CDR-L1 of SEQ ID NO:167, CDR-L2 of SEQ ID NO:177 and CDR-L3 of SEQ ID NO:192 (7A12);

(xv) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:149, CDR-H2 of SEQ ID NO:152 and CDR-H3 of SEQ ID NO:158, and light chain variable domains comprising CDR-L1 of SEQ ID NO:168, CDR-L2 of SEQ ID NO:178 and CDR-L3 of SEQ ID NO:193 (7G12);

(xvi) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:149, CDR-H2 of SEQ ID NO:152 and CDR-H3 of SEQ ID NO:158, and light chain variable domains comprising CDR-L1 of SEQ ID NO:169, CDR-L2 of SEQ ID NO:179 and CDR-L3 of SEQ ID NO:194 (7B8);

(xvii) The heavy chain variable domain comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:153 and CDR-H3 of SEQ ID NO:156, and the light chain variable domain (3B2g1m1) comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:172 and CDR-L3 of SEQ ID NO:183;

(xviii) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:154 and CDR-H3 of SEQ ID NO:156, and light chain variable domains (3B2g1m2) comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:172 and CDR-L3 of SEQ ID NO:183;

(xvix) includes heavy chain variable domains of CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:155 and CDR-H3 of SEQ ID NO:156, and light chain variable domains (3B2g1m4) including CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:172 and CDR-L3 of SEQ ID NO:183;

(xx) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:153 and CDR-H3 of SEQ ID NO:156, and a light chain variable domain (3B2g2m1) comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:172 and CDR-L3 of SEQ ID NO:195;

(xxi) includes heavy chain variable domains comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:154, and CDR-H3 of SEQ ID NO:156, and light chain variable domains (3B2g2m2) comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:172, and CDR-L3 of SEQ ID NO:195; and

(xxii) Heavy chain variable domains comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:155 and CDR-H3 of SEQ ID NO:156, and light chain variable domains (3B2g2m4) comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:172 and CDR-L3 of SEQ ID NO:195.

In a preferred embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence containing SEQ ID NO:172 or a CDR-L2 sequence with 1, 2, 3, 4, or 5 amino acids altered relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one embodiment, the antibody's CDR-H2 contains proline (P) at position 3, tryptophan (W) at position 4, and serine (S) or asparagine (N) at position 5.

In a more preferred embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain contains:

- Contains the CDR-H1 amino acid sequence consisting of SEQ ID NO:147 or SEQ ID NO:147.

- Contains the CDR-H2 amino acid sequence consisting of SEQ ID NO:153 or SEQ ID NO:153, and

- Contains the CDR-H3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:156 or SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence consisting of SEQ ID NO:159 or SEQ ID NO:159.

- Contains the CDR-L2 amino acid sequence consisting of SEQ ID NO:172 or SEQ ID NO:172, and

- Contains the CDR-L3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:195 or SEQ ID NO:195.

The MuSK antibody-based molecules described herein may comprise a variable light (VL) chain, a variable heavy (VH) chain, or a combination of VL and VH chains. In some embodiments, the VH chain of the MuSK antibody-based molecule comprises any of the VH amino acid sequences provided in Table 3 below, or comprises having a content of at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, or more than 74%. The amino acid sequence must have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity. In some embodiments, the VL chain of the MuSK antibody-based molecule comprises any of the VL amino acid sequences provided in Table 3 below, or comprises an amino acid sequence having at least 60% identity or similarity to any of the VL amino acid sequences listed in Table 3. In some implementations, the similarity or identity is at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

In one embodiment, the MuSK antibody-based molecule disclosed herein comprises: (ii) a heavy chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:97 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:98; and (ii) a heavy chain variable domain comprising an amino acid sequence having at least 80% identity with any one of SEQ ID NO:99 and 252 to 259 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:100. (iii) a heavy chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:101 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:102; (iv) a heavy chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:103 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:104; (v) a heavy chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:105. (vi) A heavy chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:107 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:108; or (vii) A heavy chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:109 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:108. (ii) A light chain variable domain having at least 80% identity with the amino acid sequence of SEQ ID NO: 110; (viii) A heavy chain variable domain having at least 80% identity with the amino acid sequence of SEQ ID NO: 111 and a light chain variable domain having at least 80% identity with the amino acid sequence of SEQ ID NO: 112; (ix) A heavy chain variable domain having at least 80% identity with the amino acid sequence of SEQ ID NO: 113 and a heavy chain variable domain having at least 80% identity with the amino acid sequence of SEQ ID NO: 114. Light chain variable domain; (x) a heavy chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO:115 and a light chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO:116; (xi) a heavy chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO:117 and a light chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO:118; (xii) a heavy chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO:119. (xiii) A heavy chain variable domain containing an amino acid sequence with at least 80% identity to SEQ ID NO:120 and a light chain variable domain containing an amino acid sequence with at least 80% identity to SEQ ID NO:121; (xiv) A heavy chain variable domain containing an amino acid sequence with at least 80% identity to SEQ ID NO:123 and a light chain variable domain containing an amino acid sequence with at least 80% identity to SEQ ID NO:122. (xv) A light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 124; (xv) A heavy chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 125 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 126; (xvi) A heavy chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 127 and an amino acid sequence having at least 80% identity with SEQ ID NO: 128. The light chain variable domain; (xvii) a heavy chain variable domain comprising an amino acid sequence having at least 80% identity with any one of SEQ ID NO:131 and 260 to 263, and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:132; and (xviii) a heavy chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:133 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:134.

In some embodiments, the MuSK antibody-based molecules disclosed herein contain: (i) a heavy chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:196 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:197; (ii) a heavy chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:198 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:199; (iii) a heavy chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:200. (iv) A heavy chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:201 and an amino acid sequence having at least 80% identity with SEQ ID NO:202 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:203; (v) A heavy chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:204 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:205; (vi) A heavy chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:206 and an amino acid sequence having at least 80% identity with SEQ ID NO:204 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:205; (vii) A heavy chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO:207 and a light chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO:208; (viii) A heavy chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO:209 and a light chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO:210; (vix) A heavy chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO:211 and a light chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO:211; (x) A heavy chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO:212 and a light chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO:213; (xi) A heavy chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO:214 and a light chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO:215; (xi) A heavy chain variable domain containing an amino acid sequence having at least 80% identity with SEQ ID NO:216 and an amino acid sequence having at least 80% identity with SEQ ID NO:217. (xii) a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:218 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:219; (xiii) a heavy chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:220 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:221; (xiv) a heavy chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:222 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:219 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:219 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:221 and a light chain variable domain comprising an amino acid sequence having at least 80% identity with SEQ ID NO:2 ... O:223 A light chain variable domain having an amino acid sequence with at least 80% identity; (xv) A heavy chain variable domain having an amino acid sequence with at least 80% identity to SEQ ID NO:224 and a light chain variable domain having an amino acid sequence with at least 80% identity to SEQ ID NO:225; (xvi) A heavy chain variable domain having an amino acid sequence with at least 80% identity to SEQ ID NO:226 and a light chain variable domain having an amino acid sequence with at least 80% identity to SEQ ID NO:227; (xvii) A heavy chain variable domain having an amino acid sequence with at least 80% identity to SEQ ID NO:228. Variable domains and light chain variable domains comprising an amino acid sequence having at least 80% identity with SEQ ID NO:229; (xviii) heavy chain variable domains comprising an amino acid sequence having at least 80% identity with SEQ ID NO:230 and light chain variable domains comprising an amino acid sequence having at least 80% identity with SEQ ID NO:231; (xix) heavy chain variable domains comprising an amino acid sequence having at least 80% identity with SEQ ID NO:232 and light chain variable domains comprising an amino acid sequence having at least 80% identity with SEQ ID NO:233; (xx) containing an amino acid sequence having at least 80% identity with SEQ ID NO:233. 34. A heavy chain variable domain having an amino acid sequence with at least 80% identity and a light chain variable domain having an amino acid sequence with at least 80% identity with SEQ ID NO:235; (xxi) A heavy chain variable domain having an amino acid sequence with at least 80% identity with SEQ ID NO:236 and a light chain variable domain having an amino acid sequence with at least 80% identity with SEQ ID NO:237; (xxii) A heavy chain variable domain having an amino acid sequence with at least 80% identity with SEQ ID NO:238 and a light chain variable domain having an amino acid sequence with at least 80% identity with SEQ ID NO:239.

In a preferred embodiment, the MuSK antibody-based molecule (or anti-MuSK antibody or its antigen-binding fragment) disclosed herein comprises a heavy chain variable domain (VH) containing an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and a light chain variable domain (VL) containing an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a preferred embodiment, the MuSK antibody-based molecule disclosed herein comprises a heavy chain variable domain containing the amino acid sequence SEQ ID NO:234 and a light chain variable domain containing the amino acid sequence SEQ ID NO:235.

In a preferred embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235.

The heavy chain variable structure domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In a more preferred embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235.

The heavy chain variable structure domain contains:

- Contains the CDR-H1 amino acid sequence consisting of SEQ ID NO:147 or SEQ ID NO:147.

- Contains the CDR-H2 amino acid sequence consisting of SEQ ID NO:153 or SEQ ID NO:153, and

- Contains the CDR-H3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:156 or SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence consisting of SEQ ID NO:159 or SEQ ID NO:159.

- Contains the CDR-L2 amino acid sequence consisting of SEQ ID NO:172 or SEQ ID NO:172, and

- Contains the CDR-L3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:195 or SEQ ID NO:195.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises a wild-type human IgG constant Fc region, a heavy chain variable domain, and a light chain variable domain, wherein the wild-type human IgG constant Fc region contains at least 80% sequence identity with SEQ ID NO:266 or SEQ ID NO:267, wherein the heavy chain variable domain contains an amino acid sequence having at least 80% identity or similarity with SEQ ID NO:234 and the light chain variable domain contains an amino acid sequence having at least 80% identity or similarity with SEQ ID NO:235, and wherein the heavy chain variable domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one embodiment, the antibody's CDR-H2 contains proline (P) at position 3, tryptophan (W) at position 4, and serine (S) or asparagine (N) at position 5.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises a constant Fc region of wild-type human IgG, a heavy chain variable domain, and a light chain variable domain, wherein the constant Fc region of wild-type human IgG comprises SEQ ID NO:266 or SEQ ID NO:267, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234, and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235.

The heavy chain variable structure domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one embodiment, the antibody's CDR-H2 contains proline (P) at position 3, tryptophan (W) at position 4, and serine (S) or asparagine (N) at position 5.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises a constant Fc region of wild-type human IgG, a heavy chain variable domain, and a light chain variable domain, wherein the constant Fc region of wild-type human IgG comprises SEQ ID NO:266 or SEQ ID NO:267, the heavy chain variable domain comprises SEQ ID NO:234, and the light chain variable domain comprises SEQ ID NO:235.

The heavy chain variable structure domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one embodiment, the antibody's CDR-H2 contains proline (P) at position 3, tryptophan (W) at position 4, and serine (S) or asparagine (N) at position 5.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises a constant Fc region of wild-type human IgG, a heavy chain variable domain, and a light chain variable domain, wherein the constant Fc region of wild-type human IgG comprises SEQ ID NO:266 or SEQ ID NO:267, the heavy chain variable domain comprises SEQ ID NO:234, and the light chain variable domain comprises SEQ ID NO:235.

The heavy chain variable structure domain contains:

- Contains the CDR-H1 amino acid sequence consisting of SEQ ID NO:147 or SEQ ID NO:147.

- Contains the CDR-H2 amino acid sequence consisting of SEQ ID NO:153 or SEQ ID NO:153, and

- Contains the CDR-H3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:156 or SEQ ID NO:156 and

The variable structural domain of the light chain includes:

- Contains a CDR-L1 amino acid sequence consisting of SEQ ID NO:159 or SEQ ID NO:159.

- Contains the CDR-L2 amino acid sequence consisting of SEQ ID NO:172 or SEQ ID NO:172, and

- Contains the CDR-L3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:195 or SEQ ID NO:195.

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises a constant Fc region of wild-type human IgG, a heavy chain variable domain, and a light chain variable domain.

The wild-type human IgG constant Fc region contains at least 80% sequence identity with SEQ ID NO:266 or SEQ ID NO:267, wherein a) one or more of the following mutations (all numbered according to the EU numbering system) have been introduced into the full-length heavy chain: N297A substitution; N297Q substitution; L234A substitution; L234D substitution; L234E substitution; L234G substitution; L234H substitution; L234F substitution; L234K substitution; L234 Replace with Q; Replace with L234R; Replace with L234S; Replace with L234T; Replace with L235A; Replace with L235D; Replace with L235E; Replace with L235F; Replace with L235G; Replace with L235V; Replace with L235H; Replace with L235I; Replace with L235K; Replace with L235R; Replace with L235S; Replace with L235T; Replace with L235Q; Replace with L237A; Replace with S239D; Replace with E233P; Replace with L234V; C236 is missing; Replacement for G236E; G236R; G236K; G237A; P238A; F243L; D265A; S267E; H268A; R292P; Y300L; K322A; K322Q; A327Q; L328F; L328R; P329A; P329G; A330L; A330S; P331S; I33 2E substitution; P396L substitution; or each of the combinations of mutations described above in the fourth embodiment of this application, preferably the mutation is L234A or L235A, more preferably the mutation is L234A and L235A, and wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235, and

The heavy chain variable structure domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises a constant Fc region of wild-type human IgG, a heavy chain variable domain, and a light chain variable domain.

The wild-type human IgG constant Fc region contains SEQ ID NO:266 or SEQ ID NO:267, wherein L234A and/or L235A, according to the EU numbering system, are substituted into the Fc region, and

The heavy chain variable domain contains an amino acid sequence that has at least 80% identity or similarity to SEQ ID NO:234, and the light chain variable domain contains an amino acid sequence that has at least 80% identity or similarity to SEQ ID NO:235.

The heavy chain variable structure domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one embodiment, the antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) comprises a constant Fc region of wild-type human IgG, a heavy chain variable domain, and a light chain variable domain, wherein the constant Fc region of wild-type human IgG comprises SEQ ID NO:266 or SEQ ID NO:267, wherein L234A and/or L235A, numbered according to the EU numbering system, are introduced into the Fc region, and wherein the heavy chain variable domain comprises SEQ ID NO:234 and the light chain variable domain comprises SEQ ID NO:235.

The heavy chain variable structure domain contains:

- Contains the CDR-H1 amino acid sequence consisting of SEQ ID NO:147 or SEQ ID NO:147.

- Contains the CDR-H2 amino acid sequence consisting of SEQ ID NO:153 or SEQ ID NO:153, and

- Contains the CDR-H3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:156 or SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence consisting of SEQ ID NO:159 or SEQ ID NO:159.

- Contains the CDR-L2 amino acid sequence consisting of SEQ ID NO:172 or SEQ ID NO:172, and

- Contains the CDR-L3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:195 or SEQ ID NO:195.

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one embodiment, the anti-MuSK antibody or its antigen-binding fragment comprises:

a) A full-length heavy chain comprising an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:268, and

b) A full-length light chain comprising an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:269, and

c) One or more of the following mutations (all numbered according to the EU numbering system) have been introduced into the full-length heavy chain: N297A substitution; N297Q substitution; L234A substitution; L234D substitution; L234E substitution; L234G substitution; L234H substitution; L234F substitution; L234K substitution; L234Q substitution; L234R substitution; L234S substitution; L234T substitution; L235A substitution; L235D substitution; L235E substitution; L235F substitution; L235G substitution; L235V substitution; L235H substitution; L235I substitution; L235K substitution; L235R substitution; L235S substitution; L235T substitution; L235Q substitution; L237A substitution; S239D substitution; E233P substitution. Replacement; L234V replacement; C236 deletion; G236E replacement; G236R replacement; G236K replacement; G237A replacement; P238A replacement; F243L replacement; D265A replacement; S267E replacement; H268A replacement; R292P replacement; Y300L replacement; K322A replacement; K322Q replacement; A327Q replacement; L328F replacement; L328R replacement; P329A replacement; P329G replacement; A330L replacement; A330S replacement; P331S replacement; I332E replacement; P396L replacement; or each of the combinations of mutations described above in the fourth embodiment of this application, preferably the mutation is L234A or L235A, more preferably the mutations are L234A and L235A.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one embodiment, the anti-MuSK antibody or its antigen-binding fragment comprises:

a) Full-length heavy chain, which includes SEQ ID NO:268 and

b) A full-length light chain containing SEQ ID NO:269, and

c) The full-length heavy chain contains L234A and L235A mutations numbered according to the EU numbering system.

In one embodiment, the anti-MuSK antibody or its antigen-binding fragment comprises:

a) A full-length heavy chain containing an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:270 and

b) A full-length light chain comprising an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:271, and

c) One or more of the following mutations (all numbered according to the EU numbering system) have been introduced into the full-length heavy chain: N297A substitution; N297Q substitution; L234A substitution; L234D substitution; L234E substitution; L234G substitution; L234H substitution; L234F substitution; L234K substitution; L234Q substitution; L234R substitution; L234S substitution; L234T substitution; L235A substitution; L235D substitution; L235E substitution; L235F substitution; L235G substitution; L235V substitution; L235H substitution; L235I substitution; L235K substitution; L235R substitution; L235S substitution; L235T substitution; L235Q substitution; L237A substitution; S239D substitution; E233P Replacement; L234V replacement; C236 deletion; G236E replacement; G236R replacement; G236K replacement; G237A replacement; P238A replacement; F243L replacement; D265A replacement; S267E replacement; H268A replacement; R292P replacement; Y300L replacement; K322A replacement; K322Q replacement; A327Q replacement; L328F replacement; L328R replacement; P329A replacement; P329G replacement; A330L replacement; A330S replacement; P331S replacement; I332E replacement; P396L replacement; or each of the combinations of mutations described above in the fourth embodiment of this application, preferably the mutation is L234A or L235A, more preferably the mutations are L234A and L235A.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one embodiment, the anti-MuSK antibody or its antigen-binding fragment comprises:

a) Full-length heavy chain, comprising SEQ ID NO:270 and

b) A full-length light chain containing SEQ ID NO:271, and

c) The full-length heavy chain contains L234A and L235A mutations numbered according to the EU numbering system.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides encoding the MuSK antibody-based molecules described herein. In one embodiment, the polynucleotide encoding the MuSK antibody of the invention comprises nucleotide sequences encoding any one, two, three, four, five, or six of the aforementioned CDRs, including heavy chain CDRs of SEQ ID NO: 1 to 48, 135 to 140, 147 to 158, 240 to 251 and light chain CDRs of SEQ ID NO: 49 to 96, 141 to 146, and 159 to 195.

Therefore, the present invention provides a polynucleotide for treating neuromuscular diseases in human subjects, the polynucleotide comprising a nucleotide sequence encoding an anti-MuSK antibody or antigen-binding fragment or its VH, VL or CDR domain.

In one embodiment, the polynucleotide comprises a nucleotide sequence encoding a VH domain, wherein the VH domain comprises: (i) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:1, CDR-H2 of SEQ ID NO:17, and CDR-H3 of SEQ ID NO:33; and (ii) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18, and CDR-H3 of SEQ ID NO:33. (iii) a heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:3, CDR-H2 of SEQ ID NO:19, and CDR-H3 of SEQ ID NO:35; (iv) a heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:4, CDR-H2 of SEQ ID NO:20, and CDR-H3 of SEQ ID NO:36; (v) a heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:34, CDR-H2 of SEQ ID NO:20, and CDR-H3 of SEQ ID NO:36; (vi) A heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:37; (vii) A heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:6, CDR-H2 of SEQ ID NO:22, and CDR-H3 of SEQ ID NO:38; (vii) A heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:7, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:38. (viii) CDR-H2 of SEQ ID NO:23 and CDR-H3 of SEQ ID NO:39; (viii) Heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:8, CDR-H2 of SEQ ID NO:24 and CDR-H3 of SEQ ID NO:40; (ix) Heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:9, CDR-H2 of SEQ ID NO:25 and CDR-H3 of SEQ ID NO:41 (x) Heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:10, CDR-H2 of SEQ ID NO:26, and CDR-H3 of SEQ ID NO:42; (xi) Heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:11, CDR-H2 of SEQ ID NO:27, and CDR-H3 of SEQ ID NO:43; (xii) Heavy chain variable structural domains comprising S (xiii) A heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:12, CDR-H2 of SEQ ID NO:28, and CDR-H3 of SEQ ID NO:44; (xiv) A heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:14, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:45; (xv) CDR-H2 of SEQ ID NO:30 and CDR-H3 of SEQ ID NO:46; (xv) Heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:15, CDR-H2 of SEQ ID NO:31 and CDR-H3 of SEQ ID NO:47; (xvi) Heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:16, CDR-H2 of SEQ ID NO:32 and CDR-H3 of SEQ ID NO:47. CDR-H3 of SEQ ID NO:48; (xvii) heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:135, CDR-H2 of SEQ ID NO:137 and CDR-H3 of SEQ ID NO:139; and (xviii) heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:136, CDR-H2 of SEQ ID NO:138 and CDR-H3 of SEQ ID NO:140.

In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a VH domain, wherein the VH domain comprises: (ii.a) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18, and CDR-H3 (X2m1) of SEQ ID NO:240; and (ii.b) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18, and CDR-H3 of SEQ ID NO:240. -H2 and CDR-H3(X2m2) of SEQ ID NO:241; (ii.c) heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 and CDR-H3(X2m3) of SEQ ID NO:242; (ii.d) heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 and CDR-H3(X2m3) of SEQ ID NO:242; (ii.e) CDR-H3(X2m4) of SEQ ID NO:243; (ii.e) heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18, and CDR-H3(X2m5) of SEQ ID NO:244; (ii.f) heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18, and CDR-H3(X2m5) of SEQ ID NO:245. R-H3(X2m6); (ii.g) heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 and CDR-H3(X2m7) of SEQ ID NO:246; (ii.h) heavy chain variable structural domains comprising CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:18 and CDR-H3(X2m8) of SEQ ID NO:247.

In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a VH domain, wherein the VH domain comprises: (xvii.a) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:135, CDR-H2 of SEQ ID NO:137, and CDR-H3 (X17m1) of SEQ ID NO:248; (xvii.b) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:135, CDR-H2 of SEQ ID NO:137, and CDR-H3 of SEQ ID NO:248. CDR-H3(X17m2) of SEQ ID NO:249; (xvii.c) heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:135, CDR-H2 of SEQ ID NO:137 and CDR-H3(X17m3) of SEQ ID NO:250; (xvii.d) heavy chain variable structural domain comprising CDR-H1 of SEQ ID NO:135, CDR-H2 of SEQ ID NO:137 and CDR-H3(X17m6) of SEQ ID NO:251.

In one embodiment, the polynucleotide comprises a nucleotide sequence encoding a VH domain, wherein the VH domain comprises: (xix) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:150, and CDR-H3 of SEQ ID NO:156; (xx) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:148, CDR-H2 of SEQ ID NO:151, and CDR-H3 of SEQ ID NO:157; and (xxi) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:149, CDR-H2 of SEQ ID NO:152, and CDR-H3 of SEQ ID NO:158.

In one embodiment, the polynucleotide comprises a nucleotide sequence encoding a VH domain, wherein the VH domain comprises: (xxii) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:153, and CDR-H3 of SEQ ID NO:156; (xxiii) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:154, and CDR-H3 of SEQ ID NO:156; and (xxiv) a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:155, and CDR-H3 of SEQ ID NO:156.

In one embodiment, the polynucleotide comprises a nucleotide sequence encoding a VL domain, wherein the VL domain comprises: (i) a light chain variable domain comprising CDR-L1 of SEQ ID NO:49, CDR-L2 of SEQ ID NO:65, and CDR-L3 of SEQ ID NO:81; and (ii) a light chain variable domain comprising CDR-L1 of SEQ ID NO:50, CDR-L2 of SEQ ID NO:66, and CDR-L3 of SEQ ID NO:81. (iii) a light chain variable structural domain comprising CDR-L1 of SEQ ID NO:51, CDR-L2 of SEQ ID NO:67, and CDR-L3 of SEQ ID NO:83; (iv) a light chain variable structural domain comprising CDR-L1 of SEQ ID NO:52, CDR-L2 of SEQ ID NO:68, and CDR-L3 of SEQ ID NO:84; (v) a light chain variable structural domain, It includes CDR-L1 of SEQ ID NO:53, CDR-L2 of SEQ ID NO:69, and CDR-L3 of SEQ ID NO:85; (vi) a light chain variable structural domain, which includes CDR-L1 of SEQ ID NO:54, CDR-L2 of SEQ ID NO:70, and CDR-L3 of SEQ ID NO:86; (vii) a light chain variable structural domain, which includes CDR-L1 of SEQ ID NO:55, S (viii) A light chain variable structural domain comprising CDR-L1 of SEQ ID NO:71, CDR-L2 of SEQ ID NO:72, and CDR-L3 of SEQ ID NO:87; (ix) A light chain variable structural domain comprising CDR-L1 of SEQ ID NO:57, CDR-L2 of SEQ ID NO:73, and CDR-L3 of SEQ ID NO:88. (x) A light chain variable structural domain comprising CDR-L1 of SEQ ID NO:58, CDR-L2 of SEQ ID NO:74, and CDR-L3 of SEQ ID NO:90; (xi) A light chain variable structural domain comprising CDR-L1 of SEQ ID NO:59, CDR-L2 of SEQ ID NO:75, and CDR-L3 of SEQ ID NO:91; (xii) A light chain variable structural domain, It includes CDR-L1 of SEQ ID NO:60, CDR-L2 of SEQ ID NO:76, and CDR-L3 of SEQ ID NO:92; (xiii) a light chain variable structural domain, which includes CDR-L1 of SEQ ID NO:61, CDR-L2 of SEQ ID NO:77, and CDR-L3 of SEQ ID NO:93; (xiv) a light chain variable structural domain, which includes CDR-L1 of SEQ ID NO:62, S (xv) CDR-L2 of SEQ ID NO:78 and CDR-L3 of SEQ ID NO:94; (xvi) Light chain variable structural domains comprising CDR-L1 of SEQ ID NO:63, CDR-L2 of SEQ ID NO:79 and CDR-L3 of SEQ ID NO:95; (xvi) Light chain variable structural domains comprising CDR-L1 of SEQ ID NO:64, CDR-L2 of SEQ ID NO:80 and CDR-L3 of SEQ ID NO:95. CDR-L3 of SEQ ID NO:96; (xvii) light chain variable structural domain comprising CDR-L1 of SEQ ID NO:141, CDR-L2 of SEQ ID NO:143 and CDR-L3 of SEQ ID NO:145; and (xviii) light chain variable structural domain comprising CDR-L1 of SEQ ID NO:142, CDR-L2 of SEQ ID NO:144 and CDR-L3 of SEQ ID NO:146.

In one embodiment, the polynucleotide comprises a nucleotide sequence encoding a VL domain, wherein the VL domain comprises: (xix) a light chain variable domain comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:170, and CDR-L3 of SEQ ID NO:180; (xx) a light chain variable domain comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:171, and CDR-L3 of SEQ ID NO:181; (xxi) a light chain variable domain comprising CDR-L1 of SEQ ID NO:160, CDR-L2 of SEQ ID NO:172, and CDR-L3 of SEQ ID NO:182; (xxii) a light chain variable domain comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:172, and CDR-L3 of SEQ ID NO:182; (xxiii) light chain variable structural domains comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:171, and CDR-L3 of SEQ ID NO:184; (xxiv) light chain variable structural domains comprising CDR-L1 of SEQ ID NO:159 and CDR-L2 of SEQ ID NO:173. (xxv) light chain variable structural domains comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:173, and CDR-L3 of SEQ ID NO:186; (xxvi) light chain variable structural domains comprising CDR-L1 of SEQ ID NO:161, CDR-L2 of SEQ ID NO:174, and CDR-L3 of SEQ ID NO:185; CDR-L3 of SEQ ID NO:187; (xxvii) light chain variable structural domains comprising CDR-L1 of SEQ ID NO:162, CDR-L2 of SEQ ID NO:174, and CDR-L3 of SEQ ID NO:188; (xxviii) light chain variable structural domains comprising CDR-L1 of SEQ ID NO:163, CDR-L2 of SEQ ID NO:174, and CDR-L3 of SEQ ID NO:188. CDR-L3 of SEQ ID NO:188; (xxix) light chain variable structural domains comprising CDR-L1 of SEQ ID NO:164, CDR-L2 of SEQ ID NO:174, and CDR-L3 of SEQ ID NO:189; (xxx) light chain variable structural domains comprising CDR-L1 of SEQ ID NO:165, CDR-L2 of SEQ ID NO:175, and CDR-L3 of SEQ ID NO:190. CDR-L3 of SEQ ID NO:166; (xxxi) light chain variable structural domains comprising CDR-L1 of SEQ ID NO:166, CDR-L2 of SEQ ID NO:176, and CDR-L3 of SEQ ID NO:191; (xxxi) light chain variable structural domains comprising CDR-L1 of SEQ ID NO:167, CDR-L2 of SEQ ID NO:177, and CDR-L3 of SEQ ID NO:192. L3; (xxxii) a light chain variable structural domain comprising CDR-L1 of SEQ ID NO:168, CDR-L2 of SEQ ID NO:178 and CDR-L3 of SEQ ID NO:193; (xxxiii) a light chain variable structural domain comprising CDR-L1 of SEQ ID NO:169, CDR-L2 of SEQ ID NO:179 and CDR-L3 of SEQ ID NO:194.

In one embodiment, the polynucleotide comprises a nucleotide sequence encoding a VL domain, wherein the VL domain comprises: (xxxiv) a light chain variable domain comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:172, and CDR-L3 of SEQ ID NO:183; and (xxxv) a light chain variable domain comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:172, and CDR-L3 of SEQ ID NO:195.

In one embodiment, the isolated polynucleotide encoding the MuSK antibody-based molecule encodes any of the VH and/or VL domain sequences provided in Table 3 below. The nucleic acid molecules described herein include isolated polynucleotides, portions of expression vectors, or portions of linear DNA sequences, including linear DNA sequences for in vitro transcription/translation, and vectors compatible with prokaryotic, eukaryotic, or filamentous phage expression, secretion, and/or display of the antibodies or their binding fragments described herein.

In a preferred embodiment, the polynucleotide comprises a nucleotide sequence encoding a VH containing an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234. In another more preferred embodiment, the polynucleotide comprises a nucleotide sequence encoding a VL containing an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235.

In a preferred embodiment, the polynucleotide comprises a nucleotide sequence encoding an antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK), said molecule comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235.

The heavy-chain variable structural domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a more preferred embodiment, the polynucleotide comprises a nucleotide sequence encoding an antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK), comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235, and wherein the heavy chain variable domain contains:

- Contains the CDR-H1 amino acid sequence consisting of SEQ ID NO:147 or SEQ ID NO:147.

- Contains the CDR-H2 amino acid sequence consisting of SEQ ID NO:153 or SEQ ID NO:153, and

- Contains the CDR-H3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:156 or SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence consisting of SEQ ID NO:159 or SEQ ID NO:159.

- Contains the CDR-L2 amino acid sequence consisting of SEQ ID NO:172 or SEQ ID NO:172, and

- Contains the CDR-L3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:195 or SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a more preferred embodiment, the polynucleotide comprises or is composed of a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:264. In an even more preferred embodiment, the polynucleotide comprises or is composed of SEQ ID NO:264.

In another, more preferred embodiment, the polynucleotide comprises or is composed of a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:265. In an even more preferred embodiment, the polynucleotide comprises or is composed of SEQ ID NO:265.

In a more preferred embodiment, the polynucleotide comprises a nucleotide sequence encoding an antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK), comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain is encoded by a nucleotide sequence having at least 80% identity with SEQ ID NO:264 and the light chain variable domain is encoded by a nucleotide sequence having at least 80% identity with SEQ ID NO:265. In one embodiment, the identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one implementation, the polynucleotide comprises:

a) A nucleotide sequence having at least 80% identity with SEQ ID NO:276 encoding the full-length heavy chain, and

b) A nucleotide sequence that has at least 80% identity with SEQ ID NO:278 encoding the full-length light chain.

In one implementation, the identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one implementation, the polynucleotide comprises:

a) A nucleotide sequence having at least 80% identity with SEQ ID NO:277 encoding the heavy chain variable domain, and

b) A nucleotide sequence having at least 80% identity with SEQ ID NO:279 encoding a light chain variable domain.

In one implementation, the identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one implementation, the polynucleotide comprises:

a) A nucleotide sequence having at least 80% identity with SEQ ID NO:276 encoding the full-length heavy chain, wherein said nucleotide sequence comprises a nucleotide sequence having at least 80% identity with SEQ ID NO:277 encoding the variable domain of the heavy chain, and

b) A nucleotide sequence having at least 80% identity with SEQ ID NO:278 encoding the full-length light chain, wherein the nucleotide sequence comprises a nucleotide sequence having at least 80% identity with SEQ ID NO:279 encoding the variable domain of the light chain.

In one implementation, the identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one implementation, the polynucleotide comprises:

a) Nucleotide sequence SEQ ID NO:276, and

b) Nucleotide sequence SEQ ID NO:278.

The polynucleotides of the present invention can be generated and assembled into complete single-stranded or double-stranded molecules by chemical synthesis, such as solid-phase polynucleotide synthesis on an automated polynucleotide synthesizer. Alternatively, the polynucleotides can be generated by other techniques such as PCR, followed by conventional cloning. Techniques for generating or obtaining polynucleotides of a given sequence are well known in the art.

Polynucleotides may contain at least one non-coding sequence, such as a promoter or enhancer sequence, intron, polyadenylation signal, cis sequence that promotes RepA binding, etc. The polynucleotide sequence may also contain additional sequences encoding, for example, adapter sequences, marker or tag sequences (e.g., histidine tags or HA tags) to facilitate the purification or detection of proteins, signal sequences, fusion protein couplers (e.g., RepA, Fc moieties), or phage coat proteins (e.g., pIX or pIII).

carrier

In another aspect, a vector (preferably an expression vector) for treating neuromuscular disorders in human subjects is provided, comprising a polynucleotide encoding a molecule (or an anti-MuSK antibody or its antigen-binding fragment) based on a MuSK antibody as described herein.

Such vectors include, but are not limited to, plasmid vectors, viral vectors, including but not limited to vaccinia vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vectors for baculovirus expression, transposon-based vectors, or any other such vectors, which are suitable for introducing the polynucleotides described herein into a given organism or genetic background in any way to promote the expression of encoded antibody peptides. In one embodiment, a polynucleotide encoding a heavy chain variable domain, alone or together with a polynucleotide encoding a light chain variable domain as described herein, is combined with a promoter, a translation initiation region (e.g., a ribosome-binding sequence and a start codon), a 3' untranslated region, a polyadenylation signal, a stop codon, and a transcription termination sequence to form one or more expression vector constructs.

In one embodiment, the vector is an adenovirus-associated virus (AAV) vector. Many therapeutic AAV vectors suitable for delivering polynucleotides encoding the antibodies described herein to the central nervous system are known in the art. See, for example, Deverman et al., “Gene Therapy for Neurological Disorders: Progress and Prospects,” Nature Rev. 17:641-659 (2018), Nature Rev. 17:641-659 (2018), which is incorporated herein by reference in its entirety. Suitable AAV vectors include serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, either in their natural form or modified for enhanced tropism. Known CNS-tonic AAV vectors particularly suitable for the therapeutic expression of the MuSK antibodies described herein include AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9, either in their natural form or modified for enhanced tacticity. In one embodiment, the AAV vector is an AAV2 vector. In another embodiment, the AAV vector is an AAV5 vector (Vitale et al., “Anti-tau Conformational scFv MC1 Antibody Efficiently Reduces Pathological Tau Species in Adult JNPL3 Mice,” Acta Neuropathol. Commun. 6:82 (2018), which is incorporated herein by reference in its entirety). In another embodiment, the AAV vector is the AAV9 vector (Haiyan et al., “Targeting Root Cause by Systemic scAAV9-hIDS Gene Delivery: Functional Correction and Reversal of Severe MPSII in Mice,” Mol. Ther. Methods Clin. Dev. 10:327-340 (2018), which is incorporated herein by reference in its entirety). In another embodiment, the AAV vector is the AAVrh10 vector (Liu et al., “Vectored Intracerebral Immunizations with the Anti-Tau Monoclonal Antibody PHF1 Markedly Reduces Tau Pathology in Mutant Transgenic Mice,” J. Neurosci. 36(49):12425-35 (2016), which is incorporated herein by reference in its entirety).

In another embodiment, the AAV vector is a heterozygous vector containing the genome of one serotype (e.g., AAV2) and the capsid protein of another serotype (e.g., AAV1 or AAV3 to 9) to control tropism. See, for example, Broekmanet et al., “Adeno-associated Virus Vectors Serotyped with AAV8 Capsid are More Efficient than AAV-1 or-2 Serotypes for Widespread Gene Delivery to the Neonatal Mouse Brain,” Neuroscience 138:501-510 (2006), which is incorporated herein by reference in its entirety. In one embodiment, the AAV vector is an AAV2/8 heterozygous vector (Ising et al., “AAV-mediated Expression of Anti-Tau ScFv Decreases Tau Accumulation in a Mouse Model of Tauopathy,” J. Exp. Med. 214(5):1227 (2017), which is incorporated herein by reference in its entirety). In another embodiment, the AAV vector is an AAV2/9 heterozygous vector (Simon et al., “A Rapid Gene Delivery-Based Mouse Model for Early-Stage Alzheimer Disease-Type Tauopathy,” J. Neuropath. Exp. Neurol. 72(11):1062-71 (2013), which is incorporated herein by reference in its entirety).

In another embodiment, the AAV vector is an AAV vector modified or selected for the following: enhanced CNS transduction after intraplasmic administration, such as AAV-DJ (Grimm et al., J. Viol. 82:5887-5911 (2008), which is incorporated herein by reference in its entirety); enhanced transduction of neural stem cells and progenitor cells, such as SCH9 and AAV4.18 (Murlidharan et al., J. Virol. 89:3976-3987 (2015) and Ojala et al., Mol. Ther. 26:304-319 (2018), which is incorporated herein by reference in its entirety); enhanced retrograde transduction, such as rAAV2-retro (Muller et al.). (Nat. Biotechnol. 21:1040-1046 (2003), which is incorporated herein by reference in its entirety); selective transduction to brain endothelial cells, such as AAV-BRI (Korbelin et al., EMBO Mol. Med. 8:609-625 (2016), which is incorporated herein by reference in its entirety); or enhanced adult CNS transduction following IV administration, such as AAV-PHP.B and AAVPHP.eB (Deverman et al., Nat. Biotechnol. 34:204-209 (2016) and Chan et al., Nat. Neurosci. 20:1172-1179 (2017), which is incorporated herein by reference in its entirety).

According to this embodiment, the expression vector construct encoding a MuSK antibody-based molecule comprises a polynucleotide encoding a heavy chain polypeptide, a functional fragment thereof, a variant thereof, or a combination thereof. Alternatively, the expression construct may comprise a nucleic acid sequence encoding a light chain polypeptide, a functional fragment thereof, a variant thereof, or a combination thereof. In one embodiment, the expression vector construct comprises nucleic acid sequences encoding a heavy chain polypeptide, a functional fragment thereof, or a variant thereof, and a light chain polypeptide, a functional fragment thereof, or a variant thereof.

In one embodiment, the expression construct further comprises a promoter sequence suitable for driving the expression of a MuSK antibody-based molecule. Suitable promoter sequences include, but are not limited to, the elongation factor 1-α promoter (EF1a), the phosphoglycerate kinase-1 promoter (PGK), the cytomegalovirus immediate early gene promoter (CMV), the chimeric liver-specific promoter (LSP), the cytomegalovirus enhancer/chicken β-actin promoter (CAG), the tetracycline-responsive promoter (TRE), the thyroxine transporter promoter (TTR), the simian virus 40 promoter (SV40), and the CK6 promoter. Other promoters known in the art suitable for driving gene expression in mammalian cells are also suitable for inclusion in the expression constructs disclosed herein.

In one implementation, the expression construct (or expression vector) also encodes an adapter sequence. The adapter sequence may encode an amino acid sequence that spatially separates and/or connects one or more components of the expression construct (the heavy and light chain components of the encoded antibody).

In a preferred embodiment, the expression vector comprises: a polynucleotide encoding an antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) and contains a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain contains an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain contains an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235, and wherein the heavy chain variable domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a more preferred embodiment, the expression vector comprises nucleotides encoding an antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) and contains a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain contains an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain contains an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235, and wherein the heavy chain variable domain contains:

- Contains the CDR-H1 amino acid sequence consisting of SEQ ID NO:147 or SEQ ID NO:147.

- Contains the CDR-H2 amino acid sequence consisting of SEQ ID NO:153 or SEQ ID NO:153, and

- Contains the CDR-H3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:156 or SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence consisting of SEQ ID NO:159 or SEQ ID NO:159.

- Contains the CDR-L2 amino acid sequence consisting of SEQ ID NO:172 or SEQ ID NO:172, and

- Contains the CDR-L3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:195 or SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one embodiment, the expression vector comprises a polynucleotide encoding an antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) and includes:

a) A nucleotide sequence having at least 80% identity with SEQ ID NO:276 encoding the full-length heavy chain, and

b) A nucleotide sequence that has at least 80% identity with SEQ ID NO:278 encoding the full-length light chain.

In one implementation, the identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one embodiment, the expression vector comprises a polynucleotide encoding an antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) and includes:

a) A nucleotide sequence having at least 80% identity with SEQ ID NO:277 encoding the heavy chain variable domain, and

b) A nucleotide sequence having at least 80% identity with SEQ ID NO:279 encoding a light chain variable domain.

In one implementation, the identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a more preferred embodiment, the expression vector comprises a polynucleotide encoding an antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) and includes:

c) A nucleotide sequence having at least 80% identity with SEQ ID NO:276 encoding the full-length heavy chain, wherein said nucleotide sequence comprises a nucleotide sequence having at least 80% identity with SEQ ID NO:277 encoding the variable domain of the heavy chain, and

d) A nucleotide sequence having at least 80% identity with SEQ ID NO:278 encoding the full-length light chain, wherein the nucleotide sequence comprises a nucleotide sequence having at least 80% identity with SEQ ID NO:279 encoding the variable domain of the light chain.

In one implementation, the identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a more preferred embodiment, the expression vector comprises a polynucleotide encoding an antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK) and includes:

a) Nucleotide sequence SEQ ID NO:276, and

b) Nucleotide sequence SEQ ID NO:278.

host cells

Another aspect of the invention is a host cell or cell-free expression system for treating neuromuscular diseases in human subjects, wherein the cells contain an expression vector encoding a MuSK antibody (or an antigen-binding fragment thereof) and optionally produce the MuSK antibody described herein.

The MuSK antibody-based molecules described herein can optionally be generated from cell lines, mixed cell lines, immortalized cells, or clonal populations of immortalized cells, as is known in the art (see, for example,

Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook et a l., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, aL aboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan et al., eds., CurrentProtocols in Immunology, John Wiley&S ons, lnc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley&Sons, NY, N.Y., (1997-2001),

(It is incorporated here by reference).

In some implementations, the host cell selected for expression may be of mammalian origin. Suitable mammalian host cells include, but are not limited to, COS-1 cells, COS-7 cells, HEK293 cells, BHK21 cells, CHO cells, BSC-1 cells, HeG2 cells, SP2/0 cells, HeLa cells, mammalian myeloma cells, mammalian lymphoma cells, or any derived, immortalized, or transformed cells thereof. Other suitable host cells include, but are not limited to, yeast cells, insect cells, and plant cells. Alternatively, the host cell may be selected from species or organisms that cannot glycosylate the polypeptide, such as prokaryotic cells or organisms, such as BL21, BL21(DE3), BL21-GOLD(DE3), XL1-Blue, JM109, HMS174, HMS174(DE3), and any natural or modified strains of E. coli, Klebsiella, or Pseudomonas.

The MuSK-based antibody molecules described herein can be prepared using any of a variety of techniques employing the isolated polynucleotides, vectors, and host cells described above. Generally, antibodies can be generated using cell culture techniques, including the production of monoclonal antibodies using conventional techniques, or by transfecting antibody genes, heavy chains, and/or light chains into suitable bacterial or mammalian cell hosts to allow antibody production, where the antibodies can be recombinant. In one embodiment, the MuSK-based antibody molecules described herein are monoclonal antibodies or their functional binding fragments. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells, and recover antibodies from the culture medium. Transfection of host cells can be performed using a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as by electroporation, calcium phosphate precipitation, DEAE-glucan transfection, etc. Although the antibodies described herein can be expressed in prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, particularly mammalian cells, is sometimes preferred because such eukaryotic cells (and particularly mammalian cells) are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active antibodies.

As described above, exemplary mammalian host cells for expressing the recombinant antibodies of the present invention include Chinese hamster ovary (CHO) cells (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980), which is incorporated herein by reference in its entirety). Other suitable mammalian host cells include, but are not limited to, NSO myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient for the antibody to be expressed in the host cell or, more preferably, for the antibody to be secreted into the culture medium in which the host cell is cultured.

Host cells can also be used to generate functional antibody fragments, such as Fab fragments or scFv molecules. It is understood that variations of the above operations are also within the scope of this invention. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of the light and/or heavy chains of the antibodies described herein. Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that are not essential for binding to the target antigen. Molecules expressed from such truncated DNA molecules are also included in the antibodies described herein.

Antibodies and antibody-binding fragments are recovered and purified from recombinant cell cultures using known methods, including but not limited to protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High-performance liquid chromatography (“HPLC”) can also be used for purification.

In a preferred embodiment, the host cell expresses an antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK), comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235.

The heavy chain variable structure domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a more preferred embodiment, the host cell expresses an antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK), comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235.

The heavy chain variable structure domain contains:

- Contains the CDR-H1 amino acid sequence consisting of SEQ ID NO:147 or SEQ ID NO:147.

- Contains the CDR-H2 amino acid sequence consisting of SEQ ID NO:153 or SEQ ID NO:153, and

- Contains the CDR-H3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:156 or SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence consisting of SEQ ID NO:159 or SEQ ID NO:159.

- Contains the CDR-L2 amino acid sequence consisting of SEQ ID NO:172 or SEQ ID NO:172, and

- Contains the CDR-L3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:195 or SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one implementation, the host cell expresses an antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK), comprising:

a) A nucleotide sequence having at least 80% identity with SEQ ID NO:276 encoding the full-length heavy chain, and

b) A nucleotide sequence that has at least 80% identity with SEQ ID NO:278 encoding the full-length light chain.

In one implementation, the identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one implementation, the host cell expresses an antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK), comprising:

a) A nucleotide sequence having at least 80% identity with SEQ ID NO:277 encoding the heavy chain variable domain, and

b) A nucleotide sequence having at least 80% identity with SEQ ID NO:279 encoding a light chain variable domain.

In one implementation, the identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one implementation, the host cell expresses an antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK), comprising:

a) A nucleotide sequence having at least 80% identity with SEQ ID NO:276 encoding the full-length heavy chain, wherein said nucleotide sequence comprises a nucleotide sequence having at least 80% identity with SEQ ID NO:277 encoding the variable domain of the heavy chain, and

b) A nucleotide sequence having at least 80% identity with SEQ ID NO:278 encoding the full-length light chain, wherein the nucleotide sequence comprises a nucleotide sequence having at least 80% identity with SEQ ID NO:279 encoding the variable domain of the light chain.

In one implementation, the identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one implementation, the host cell expresses an antibody-based molecule that binds to human muscle-specific tyrosine protein kinase (MuSK), comprising:

a) Nucleotide sequence SEQ ID NO:276, and

b) Nucleotide sequence SEQ ID NO:278.

Compositions containing molecules based on MuSK antibodies

The MuSK antibody-based molecules of the present invention or the polynucleotides encoding MuSK antibody-based molecules can be advantageously applied as compositions.

Therefore, in another aspect, compositions are provided for treating neuromuscular disorders in human subjects, said compositions comprising antibodies or antigen-binding fragments, polynucleotides, expression vectors, or host cells or cell-free expression systems as defined herein.

In one embodiment, the composition is a pharmaceutical composition. In another embodiment, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier or excipient.

In one embodiment, such a composition is a pharmaceutical composition comprising an active therapeutic agent (i.e., a MuSK antibody) and one or more other pharmaceutically acceptable components. See Remington: The Science and Practice of Pharmacy ( ^21st Edition) (2005) (Troy, DB et al. (Eds.) Lippincott Williams & Wilkins (Publs.), Baltimore MD), which is incorporated herein by reference in its entirety. Preferred forms depend on the intended method of administration and therapeutic application. Depending on the desired formulation, the composition may also comprise a pharmaceutically acceptable nontoxic carrier, excipient, diluent, filler, salt, buffer, detergent (e.g., a nonionic detergent, such as Tween-20 or Tween-80), stabilizer (e.g., a sugar or a protein-free amino acid), preservative, tissue fixative, solubilizer, and/or other substances suitable for inclusion in the pharmaceutical composition, and these are typically used to formulate pharmaceutical compositions for administration to animals or humans. Diluents are selected so as not to affect the bioactivity of the composition. Examples of such diluents include distilled water, physiological phosphate-buffered saline, Ringer's solution, dextran solution, and Hank's solution. Additionally, pharmaceutical compositions or formulations may contain other carriers, or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of this invention include water, saline, phosphate-buffered saline, ethanol, dextran, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (e.g., olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil), carboxymethyl cellulose colloidal solutions, tragacanth gum, and injectable organic esters (e.g., ethyl oleate), and/or various buffer solutions. Other carriers are well known in the pharmaceutical industry.

Pharmaceutical carriers include sterile aqueous solutions or dispersions, as well as sterile powders for the provisional preparation of sterile injectable solutions or dispersions. The use of such media and reagents for pharmaceutically active substances is known in the art. Their use in the pharmaceutical compositions of the present invention is contemplated unless any conventional media or reagent is incompatible with the active compound.

The composition may also contain large, slowly metabolizing macromolecules, such as proteins, polysaccharides (e.g., chitosan), polylactic acid, polyglycolic acid and copolymers (e.g., latex-functionalized agarose, agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers and lipid aggregates (e.g., oil droplets or liposomes). The suitability of the carrier and other components of the pharmaceutical composition is determined based on the absence of a significant negative impact on the desired biological properties of the active antibody-based molecules of the present invention (e.g., less than a significant effect on antigen binding (e.g., 10% or less relative inhibition, 5% or less relative inhibition, etc.)).

The pharmaceutical compositions of the present invention may further comprise pharmaceutically acceptable antioxidants, such as (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

The pharmaceutical compositions of the present invention may also contain isotonic agents, such as sugars, polyols, such as mannitol, sorbitol, glycerol, or sodium chloride.

The pharmaceutical compositions of the present invention may also contain one or more excipients suitable for the chosen route of administration, such as preservatives, wetting agents, emulsifiers, dispersants, or buffers, which may improve the shelf life or efficacy of the pharmaceutical composition. The antibodies of the present invention can be prepared with a carrier that protects the antibody from rapid release, such as a controlled-release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, alone or with waxes, or other substances known in the art. Methods for preparing such formulations are generally known to those skilled in the art. See, for example, *SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS*, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In one embodiment, the antibody of the present invention can be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions, as well as sterile powders for the provisional preparation of sterile injectable solutions or dispersions. The use of such media and reagents for pharmaceutically active substances is known in the art.

Injectable pharmaceutical compositions must generally be sterile and stable under the conditions of preparation and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for achieving high drug concentrations. The carrier can be an aqueous or non-aqueous solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters, such as ethyl oleate. Appropriate flowability can be maintained, for example, by using coatings (e.g., lecithin), in the case of dispersions by maintaining the desired particle size, and by using surfactants. In many cases, isotonic agents, such as sugars, polyols (e.g., glycerol, mannitol, sorbitol), or sodium chloride, will be preferably included in the composition. Extended absorption of the injectable composition can be achieved by including agents that delay absorption, such as monostearate and gelatin, in the composition. Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound with, for example, one or a combination of the ingredients listed above (as needed) into a suitable solvent, followed by sterile microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile carrier containing a basic dispersion medium and other desired components. Examples of preparation methods for sterile powders used to prepare sterile injectable solutions include vacuum drying and freeze-drying (lyophilization), which produce a powder containing the active ingredient plus any additional desired components from its previously sterile filtered solution.

For parenteral administration, the pharmaceutical preparations of the present invention are typically formulated together with a pharmaceutical carrier in an injectable dose of a physiologically acceptable diluent, such as water, oil, saline, glycerin, or ethanol. Additionally, excipients may be present in the composition, such as wetting agents or emulsifiers, surfactants, pH buffers, etc. Other components of the pharmaceutical composition are of petroleum, animal, plant, or synthetic origin. Peanut oil, soybean oil, and mineral oil are examples of suitable substances. Generally, glycols (e.g., propylene glycol or polyethylene glycol) are preferred liquid carriers, particularly for injectable solutions. The pharmaceutical preparations of the present invention can be administered in the form of reservoir injections or implants, formulated in a manner that allows for sustained release of the active ingredient.

Typically, compositions are prepared as injectable formulations, as liquid solutions or suspensions; they can also be prepared in solid forms suitable for dissolving or suspending in a liquid carrier prior to injection. Formulations may also be emulsified or encapsulated in liposomes or microparticles, such as polylactide, polyglycolic acid, or copolymers, to enhance the excipient effect (Langer, et al., Science 249:1527 (1990); Hanes, et al., Advanced Drug Delivery Reviews 28:97-119 (1997), which are incorporated herein by reference in their entirety). Other formulations suitable for different routes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications.

In one embodiment, the composition comprises an anti-MuSK antibody (or antigen-binding fragment) containing: a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:153, and CDR-H3 of SEQ ID NO:156, and a light chain variable domain (3B2g2m1) comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:172, and CDR-L3 of SEQ ID NO:195.

In one embodiment, the composition comprises an anti-MuSK antibody (or antigen-binding fragment) containing: a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:153, and CDR-H3 of SEQ ID NO:156, and a light chain variable domain (3B2g1m1) comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:172, and CDR-L3 of SEQ ID NO:183.

In one embodiment, the anti-MuSK antibody (or antigen-binding fragment) comprises: a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:154, and CDR-H3 of SEQ ID NO:156, and a light chain variable domain (3B2g1m2) comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:172, and CDR-L3 of SEQ ID NO:183.

In one embodiment, the anti-MuSK antibody (or antigen-binding fragment) comprises: a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:154, and CDR-H3 of SEQ ID NO:156, and a light chain variable domain (3B2g2m2) comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:172, and CDR-L3 of SEQ ID NO:195.

In one embodiment, the anti-MuSK antibody (or antigen-binding fragment) comprises: a heavy chain variable domain comprising CDR-H1 of SEQ ID NO:147, CDR-H2 of SEQ ID NO:150, and CDR-H3 of SEQ ID NO:156, and a light chain variable domain comprising CDR-L1 of SEQ ID NO:159, CDR-L2 of SEQ ID NO:172, and CDR-L3 of SEQ ID NO:183 (3B2).

In a preferred embodiment, the composition comprises an antibody or antigen-binding fragment that binds to human muscle-specific tyrosine protein kinase (MuSK), comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235.

The heavy-chain variable structural domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a more preferred embodiment, the composition comprises an antibody or antigen-binding fragment that binds to human muscle-specific tyrosine protein kinase (MuSK), comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235.

The heavy-chain variable structural domain contains:

- Contains the CDR-H1 amino acid sequence consisting of SEQ ID NO:147 or SEQ ID NO:147.

- Contains the CDR-H2 amino acid sequence consisting of SEQ ID NO:153 or SEQ ID NO:153, and

- Contains the CDR-H3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:156 or SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence consisting of SEQ ID NO:159 or SEQ ID NO:159.

- Contains the CDR-L2 amino acid sequence consisting of SEQ ID NO:172 or SEQ ID NO:172, and

- Contains the CDR-L3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:195 or SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a preferred embodiment, the composition comprises an antibody or antigen-binding fragment that binds to human muscle-specific tyrosine protein kinase (MuSK), comprising a constant Fc region of wild-type human IgG, a heavy chain variable domain, and a light chain variable domain, wherein the constant Fc region of wild-type human IgG comprises SEQ ID NO:266 or SEQ ID NO:267, the heavy chain variable domain, and the light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234, and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235.

The heavy-chain variable structural domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a more preferred embodiment, the composition comprises an antibody or antigen-binding fragment that binds to human muscle-specific tyrosine protein kinase (MuSK), comprising a constant Fc region of wild-type human IgG, a heavy chain variable domain, and a light chain variable domain, wherein the constant Fc region of wild-type human IgG comprises SEQ ID NO:266 or SEQ ID NO:267, the heavy chain variable domain, and the light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234, and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235.

The heavy-chain variable structural domain contains:

- Contains the CDR-H1 amino acid sequence consisting of SEQ ID NO:147 or SEQ ID NO:147.

- Contains the CDR-H2 amino acid sequence consisting of SEQ ID NO:153 or SEQ ID NO:153, and

- Contains the CDR-H3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:156 or SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence consisting of SEQ ID NO:159 or SEQ ID NO:159.

- Contains the CDR-L2 amino acid sequence consisting of SEQ ID NO:172 or SEQ ID NO:172, and

- Contains the CDR-L3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:195 or SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a preferred embodiment, the composition comprises an antibody or antigen-binding fragment that binds to human muscle-specific tyrosine protein kinase (MuSK), comprising a constant Fc region of wild-type human IgG (in which L234A and/or L235A substitutions according to the EU numbering system are introduced), a heavy chain variable domain, and a light chain variable domain, wherein the wild-type human IgG constant Fc region comprises SEQ ID NO:266 or SEQ ID NO:267, the heavy chain variable domain, and the light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235, and

The heavy-chain variable structural domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a more preferred embodiment, the composition comprises an antibody or antigen-binding fragment that binds to human muscle-specific tyrosine protein kinase (MuSK), comprising a constant Fc region of wild-type human IgG (in which L234A and/or L235A substitutions according to the EU numbering system are introduced), a heavy chain variable domain, and a light chain variable domain, wherein the wild-type human IgG constant Fc region comprises SEQ ID NO:266 or SEQ ID NO:267, the heavy chain variable domain, and the light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235, and

The heavy-chain variable structural domain contains:

- Contains the CDR-H1 amino acid sequence consisting of SEQ ID NO:147 or SEQ ID NO:147.

- Contains the CDR-H2 amino acid sequence consisting of SEQ ID NO:153 or SEQ ID NO:153, and

- Contains the CDR-H3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:156 or SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence consisting of SEQ ID NO:159 or SEQ ID NO:159.

- Contains the CDR-L2 amino acid sequence consisting of SEQ ID NO:172 or SEQ ID NO:172, and

- Contains the CDR-L3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:195 or SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one embodiment, the composition comprises an antibody or antigen-binding fragment that binds to human muscle-specific tyrosine protein kinase (MuSK), comprising:

a) A full-length heavy chain containing an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:268 and

b) A full-length light chain containing an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:269, and

c) One or more of the following mutations (all numbered according to the EU numbering system) have been introduced into the full-length heavy chain: N297A substitution; N297Q substitution; L234A substitution; L234D substitution; L234E substitution; L234G substitution; L234H substitution; L234F substitution; L234K substitution; L234Q substitution; L234R substitution; L234S substitution; L234T substitution; L235A substitution; L235D substitution; L235E substitution; L235F substitution; L235G substitution; L235V substitution; L235H substitution; L235I substitution; L235K substitution; L235R substitution; L235S substitution; L235T substitution; L235Q substitution; L237A substitution; S239D substitution; E233P substitution. Replacement; L234V replacement; C236 deletion; G236E replacement; G236R replacement; G236K replacement; G237A replacement; P238A replacement; F243L replacement; D265A replacement; S267E replacement; H268A replacement; R292P replacement; Y300L replacement; K322A replacement; K322Q replacement; A327Q replacement; L328F replacement; L328R replacement; P329A replacement; P329G replacement; A330L replacement; A330S replacement; P331S replacement; I332E replacement; P396L replacement; or each of the combinations of mutations described above in the fourth embodiment of this application, preferably the mutation is L234A or L235A, more preferably the mutations are L234A and L235A.

In one embodiment, the composition comprises an antibody or antigen-binding fragment that binds to human muscle-specific tyrosine protein kinase (MuSK), comprising:

a) A full-length heavy chain, comprising SEQ ID NO:268, and

b) A full-length light chain containing SEQ ID NO:269, and

c) The full-length heavy chain contains L234A and L235A mutations numbered according to the EU numbering system.

In one implementation, the binding to the same ligand is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or becomes undetectable compared to the binding of an antibody to an effector ligand that does not have any amino acid substitutions in its constant Fc region of human IgG.

In one embodiment, the composition comprises an antibody or antigen-binding fragment that binds to human muscle-specific tyrosine protein kinase (MuSK), comprising:

a) A full-length heavy chain comprising an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:270, and

b) A full-length light chain comprising an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:271, and

c) One or more of the following mutations (all numbered according to the EU numbering system) have been introduced into the full-length heavy chain: N297A substitution; N297Q substitution; L234A substitution; L234D substitution; L234E substitution; L234G substitution; L234H substitution; L234F substitution; L234K substitution; L234Q substitution; L234R substitution; L234S substitution; L234T substitution; L235A substitution; L235D substitution; L235E substitution; L235F substitution; L235G substitution; L235V substitution; L235H substitution; L235I substitution; L235K substitution; L235R substitution; L235S substitution; L235T substitution; L235Q substitution; L237A substitution; S239D substitution; E233P substitution. Replacement; L234V replacement; C236 deletion; G236E replacement; G236R replacement; G236K replacement; G237A replacement; P238A replacement; F243L replacement; D265A replacement; S267E replacement; H268A replacement; R292P replacement; Y300L replacement; K322A replacement; K322Q replacement; A327Q replacement; L328F replacement; L328R replacement; P329A replacement; P329G replacement; A330L replacement; A330S replacement; P331S replacement; I332E replacement; P396L replacement; or each of the combinations of mutations described above in the fourth embodiment of this application, preferably the mutation is L234A or L235A, more preferably the mutations are L234A and L235A.

In one embodiment, the composition comprises an antibody or antigen-binding fragment that binds to human muscle-specific tyrosine protein kinase (MuSK), comprising:

a) A full-length heavy chain containing SEQ ID NO:270, and

b) A full-length light chain containing SEQ ID NO:271, and

c) The full-length heavy chain contains L234A and L235A mutations numbered according to the EU numbering system.

In one implementation, the binding to the same ligand is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or becomes undetectable compared to the binding of an antibody to an effector ligand that does not have any amino acid substitutions in its constant Fc region of human IgG.

The MuSK antibody-based molecules of this invention can be administered parenterally, topically, orally, or intranasally for therapeutic purposes. Intramuscular injection (e.g., into the muscles of the arm or leg) and intravenous infusion are preferred methods of administration of the molecules of this invention. In some methods, such molecules are administered as a slow-release composition or device, such as the Medipad ^™ device (Elan Pharm. Technologies, Dublin, Ireland). In some methods, the antibodies disclosed herein are injected directly into a specific tissue, such as intracranial injection.

In one embodiment, the pharmaceutical composition of the present invention is administered parenterally. The phrase "parenterally" as used herein and its variations refer to administration methods other than enteric and surface administration, typically by injection, and including transdermal, intravenous, intramuscular, intraarterial, intrasheath, intracapsular, intracranial, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injections, subcutaneous, and infusions. In one embodiment, the pharmaceutical composition is administered via intravenous or subcutaneous injection or infusion.

In one embodiment, the anti-MuSK antibody or its antigen-binding fragment (or polynucleotide, expression vector, host cell, or composition) according to the present invention is administered in combination with an anticholinergic compound. The anticholinergic compound is a compound capable of inhibiting the action of the neurotransmitter acetylcholine at synapses or nerve effector junctions (e.g., neuromuscular junctions). Preferably, the anticholinergic compound is a compound capable of inhibiting the activity of muscarinic acetylcholine receptors.

Anticholinergic compounds can also be formulated in compositions that are anti-MuSK antibodies or antigen-binding fragments. The types of anti-MuSK antibody or antigen-binding fragment compositions disclosed herein can also be used in compositions containing anticholinergic compounds. Both compounds can be present in a single composition. Alternatively, they can be formulated in separate compositions.

The use of compounds that induce mechanisms promoting NMJ (neuromuscular junction) stability and/or repair is attractive for the treatment of any neuromuscular disease, especially when such NMJ is affected. In a preferred embodiment, the use of two different compounds, each activating and inducing mechanisms promoting NMJ stability and/or repair, is even more attractive because such combination therapy has been shown to be synergistic. Therefore, the combination is highly beneficial for the treatment of neuromuscular diseases, particularly those with affected NMJs, such as ALS.

In one embodiment, the anti-MuSK antibody or its antigen-binding fragment, polynucleotide, expression vector, host cell, or composition used according to any of the preceding claims, wherein the neuromuscular disease is characterized by impaired neuromuscular transmission and/or NMJ denervation.

Impaired neuromuscular transmission is characterized by at least one of the following:

a. Excessive stimulation by muscarinic substances,

b. Motor neuron death,

c. NMJ denervation and

d. Impaired synaptic transmission.

In one implementation, impaired neuromuscular transmission or impaired synaptic transmission may be characterized by insufficient MuSK signaling, insufficient MuSK dimerization, insufficient MuSK phosphorylation, insufficient MuSK signaling, and/or insufficient acetylcholine receptor aggregation.

In one implementation, impaired neuromuscular transmission or impaired synaptic transmission may be characterized by poor motor performance, reduced grip strength, poor contractile properties of muscles at the NMJ, poor resistance to muscle fatigue, and reduced muscle mass.

In one implementation, neuromuscular disorders are analyzed, evaluated, or diagnosed through electrophysiological assessment; pharmacodynamic assessment; levels of neurofilaments (e.g., neurofilament light chains, NFL) in serum, plasma, and/or cerebrospinal fluid (CSF); or NMJ biopsy.

Neuromuscular disorders can be selected from the following: amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), myasthenia gravis (MG), congenital myasthenia gravis, Lambert-Eaton myasthenic syndrome (LEMS), Lyme disease, poliomyelitis, post-poliomyelitis, heavy metal poisoning, Kennedy syndrome, adult-onset Tay-Sachs disease, hereditary spastic paraplegia, multifocal neuropathy, cervical spondylosis, extramedullary tumors with compressive radiculopathy and myelopathy, inclusion body myositis, progressive bulbar palsy, progressive muscular atrophy, motor neuron syndrome, and thyrotoxic myopathy. ALS is the preferred neuromuscular disorder.

In one implementation, the anti-MuSK antibody or its antigen-binding fragment as defined herein may be administered to asymptomatic ALS subjects. This means that such an antibody or antigen-binding fragment may be administered to the subject prior to the onset of ALS. The same applies to other neuromuscular disorders.

In this context, an asymptomatic ALS subject may be someone who has been diagnosed as susceptible to neuromuscular disorders or diseases (such as ALS). Identifying an individual (or subject) with a neuromuscular disorder may mean identifying them using diagnostic methods. Such a subject may be a symptomatic subject diagnosed during or after an onset of the disease, or someone susceptible to neuromuscular disorders or diseases (i.e., an asymptomatic subject diagnosed before an onset of the disease, which is synonymous with pre-onset).

Neuromuscular disorders can be caused by genetic defects. These defects are wholly or partially caused by variations in the genomic DNA sequence relative to the genomic DNA sequence of a corresponding individual or subject without the defect. Genetic defects can be caused by mutations in a single gene (monogenous disorders), mutations in multiple genes (multifactorial genetic disorders), a combination of gene mutations and environmental factors, or chromosomal damage (changes in the number or structure of the entire chromosome, or changes in the structure of the gene carrying the mutation). Types of gene mutations include base substitutions, deletions, and insertions.

In one embodiment, the subject is identified as having (or being predisposed to) a neuromuscular disease caused by a genetic defect. In one embodiment, the neuromuscular disease is ALS, and the genetic defect is in the SOD1 gene. Individuals or subjects predisposed to ALS include those with one or more risk factors for developing ALS (including aging, a personal or family history of the disease, or a genetic predisposition to one or more SOD-1 related diseases). A potential genetic cause or predisposition to ALS is a mutation in the human SOD1 gene. Therefore, subjects with ALS, predisposed to ALS, or susceptible to ALS can be identified by genetic testing of the subject's SOD1 gene using assays known in the art (e.g., gene sequencing). At least 180 mutations in human SOD1 known in the art are associated with ALS. In one embodiment, the SOD1 mutation is selected from one or more of the following:

A4V, H46R, G93S, A4T, G141X, D133A, V148G, N139K, G85R, G93A, V14G, C6S, I113T, D49K, G37R,

A89V, E100G, D90A, T137A, E100K, G41A, G41D, G41S, G13R, G72S, L8V, F20C, Q22L, H48R, T54R,

S591, V87A, T88δTAD, A89T, V97M, S105δSL, VI 18L, D124G, LI 14F, D90A, G12RG147R and G37R

In one implementation, the mutation in the SOD1 gene is G37R.

Therefore, when the subject has one of the following SOD1 mutations, asymptomatic individuals or subjects can be identified (before the onset of disease):

A4V, H46R, G93S, A4T, G141X, D133A, V148G, N139K, G85R, G93A, V14G, C6S, I 113T, D49K, G37R, A89V, E100G, D90A, T137A, E100K, G41A, G41D , G41S, G13R, G72S, L8V, F20C, Q22L, H48R, T54R, S591, V87A, T88δTAD, A89T, V97M, S105δSL, VI 18L, D124G, LI 14F, D90A, G12RG147R and G37R.

Analysis of susceptibility to ALS (i.e., asymptomatic subjects at risk of developing ALS) can be performed by analyzing the subjects' family history of ALS. Family history analysis may include a review of three generations of ALS pedigree, medical records and autopsy reports of family members, and identification of autosomal dominant patterns of SOD1 mutations.

Identification of individuals or subjects with asymptomatic ALS (but predisposition to the disease) can also be performed using ALS biomarkers. For example, ALS-specific biomarkers may include circulating microRNAs, circular RNA (circRNA) or messenger RNA (mRNA), TDP-42 aggregates, 8-oxodG, 15-F2t-isoprostan (IsoP), plasma TNF-α, IL-10, TRAIL, plasma IL-1β, CSF TRAIL, pro-inflammatory T helper (Th)17 cells, Th1 cells, anti-inflammatory Th2 cells, regulatory T cells (Treg), pro-inflammatory IL-1β, IL-6, IFN-γ, anti-inflammatory IL-10, cholesterol, LDL cholesterol, apolipoprotein B, HDL cholesterol, apolipoprotein AI, and plasma creatinine. The following markers are considered to indicate ALS susceptibility: PCR, plasma ferritin, transferrin, hepcidin, chitosan trisaccharidase-1 (CHIT1), chitinase-3-like protein 2 (CHI3L2/YKL39), total tTu, phosphorylated tTu (pTau), amyloid-beta (Ab), novel INHAT repressor (NIR), ubiquitin C-terminal hydrolase L1 (UCHL1), microtubule-associated protein 2, capped actin, coagulant-like protein (CAPG), or glycoprotein nonmetastatic melanoma protein B (GPNMB). A subject is considered susceptible to ALS when at least one of these marker measurements deviates from that of a normal age-matched but ALS-free individual by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. Subject susceptibility to ALS can also be analyzed using imaging. For example, such imaging analysis could be MRI assessment of skeletal muscle, imaging-derived functional muscle scores, or tongue ultrasound prediction of medullary progression, with or without MRI.

In one embodiment, the anticholinergic compound is administered separately, sequentially, or simultaneously with an antiMuSK antibody or its antigen-binding fragment, polynucleotide, expression vector, host cell, cell-free expression system, or combination thereof.

In one embodiment, the anticholinergic compound is administered during a disease onset or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days or 1, 2, 3, 4, 5, 6, 7 weeks after the onset of the disease; or within 1, 2, 3, 4, 5, 6, 7 months after the onset of the disease. In one embodiment, the anticholinergic compound is administered during a disease onset or within one week after the onset of the disease. Unexpectedly, attractive results were obtained when the anticholinergic compound was administered as soon as possible during or after a disease onset. Those skilled in the art will understand that the anticholinergic compound is preferably not used to reduce or alleviate symptoms associated with neuromuscular disorders (e.g., ALS). In one embodiment, the anticholinergic compound is not used to reduce or alleviate urinary urgency. In one embodiment, the anticholinergic compound is not used to reduce or alleviate urinary urgency in neuromuscular disorders (e.g., ALS).

In one embodiment, the anticholinergic compound is administered at the onset of the disease, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the onset of the disease; within 1, 2, 3, 4, 5, 6, or 7 weeks; or within 1, 2, 3, 4, 5, 6, or 12 months after the onset of the disease, but prior to diagnosis. In some preferred embodiments, administration is performed between the diagnosis and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days before the diagnosis; within 1, 2, 3, 4, 5, 6, or 7 weeks; or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months prior to diagnosis.

In one implementation, an anti-MuSK antibody or its antigen-binding fragment, polynucleotide, expression vector, host cell, or composition thereof is administered before or during the onset of the disease. "Before the onset of the disease" can mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days prior to the onset of the disease, or 1, 2, 3, 4, 5, 6, 7, or 8 weeks prior to the onset of the disease, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 months prior to the onset of the disease. In this context, "pre-onset subject" can mean a subject without symptoms of the neuromuscular disorder (e.g., ALS).

Therefore, in another aspect of the invention, an anti-MuSK antibody or its antigen-binding fragment (polynucleotide, expression vector, host cell, cell-free expression system, or composition) for treating ALS in human subjects is provided, wherein the antibody or antigen-binding fragment is administered prior to disease onset, preferably within 1, 2, 3, 4, 5, or 6 months prior to disease onset. In this context, a human subject prior to onset may mean a human subject without symptoms of ALS. In one embodiment, the subject is diagnosed as predisposed to neuromuscular disorders or diseases, such as ALS.

In one embodiment, the antibody or antigen-binding fragment binds to the MuSK coil (Fz)-like domain sequence of SEQ ID NO:129.

In one embodiment, the antibody or its antigen-binding fragment comprises a constant Fc region of wild-type human IgG, which contains at least 80% sequence identity with SEQ ID NO:266 or SEQ ID NO:267.

In one implementation, the antibody or antigen-binding fragment is an agonist MuSK antibody and/or the effector function has been reduced or eliminated.

In one implementation, the reduction or elimination of effector function is achieved by introducing one or more of the following mutations (all numbered according to the EU numbering system) into the constant Fc region of the human IgG of the antibody-based molecule described herein, SEQ ID NO:266 or SEQ ID NO:267: N297A substitution; N297Q substitution; L234A substitution; L234D substitution; L234E substitution; L234G substitution; L234H substitution; L234F substitution; L234K substitution; L234Q substitution; L234R substitution; L234S substitution; L234T substitution; L235A substitution; L235D substitution; L235E substitution; L235F substitution; L235G substitution; L235V substitution; L235H substitution; L23 Replace 5I; Replace L235K; Replace L235R; Replace L235S; Replace L235T; Replace L235Q; Replace L237A; Replace S239D; Replace E233P; Replace L234V; C236 is missing; Replace G236E; Replace G236R; Replace G236K; Replace G237A; Replace P238A; Replace F243L; Replace D265A; Replace S267E; Replace H268A; Replace R292P; Replace Y300L; Replace K322A; Replace K322Q; Replace A327Q; Replace L328F; Replace L328R; Replace P329A; Replace P329G; Replace A330L; Replace A330S; Replace P331S; Replace I332E or P396L.

In a preferred embodiment, an L234A or L235A substitution is introduced into the human IgG constant Fc region of the antibody-based molecule described herein. In a more preferred embodiment, L234A and L235A substitutions are introduced into the human IgG constant Fc region of the antibody-based molecule described herein. This embodiment produces an antibody-based molecule having the heavy chain represented by SEQ ID NO:268 or SEQ ID NO:270.

In one implementation, the antibody or antigen-binding fragment comprises:

a) A heavy chain variable domain (VH) containing an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and

b) A light chain variable domain (VL) containing an amino acid sequence that has at least 80% identity or similarity to SEQ ID NO:235.

In one implementation, the antibody or antigen-binding fragment comprises a heavy chain variable domain (VH) and a light chain variable domain (VL):

The VH therein contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The VL contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain variable domain (VH) and a light chain variable domain (VL):

- wherein the VH comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234, and the VL comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235, and

- wherein the VH contains:

○ Contains the CDR-H1 amino acid sequence of SEQ ID NO:147 or having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

○ Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

○ Contains a CDR-H3 amino acid sequence (3B2g2m1) having 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:156, and - wherein the VL contains:

○ Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

○ Contains the CDR-L2 amino acid sequence of SEQ ID NO:172 or having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

○ Contains SEQ ID NO:195 or a CDR-L3 amino acid sequence (3B2g2m1) with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one implementation, the antibody or antigen-binding fragment comprises:

a) A heavy chain variable domain (VH) comprising an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234, and

b) A light chain variable domain (VL) containing an amino acid sequence that has at least 80% identity or similarity to SEQ ID NO:235.

In one implementation, the antibody or antigen-binding fragment comprises:

a) A full-length heavy chain, comprising SEQ ID NO:268, and

b) A full-length light chain containing SEQ ID NO:269, and

c) The full-length heavy chain contains L234A and L235A mutations numbered according to the EU numbering system.

Surprisingly, attractive results were obtained when anti-MuSK antibodies (expression vectors or polynucleotides or antigen-binding fragments of host cells) were administered before the onset of the disease. In one embodiment, the subject therefore did not have symptoms of the neuromuscular disease (e.g., ALS). In such an embodiment, the subject has been diagnosed as predisposed to a neuromuscular disease (e.g., ALS) based on his/her family history, genetic background, or based on elevated levels of neurofilaments (e.g., neurofilament light chains (NFL)) identified in his/her serum or cerebrospinal fluid (CSF), or based on a positive gene test for an ALS-related gene mutation, or based on changes in the levels of ALS biomarkers, or a combination thereof. However, he/she has not yet developed any obvious symptoms; he/she is asymptomatic. In a preferred embodiment, the antibody is administered to the subject as early as possible after diagnosis of a genetic defect or ALS-related genetic mutation but before any visible symptoms have occurred (i.e., an asymptomatic subject). In a more preferred embodiment, the treatment is administered to the subject as early as possible after a diagnosis of a genetic defect or ALS-related genetic mutation has been made but no visible symptoms have yet occurred (and the subject has a family history of ALS). In this context, "immediately" may mean within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or within 1, 2, 3, 4, 5, 6, or 7 days, or within 1, 2, 3, or 4 weeks. In this context, such treatment ultimately demonstrates limited or no toxicity and/or side effects, or any harmful effects, in the absence of a confirmed ALS diagnosis.

In another aspect of the invention, a combination is provided comprising an anti-MuSK antibody or its antigen-binding fragment as described herein and an anticholinergic compound. This combination is preferably used for the treatment of neuromuscular diseases in human subjects.

The neuromuscular disease is characterized by impaired neuromuscular transmission and/or denervation at the NMJ (neuromuscular junction). Neuromuscular disease is characterized by at least one of the following:

a. Excessive stimulation by muscarinic substances,

b. Motor neuron death,

c. Neuromuscular junction (NMJ) denervation and

d. Impaired synaptic transmission.

In one embodiment, the neuromuscular disease is selected from the following: amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), myasthenia gravis (MG), congenital myasthenia gravis, Lambert-Eaton myasthenic syndrome (LEMS), Lyme disease, poliomyelitis, post-poliomyelitis, heavy metal poisoning, Kennedy syndrome, adult-onset Ty Sachs disease, hereditary spastic paraplegia, multifocal neuropathy, cervical spondylosis, extramedullary tumors with compressive radiculopathy and myelopathy, inclusion body myositis, progressive bulbar palsy, progressive muscular atrophy, motor neuron syndrome, and thyrotoxic myopathy. In a preferred embodiment, the neuromuscular disease is ALS.

In this context, the combination does not require the anti-MuSK antibody or its antigen-binding fragment and the anticholinergic compound described herein to be physically present together in a composition.

In one embodiment, the anticholinergic compound is administered alone, sequentially, or simultaneously. In one embodiment, the antibody or antigen-binding fragment is administered before the onset of the disease, or within 1, 2, 3, 4, 5, or 6 months before the onset of the disease. In one embodiment, the antibody or antigen-binding fragment is administered before the onset of the disease, or preferably within 1, 2, 3, 4, 5, or 6 months before the onset of the disease, and/or wherein the anticholinergic compound is administered at the onset of the disease or within 1, 2, 3, 4, 5, 6, or 7 weeks after the onset of the disease. In this context, a subject prior to the onset of the disease may mean a subject without symptoms of the neuromuscular condition. In one embodiment, the subject treated with the antibody has been initially diagnosed as susceptible to neuromuscular conditions or disease.

In one embodiment, an anti-MuSK antibody or its antigen-binding fragment binds to the MuSK coil (Fz)-like domain sequence of SEQ ID NO:129.

In one embodiment, the antibody or its antigen-binding fragment comprises a constant Fc region of wild-type human IgG, which contains at least 80% sequence identity with SEQ ID NO:266 or SEQ ID NO:267.

In one implementation, the antibody or antigen-binding fragment is an agonist MuSK antibody and/or the effector function has been reduced or eliminated.

In one implementation, the reduction or elimination of effector function is achieved by introducing one or more of the following mutations (all numbered according to the EU numbering system) into the constant Fc region of the human IgG of the antibody-based molecule described herein, SEQ ID NO:266 or SEQ ID NO:267: N297A substitution; N297Q substitution; L234A substitution; L234D substitution; L234E substitution; L234G substitution; L234H substitution; L234F substitution; L234K substitution; L234Q substitution; L234R substitution; L234S substitution; L234T substitution; L235A substitution; L235D substitution; L235E substitution; L235F substitution; L235G substitution; L235V substitution; L235H substitution; L23 Replace 5I; Replace L235K; Replace L235R; Replace L235S; Replace L235T; Replace L235Q; Replace L237A; Replace S239D; Replace E233P; Replace L234V; C236 is missing; Replace G236E; Replace G236R; Replace G236K; Replace G237A; Replace P238A; Replace F243L; Replace D265A; Replace S267E; Replace H268A; Replace R292P; Replace Y300L; Replace K322A; Replace K322Q; Replace A327Q; Replace L328F; Replace L328R; Replace P329A; Replace P329G; Replace A330L; Replace A330S; Replace P331S; Replace I332E or P396L.

In a preferred embodiment, an L234A or L235A substitution is introduced into the human IgG constant Fc region of the antibody-based molecule described herein. In a more preferred embodiment, L234A and L235A substitutions are introduced into the human IgG constant Fc region of the antibody-based molecule described herein. This embodiment produces an antibody-based molecule having the heavy chain represented by SEQ ID NO:268 or SEQ ID NO:270.

In one implementation, the antibody or antigen-binding fragment comprises:

a) A heavy chain variable domain (VH) comprising an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234, and

b) A light chain variable domain (VL) containing an amino acid sequence that has at least 80% identity or similarity to SEQ ID NO:235.

In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain variable domain (VH) and a light chain variable domain (VL):

The VH therein contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The VL mentioned therein includes:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain variable domain (VH) and a light chain variable domain (VL):

- wherein the VH comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234, and the VL comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235, and

- wherein the VH contains:

○ Contains the CDR-H1 amino acid sequence of SEQ ID NO:147 or having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

○ Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

○ Contains SEQ ID NO:156 or a CDR-H3 amino acid sequence (3B2g2m1) having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and - wherein the VL comprises:

○ Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

○ Contains the CDR-L2 amino acid sequence of SEQ ID NO:172 or having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

○ Contains SEQ ID NO:195 or a CDR-L3 amino acid sequence (3B2g2m1) with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one implementation, the antibody or antigen-binding fragment comprises:

a) A full-length heavy chain, comprising SEQ ID NO:268, and

b) A full-length light chain containing SEQ ID NO:269, and

c) The full-length heavy chain contains L234A and L235A mutations numbered according to the EU numbering system.

In one implementation, the antibody or antigen-binding fragment comprises:

a) A full-length heavy chain containing SEQ ID NO:270, and

b) A full-length light chain containing SEQ ID NO:271, and

c) The full-length heavy chain contains L234A and L235A mutations numbered according to the EU numbering system.

In one embodiment, the anti-MuSK antibody or its antigen fragment (polynucleotide, expression vector, host cell, cell-free expression system, or composition) is administered before the onset of the disease (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days before the onset of the disease, or 1, 2, 3, 4, 5, 6, or 7 weeks before the onset of the disease), and the anticholinergic compound is administered at the onset of the disease (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the onset of the disease, or 1, 2, 3, 4, 5, 6, or 7 weeks after the onset of the disease). In this context, a subject prior to the onset of the disease may mean a subject without symptoms of the neuromuscular disorder (e.g., ALS).

In a preferred embodiment, a disease episode includes at least one symptom selected from the following: muscle twitching, muscle spasms, cramps, muscle weakness, slurred speech and/or nasal speech, difficulty chewing or swallowing, dysphagia, dysarthria, and dyspnea. In a more preferred embodiment, the disease is ALS and a disease episode includes at least one symptom selected from the following: muscle twitching, muscle spasms, cramps, muscle weakness, slurred speech and/or nasal speech, difficulty chewing or swallowing, dysphagia, dysarthria, and dyspnea.

The onset of illness can be assessed by a physician or veterinarian. In one implementation, the onset of weight loss is considered the onset of illness.

In a preferred embodiment, the neuromuscular disease is ALS and the anti-MuSK antibody or antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235.

The heavy-chain variable structural domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a more preferred embodiment, the neuromuscular disease is ALS and the anti-MuSK antibody or antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235, and

The heavy-chain variable structural domain contains:

- Contains the CDR-H1 amino acid sequence consisting of SEQ ID NO:147 or SEQ ID NO:147.

- Contains the CDR-H2 amino acid sequence consisting of SEQ ID NO:153 or SEQ ID NO:153, and

- Contains the CDR-H3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:156 or SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence consisting of SEQ ID NO:159 or SEQ ID NO:159.

- Contains the CDR-L2 amino acid sequence consisting of SEQ ID NO:172 or SEQ ID NO:172, and

- Contains the CDR-L3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:195 or SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a preferred embodiment, the neuromuscular disease is ALS and the anti-MuSK antibody or antigen-binding fragment comprises a constant Fc region of wild-type human IgG, a heavy chain variable domain, and a light chain variable domain, wherein the constant Fc region of wild-type human IgG comprises SEQ ID NO:266 or SEQ ID NO:267, a heavy chain variable domain, and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235, and

The heavy-chain variable structural domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a more preferred embodiment, the neuromuscular disease is ALS and the anti-MuSK antibody or antigen-binding fragment comprises a wild-type human IgG constant Fc region, a heavy chain variable domain, and a light chain variable domain, wherein the wild-type human IgG constant Fc region comprises SEQ ID NO:266 or SEQ ID NO:267, a heavy chain variable domain, and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235, and

The heavy-chain variable structural domain contains:

- Contains the CDR-H1 amino acid sequence consisting of SEQ ID NO:147 or SEQ ID NO:147.

- Contains the CDR-H2 amino acid sequence consisting of SEQ ID NO:153 or SEQ ID NO:153, and

- Contains the CDR-H3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:156 or SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence consisting of SEQ ID NO:159 or SEQ ID NO:159.

- Contains the CDR-L2 amino acid sequence consisting of SEQ ID NO:172 or SEQ ID NO:172, and

- Contains the CDR-L3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:195 or SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a preferred embodiment, the neuromuscular disease is ALS and the anti-MuSK antibody or antigen-binding fragment comprises a constant Fc region of wild-type human IgG (in which L234A and/or L235A substitutions according to the EU numbering system are introduced into the Fc region), a heavy chain variable domain, and a light chain variable domain, wherein the wild-type human IgG constant Fc region comprises SEQ ID NO:266 or SEQ ID NO:267, a heavy chain variable domain, and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235, and

The heavy-chain variable structural domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a more preferred embodiment, the neuromuscular disease is ALS and the anti-MuSK antibody or antigen-binding fragment comprises a constant Fc region of wild-type human IgG (in which L234A and/or L235A substitutions according to the EU numbering system are introduced into the Fc region), a heavy chain variable domain, and a light chain variable domain, wherein the constant Fc region of wild-type human IgG comprises SEQ ID NO:266 or SEQ ID NO:267, a heavy chain variable domain, and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235, and

The heavy-chain variable structural domain contains:

- Contains the CDR-H1 amino acid sequence consisting of SEQ ID NO:147 or SEQ ID NO:147.

- Contains the CDR-H2 amino acid sequence consisting of SEQ ID NO:153 or SEQ ID NO:153, and

- Contains the CDR-H3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:156 or SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence consisting of SEQ ID NO:159 or SEQ ID NO:159.

- Contains the CDR-L2 amino acid sequence consisting of SEQ ID NO:172 or SEQ ID NO:172, and

- Contains the CDR-L3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:195 or SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one implementation, the neuromuscular disease is ALS and the anti-MuSK antibody or antigen-binding fragment contains:

a) A full-length heavy chain comprising an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:268, and

b) A full-length light chain comprising an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:269, and

c) One or more of the following mutations (all numbered according to the EU numbering system) have been introduced into the full-length heavy chain: N297A substitution; N297Q substitution; L234A substitution; L234D substitution; L234E substitution; L234G substitution; L234H substitution; L234F substitution; L234K substitution; L234Q substitution; L234R substitution; L234S substitution; L234T substitution; L235A substitution; L235D substitution; L235E substitution; L235F substitution; L235G substitution; L235V substitution; L235H substitution; L235I substitution; L235K substitution; L235R substitution; L235S substitution; L235T substitution; L235Q substitution; L237A substitution; S239D substitution; E233P substitution. Replacement; L234V replacement; C236 deletion; G236E replacement; G236R replacement; G236K replacement; G237A replacement; P238A replacement; F243L replacement; D265A replacement; S267E replacement; H268A replacement; R292P replacement; Y300L replacement; K322A replacement; K322Q replacement; A327Q replacement; L328F replacement; L328R replacement; P329A replacement; P329G replacement; A330L replacement; A330S replacement; P331S replacement; I332E replacement; P396L replacement; or each of the combinations of mutations described above in the fourth embodiment of this application, preferably the mutation is L234A or L235A, more preferably the mutations are L234A and L235A.

In one implementation, the neuromuscular disease is ALS and the anti-MuSK antibody or antigen-binding fragment contains:

a) A full-length heavy chain, comprising SEQ ID NO:268, and

b) A full-length light chain containing SEQ ID NO:269, and

c) The full-length heavy chain contains L234A and L235A mutations numbered according to the EU numbering system.

In one implementation, the neuromuscular disease is ALS and the anti-MuSK antibody or antigen-binding fragment contains:

a) A full-length heavy chain comprising an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:270, and

b) A full-length light chain comprising an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:271, and

c) One or more of the following mutations (all numbered according to the EU numbering system) have been introduced into the full-length heavy chain: N297A substitution; N297Q substitution; L234A substitution; L234D substitution; L234E substitution; L234G substitution; L234H substitution; L234F substitution; L234K substitution; L234Q substitution; L234R substitution; L234S substitution; L234T substitution; L235A substitution; L235D substitution; L235E substitution; L235F substitution; L235G substitution; L235V substitution; L235H substitution; L235I substitution; L235K substitution; L235R substitution; L235S substitution; L235T substitution; L235Q substitution; L237A substitution; S239D substitution; E233P substitution. Replacement; L234V replacement; C236 deletion; G236E replacement; G236R replacement; G236K replacement; G237A replacement; P238A replacement; F243L replacement; D265A replacement; S267E replacement; H268A replacement; R292P replacement; Y300L replacement; K322A replacement; K322Q replacement; A327Q replacement; L328F replacement; L328R replacement; P329A replacement; P329G replacement; A330L replacement; A330S replacement; P331S replacement; I332E replacement; P396L replacement; or each of the combinations of mutations described above in the fourth embodiment of this application, preferably the mutation is L234A or L235A, more preferably the mutations are L234A and L235A.

In one implementation, the neuromuscular disease is ALS and the anti-MuSK antibody or antigen-binding fragment contains:

a) A full-length heavy chain containing SEQ ID NO:270, and

b) A full-length light chain containing SEQ ID NO:271, and

c) The full-length heavy chain contains L234A and L235A mutations numbered according to the EU numbering system.

In some implementations, the anticholinergic compound is a muscarinic receptor antagonist. Muscarinic receptors, also known as muscarinic acetylcholine receptors or mAchRs, are acetylcholine receptors that form G protein receptor complexes in the cell membranes of certain neurons and other cells. Muscarinic receptors play several roles in mediating the action of the neurotransmitter acetylcholine. For example, muscarinic receptors are contained in the presynaptic membrane of somatic neurons at the neuromuscular junction, where they are involved in the regulation of acetylcholine release.

The five subtypes of muscarinic receptors, M1 through M5, are widely accepted. This classification stems from their different selectivity for certain agonists and antagonists. M1, M3, and M5 receptors are coupled to the Gq protein in the cell membrane, while M2 and M4 receptors are coupled to the Gi/o protein in the cell membrane. Unbound by this theory, the genes CHRM1-5 encode receptors M1 through M5, respectively.

The basic or constitutive activity of muscarinic receptors is defined as the physical, biological, and/or chemical activity of the receptor in the absence of acetylcholine, muscarinic receptor agonists, and muscarinic receptor antagonists.

A muscarinic receptor agonist, also known as a muscarinic receptor agonist, is defined as a compound that enhances the physical, biological, and/or chemical activity of a receptor when it comes into contact with the receptor. Enhanced activity refers to activity similar to that induced by contacting the receptor with acetylcholine.

An antagonist of a muscarinic receptor, also known as a muscarinic receptor antagonist, is defined as a neutral antagonist or a negative antagonist of a muscarinic receptor.

Muscarinic receptor neutral antagonists are compounds that compete with muscarinic receptor neutral agonists or muscarinic receptor negative antagonists for binding to the receptor, thereby blocking the action of the agonist or negative antagonist (i.e. increasing or decreasing activity), while the neutral antagonist alone does not significantly alter the receptor's basal activity.

In some implementations, the anticholinergic compound is a neutral antagonist of muscarinic receptors.

Muscarinic receptor negative antagonists are compounds that, when contacted with a receptor, reduce the physical, biological, and/or chemical activity of the receptor, even in the absence of a muscarinic receptor agonist. Reduced activity refers to activity opposite to that induced by contacting the receptor with acetylcholine.

In some implementations, the anticholinergic compound is a negative antagonist of muscarinic receptors.

A muscarinic receptor antagonist (blocking agonists, blocking negative antagonists, or reducing agents) is defined as selective for one or more muscarinic receptor subtypes M1, M2, M3, M4, and/or M5 if its effect is significant only upon contact with one or more subtypes of muscarinic receptors, and significantly less or no upon contact with another subtype. Therefore, the term "muscarinic receptor antagonist selective for muscarinic receptor M3" should be understood to mean that when the antagonist is contacted with muscarinic receptors of subtypes M1, M2, M4, or M5, a significantly lesser or no effect is obtained. In this context, "significantly smaller" can be at most 1/10, 1/20, 1/30, 1/40, 1/50, 1/60, 1/70, 1/80, 1/90, 1/100, 1/200, 1/300, 1/400, 1/500, 1/600, 1/700, 1/800, 1/900, 1/1000, 1/10000, 1/100000, or 1/1000000, or at least smaller than 1/1000000.

10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 200%, 220%, 240%, 260%, 280%, 300%, 320%, 340%, 360%, 380%, 400%, 420%, 440%, 460%, 480%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 2500% 3000%, 3500%, 4000%, 4500%, 5000%, 5500%, 6000%, 6500%, 7000%, 7500%, 8000%, 8500%, 9000%, 9500%, 10000%, 20000%, 30000%, 40000%, 50000%, 60000%, 70000%, 80000%, 90000%, 100000%, 1000000%, 10000000%, or 100000000%.

The activity of muscarinic receptors (preferably M1, M3 and M5 subtypes) can be measured using dynamic Ca2+ imaging. These receptors regulate the level of IP3, which in turn controls the release of Ca2+ from internal storage[7].

In some implementations, the anticholinergic compound is a muscarinic receptor antagonist, which:

- It is selective for muscarinic receptor M1, or

- It is selective for muscarinic receptor M3, or

- It is selective for muscarinic receptor M5, or

- It is selective for muscarinic receptors M1 and M3, or

- It is selective for muscarinic receptors M1 and M5, or

- It is selective for muscarinic receptors M3 and M5, or

- It is selective for muscarinic receptors M1, M3, and M5.

In some implementations, the anticholinergic compound is a muscarinic receptor antagonist, which:

- It is selective for muscarinic receptor M3, or

- It is selective for muscarinic receptors M1 and M3, or

- It is selective for muscarinic receptors M3 and M5, or

- It is selective for muscarinic receptors M1, M3, and M5.

In some embodiments, the anticholinergic compound is darifenacin, ipratropium bromide, tiotropium bromide, trospium, glycopyrronium, acridineium, umeclidinium, solifenacin, dicylomine, fesoterodine, fiavoxate, glycopyrrolate, propantheline, 1R,2R,4S,5S,7S)-7-[({4-fluoro-2-(thiophen-2-yl)phenyl}amino] [38]-9,9-dimethyl-3-oxa-9-azatricyclo[3.3.1.02,4]nonane-9-carboxylic acid (BS46 in [38]), N-(2-[3-([3R]-1-(cyclohexylmethyl)-3-piperidinyl]methylamino)-3-oxopropyl]amino-2-oxoethyl)-3,3,3-triphenyl-propionamide (J-115311 in [39]), in the triphenylpropionic acid moiety and piperin 3,3,3-triphenylpropionamide derivatives ([40]) with one or two amino acid residues between the pyridylmethylamine moieties, OrM3 ([41]) or (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino]carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-nitrocationic bicyclo[2.2.2]octane bromide (CHF 5407 in [41]). Unbound by this theory, these compounds can be considered muscarinic receptor antagonists.

In some implementations, the anticholinergic compounds are daffodil, ipratropium bromide, tiotropium bromide, troxodium chloride, glycopyrronium bromide, adecyl bromide, ummelium bromide, sofinacin, bicyclic amine, forsterodine, flavoxate, ganrobromide, or propantheline bromide. Not bound by this theory, daffodil, ipratropium bromide, and tiotropium bromide can be considered muscarinic receptor antagonists.

In some implementations, the anticholinergic compound is dafinacin, ipratropium bromide, tiotropium bromide, or trox chloride. Unbound from this theory, dafinacin, ipratropium bromide, and tiotropium bromide can be considered muscarinic receptor antagonists.

In a preferred embodiment, the anticholinergic compound is dafinarax. Dafinarax can be represented by the following structure:

Preferably, dafinacin is dafinacin hydrobromide. Dafinacin hydrobromide can be represented by the following structure:

In some embodiments, any of the anticholinergic compounds disclosed in the above embodiments may be present as their pharmaceutically acceptable salts. In particular, the anticholinergic compound is dafinaxine or its pharmaceutically acceptable salt.

Examples of pharmaceutically acceptable salts include, but are not limited to, alkali metal (e.g., sodium, potassium, or lithium) salts or alkaline earth metal (e.g., calcium) salts; however, any salt that is generally nontoxic and effective when applied to the object being treated is acceptable. Other salts may include, but are not limited to: (1) acid addition salts, which can be obtained by reacting the free base of the parent compound with an inorganic acid (e.g., hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid) or an organic acid (e.g., acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, carbamate sulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid, or malonic acid); or (2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion (e.g., an alkali metal ion, an alkaline earth metal ion, or an aluminum ion), or when coordinated with an organic base (e.g., ethanolamine, diethanolamine, triethanolamine, trimethylamine, N-methylglucosamine, etc.). Medicinal salts are well known to those skilled in the art, and any such medicinal salt can be considered for combination with the embodiments described herein.

Acceptable salts can be obtained using standard practices known in the art, including (but not limited to) reacting a sufficiently acidic compound with a suitable base that provides a physiologically acceptable anion. Suitable acid addition salts are formed from acids that form nontoxic salts. Illustrative but non-limiting examples include acetates, aspartates, benzoates, benzenesulfonates, bicarbonates/carbonates, bisulfates/sulfates, borates, camphorsulfonates, citrates, ethanedisulfonates, ethanesulfonates, formates, fumarates, glucohepanoates, glucuronates, hexafluorophosphates, hibenzates, hydrochlorides/chlorides, hydrobromates/bromines, hydroiodates/iodides, lactates, malates, maleates, malonates, methanesulfonates, methyl sulfates, naphthates, 2-naphthalenesulfonates, nicotinates, nitrates, orotates, oxalates, palmitates, pyrates, phosphates/hydrogen phosphates/dihydrogen phosphates, sucrose salts, stearates, succinates, tartrates, toluenesulfonates, and trifluoroacetates. Suitable base salts of the compounds described herein are formed from bases that form non-toxic salts. Illustrative but non-limiting examples include arginine, benzathine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts. Hemisalts that form both acids and bases, such as hemisulfates and hemicalcium salts, can also be formed.

The anticholinergic compounds disclosed in the above embodiments can be administered as a composition, preferably as a therapeutic composition. In some embodiments, the composition is formulated as a once-daily extended-release tablet for oral administration, said extended-release tablet comprising davidin, preferably davidin hydrobromide. Preferably, the composition comprises one or more of the following excipients: anhydrous calcium hydrogen phosphate, hydroxypropyl methylcellulose, magnesium stearate, titanium dioxide, iron oxide yellow, iron oxide red, PEG 400 and/or talc. In one embodiment, the composition is referred to as ENABLEX ^™ . ENABLEX ^™ is formulated as 7.5 mg or 15 mg davidin (davidin hydrobromide).

In a preferred embodiment, the neuromuscular disease is ALS, and the anti-MuSK antibody or antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235.

The heavy-chain variable structural domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

It also used anticholinergic compounds.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a more preferred embodiment, the neuromuscular disease is ALS, and the anti-MuSK antibody or antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235.

The heavy-chain variable structural domain contains:

- Contains the CDR-H1 amino acid sequence consisting of SEQ ID NO:147 or SEQ ID NO:147.

- Contains the CDR-H2 amino acid sequence consisting of SEQ ID NO:153 or SEQ ID NO:153, and

- Contains the CDR-H3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:156 or SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence consisting of SEQ ID NO:159 or SEQ ID NO:159.

- Contains the CDR-L2 amino acid sequence consisting of SEQ ID NO:172 or SEQ ID NO:172, and

- Contains the CDR-L3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:195 or SEQ ID NO:195.

It also used anticholinergic compounds.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a preferred embodiment, the neuromuscular disease is ALS and the anti-MuSK antibody or antigen-binding fragment comprises a constant Fc region of wild-type human IgG, a heavy chain variable domain, and a light chain variable domain, wherein the constant Fc region of wild-type human IgG comprises SEQ ID NO:266 or SEQ ID NO:267, a heavy chain variable domain, and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235, and

The heavy-chain variable structural domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In a more preferred embodiment, the neuromuscular disease is ALS and the anti-MuSK antibody or antigen-binding fragment comprises a wild-type human IgG constant Fc region, a heavy chain variable domain, and a light chain variable domain, wherein the wild-type human IgG constant Fc region comprises SEQ ID NO:266 or SEQ ID NO:267, a heavy chain variable domain, and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235, and

The heavy-chain variable structural domain contains:

- Contains the CDR-H1 amino acid sequence consisting of SEQ ID NO:147 or SEQ ID NO:147.

- Contains the CDR-H2 amino acid sequence consisting of SEQ ID NO:153 or SEQ ID NO:153, and

- Contains the CDR-H3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:156 or SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence consisting of SEQ ID NO:159 or SEQ ID NO:159.

- Contains the CDR-L2 amino acid sequence consisting of SEQ ID NO:172 or SEQ ID NO:172, and

- Contains the CDR-L3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:195 or SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a preferred embodiment, the neuromuscular disease is ALS and the anti-MuSK antibody or antigen-binding fragment comprises a constant Fc region of wild-type human IgG (in which L234A and/or L235A substitutions according to the EU numbering system are introduced into the Fc region), a heavy chain variable domain, and a light chain variable domain, wherein the wild-type human IgG constant Fc region comprises SEQ ID NO:266 or SEQ ID NO:267, a heavy chain variable domain, and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235, and

The heavy-chain variable structural domain contains:

- Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147.

- Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and

- Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159.

- Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and

- Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195.

In a more preferred embodiment, the neuromuscular disease is ALS and the anti-MuSK antibody or antigen-binding fragment comprises a constant Fc region of wild-type human IgG (in which L234A and/or L235A substitutions according to the EU numbering system are introduced into the Fc region), a heavy chain variable domain, and a light chain variable domain, wherein the constant Fc region of wild-type human IgG comprises SEQ ID NO:266 or SEQ ID NO:267, a heavy chain variable domain, and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234 and the light chain variable domain comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235, and

The heavy-chain variable structural domain contains:

- Contains the CDR-H1 amino acid sequence consisting of SEQ ID NO:147 or SEQ ID NO:147.

- Contains the CDR-H2 amino acid sequence consisting of SEQ ID NO:153 or SEQ ID NO:153, and

- Contains the CDR-H3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:156 or SEQ ID NO:156 and

The variable structural domain of the light chain contains:

- Contains a CDR-L1 amino acid sequence consisting of SEQ ID NO:159 or SEQ ID NO:159.

- Contains the CDR-L2 amino acid sequence consisting of SEQ ID NO:172 or SEQ ID NO:172, and

- Contains the CDR-L3 amino acid sequence (3B2g2m1) consisting of SEQ ID NO:195 or SEQ ID NO:195.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one implementation, the neuromuscular disease is ALS and the anti-MuSK antibody or antigen-binding fragment contains:

a) A full-length heavy chain comprising an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:268, and

b) A full-length light chain comprising an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:269, and

c) One or more of the following mutations (all numbered according to the EU numbering system) have been introduced into the full-length heavy chain: N297A substitution; N297Q substitution; L234A substitution; L234D substitution; L234E substitution; L234G substitution; L234H substitution; L234F substitution; L234K substitution; L234Q substitution; L234R substitution; L234S substitution; L234T substitution; L235A substitution; L235D substitution; L235E substitution; L235F substitution; L235G substitution; L235V substitution; L235H substitution; L235I substitution; L235K substitution; L235R substitution; L235S substitution; L235T substitution; L235Q substitution; L237A substitution; S239D substitution; E233P substitution. Replacement; L234V replacement; C236 deletion; G236E replacement; G236R replacement; G236K replacement; G237A replacement; P238A replacement; F243L replacement; D265A replacement; S267E replacement; H268A replacement; R292P replacement; Y300L replacement; K322A replacement; K322Q replacement; A327Q replacement; L328F replacement; L328R replacement; P329A replacement; P329G replacement; A330L replacement; A330S replacement; P331S replacement; I332E replacement; P396L replacement; or each of the combinations of mutations described above in the fourth embodiment of this application, preferably the mutation is L234A or L235A, more preferably the mutations are L234A and L235A.

In one implementation, the neuromuscular disease is ALS and the anti-MuSK antibody or antigen-binding fragment contains:

a) A full-length heavy chain, comprising SEQ ID NO:268, and

b) A full-length light chain containing SEQ ID NO:269, and

c) The full-length heavy chain contains L234A and L235A mutations numbered according to the EU numbering system.

In one implementation, the neuromuscular disease is ALS and the anti-MuSK antibody or antigen-binding fragment contains:

a) A full-length heavy chain comprising an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:270, and

b) A full-length light chain comprising an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:271, and

c) One or more of the following mutations (all numbered according to the EU numbering system) have been introduced into the full-length heavy chain: N297A substitution; N297Q substitution; L234A substitution; L234D substitution; L234E substitution; L234G substitution; L234H substitution; L234F substitution; L234K substitution; L234Q substitution; L234R substitution; L234S substitution; L234T substitution; L235A substitution; L235D substitution; L235E substitution; L235F substitution; L235G substitution; L235V substitution; L235H substitution; L235I substitution; L235K substitution; L235R substitution; L235S substitution; L235T substitution; L235Q substitution; L237A substitution; S239D substitution; E233P substitution. Replacement; L234V replacement; C236 deletion; G236E replacement; G236R replacement; G236K replacement; G237A replacement; P238A replacement; F243L replacement; D265A replacement; S267E replacement; H268A replacement; R292P replacement; Y300L replacement; K322A replacement; K322Q replacement; A327Q replacement; L328F replacement; L328R replacement; P329A replacement; P329G replacement; A330L replacement; A330S replacement; P331S replacement; I332E replacement; P396L replacement; or each of the combinations of mutations described above in the fourth embodiment of this application, preferably the mutation is L234A or L235A, more preferably the mutations are L234A and L235A.

In one implementation, the similarity or identity is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one implementation, the neuromuscular disease is ALS and the anti-MuSK antibody or antigen-binding fragment contains:

a) A full-length heavy chain containing SEQ ID NO:270, and

b) A full-length light chain containing SEQ ID NO:271, and

c) The full-length heavy chain contains L234A and L235A mutations numbered according to the EU numbering system.

In embodiments where a person or patient has been diagnosed with a neuromuscular disorder (such as one of those disclosed above), the MuSK antibody of the present invention, optionally in combination with an anticholinergic compound, is administered to such a patient in an amount sufficient to cure, treat, or at least partially suppress disease symptoms (e.g., as determined by biochemical, histological, and/or behavioral assessments) (including its complications and intermediate pathological phenotypes in disease development). In some embodiments, administration of the therapeutic molecules of the present invention alleviates or eliminates neuromuscular symptoms.

The effective dose of the therapeutic molecules (i.e., anti-MuSK antibodies or their antigen-binding fragments and anticholinergic compounds) provided by this invention for treating the aforementioned conditions can vary depending on many different factors, including the route of administration, target site, patient's physiological state, and other medications administered. The therapeutic dose is typically titrated to optimize its safety and efficacy. On any given date of dose administration, the dose of the MuSK antibody-based molecules described herein can be from about 0.0001 mg/kg to about 100 mg/kg of patient body weight, and more typically from about 0.01 mg/kg to about 20 mg/kg of patient body weight. For example, the dose can be 1 mg/kg of body weight or 10 mg/kg of body weight, or in the range of 1 mg/kg to 10 mg/kg of body weight. Therefore, exemplary dosages include: about 0.1 mg/kg to about 10 mg/kg body weight, about 0.1 mg/kg to about 5 mg/kg body weight, about 0.1 mg/kg to about 2 mg/kg body weight, about 0.1 mg/kg to about 1 mg/kg body weight, such as about 0.15 mg/kg body weight, about 0.2 mg/kg body weight, about 0.5 mg/kg body weight, about 1 mg/kg body weight, about 1.5 mg/kg body weight, about 2 mg/kg body weight, about 5 mg/kg body weight, or about 10 mg/kg body weight.

Physicians or veterinarians with ordinary skills in the art can readily determine and prescribe an effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian can start with a dose of the antibody-based molecule in the pharmaceutical composition at a level below that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Generally, the appropriate daily dose of the composition of the present invention will be the amount of the lowest dose of compound that effectively produces a therapeutic effect. Such an effective dose will generally depend on the factors described above. Administration can be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, and, for example, near the target site. If desired, the effective daily dose of the pharmaceutical composition can be administered as two, three, four, five, six, or more subdoses at appropriate intervals throughout the day, optionally in unit dosage form. While the antibody-based molecule of the present invention can be administered alone, it is preferred to administer the antibody-based molecule as part of the pharmaceutical composition, as described above.

For therapeutic purposes, the MuSK antibody-based molecules of the present invention (and optionally anticholinergic compounds) are typically administered at multiple times. The intervals between single doses (e.g., bolus or infusion) can be weekly, monthly, or annually. In some embodiments, the MuSK antibody-based molecules of the present invention (and optionally anticholinergic compounds) are administered to human subjects at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times over a four-month period.

In some embodiments, one or more loading doses of the pharmaceutical composition are administered to a human subject, followed by one or more maintenance doses. In some cases, three loading doses are administered, with the loading doses spaced two weeks apart, for example, on day 1, day 15, and day 29. In some cases, maintenance doses are administered every four weeks starting four weeks after the third loading dose (e.g., for one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, or ten months).

In some embodiments, a human subject is given three loading doses of the pharmaceutical composition, followed by at least one maintenance dose (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). In some cases, the three loading doses are given two weeks apart. In some cases, the three loading doses are given 14 days apart. In some cases, one or more maintenance doses are given every four weeks, starting four weeks after the third loading dose. In some cases, one or more maintenance doses are given monthly, starting one month after the third loading dose. In some cases, one or more maintenance doses are given every 28 days, starting 28 days after the third loading dose.

In some methods, the dosage is adjusted to achieve a plasma concentration of 1 ng/ml to 1000 μg/mL, preferably 1 to 1000 μg/mL, and more preferably 25 to 300 μg/mL. Alternatively, the therapeutic molecule of the present invention can be administered as a slow-release formulation, in which case a lower frequency of administration is required. The dosage and frequency vary depending on the half-life of the antibody in the patient. Generally, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. scFv molecules typically have a short serum half-life.

In another embodiment, a pharmaceutical composition comprising a recombinant nucleic acid sequence encoding a molecule (and optionally in combination with an anticholinergic compound) as described herein is administered to a subject to promote in vivo expression and formation of the antibody-based molecule to treat conditions mediated by reduced signaling and/or phosphorylation of MuSK. Expression vector constructs suitable for this embodiment of the invention have been described above.

The polynucleotide composition enables the production of MuSK antibody-based molecules in a subject within at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 60 hours after administration of the composition. The composition also enables the production of antibody-based molecules in a subject within at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after administration of the composition. The composition enables the generation of antibody-based molecules in a subject within about 1 hour to about 6 days, about 1 hour to about 5 days, about 1 hour to about 4 days, about 1 hour to about 3 days, about 1 hour to about 2 days, about 1 hour to about 1 day, about 1 hour to about 72 hours, about 1 hour to about 60 hours, about 1 hour to about 48 hours, about 1 hour to about 36 hours, about 1 hour to about 24 hours, about 1 hour to about 12 hours, or about 1 hour to about 6 hours after application of the composition to the subject.

When applied to a subject in need, the composition enables the sustained production of antibody-based molecules in the subject. The composition can result in sustained production of antibody-based molecules in the subject for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 3 days. Antibody-based molecules are produced in 1 day, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, or 60 days.

As used herein, the term "treatment" and its variations mean to improve, slow down, or reverse the progression or severity of a disease or condition, or to improve, slow down, or reverse one or more symptoms or side effects of such a disease or condition. For the purposes of this invention, "treatment" or its variations also mean a method for obtaining a beneficial or desired clinical outcome, wherein a "beneficial or desired clinical outcome" includes, but is not limited to, partially or wholly, detectably or undetectably, relieving symptoms, reducing the severity of a condition or disease, stabilizing (i.e., not worsening) a disease or condition state, delaying or slowing the progression of a disease or condition state, improving or reducing a disease or condition state, and alleviating a disease or condition.

Therefore, in one embodiment, the anti-MuSK antibody or antigen-binding fragment according to the invention, or the composition according to the invention, is used to treat neuromuscular diseases in human subjects, wherein said treatment results in stabilization of the disease. Stabilization may last for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months, or at least 1 year, 2 years, or 3 years. Each therapeutic effect further characterized herein can be considered as stabilization of the disease.

In one embodiment, the use of an anti-MuSK antibody or antigen-binding fragment (or polynucleotide, expression vector, host cell, composition) demonstrates a therapeutic effect on the treated human subjects as defined herein.

Such therapeutic effects can be at least one of the following disclosed effects.

By binding to the MuSK epitope, the anti-MuSK antibody or antigen-binding fragment of the present invention can induce agonistic MuSK activity. In the context of this application, "inducing agonistic MuSK activity" can be replaced by "activating MuSK." Agonistic MuSK activity or MuSK activation can be triggered at the molecular and/or cellular level and/or in more biologically complex systems (such as NMJs, synapses, living organisms). In the context of this application, agonistic MuSK activity can be replaced by triggering a MuSK-induced signal or by inducing MuSK activation in muscle cells at the NMJ. MuSK-induced signals (or MuSK activation or MuSK activity) can be at least one of the following: induction of MuSK dimerization, induction of MuSK tyrosine phosphorylation, induction or enhancement of AChR accumulation at NMJs (or in vitro accumulation in myotubular AChR patches), enhancement of the number or percentage of fully innervated NMJs, reduction of the enhancement of the number or percentage of fully denervated NMJs, maintenance of the number or percentage of fully innervated NMJs (disease stabilization/disease progression stabilization), improvement of synaptic transmission reliability, improvement of motor performance, prevention/stabilization or even reduction/decrease of motor neuron death, and extended lifespan of the treated subject.

The MuSK-induced signal by the anti-MuSK antibody of the present invention can be the induction of MuSK dimerization, which can be assessed by Western blotting. In the context of the present invention, in experiments using the antibody of the present invention, the agonistic activity of MuSK can be assessed when the induction of MuSK dimerization is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more compared to the same experimental setup with or without any antibody or with a negative control antibody. Alternatively, in the context of the present invention, the agonistic activity of the MuSK antibody can be assessed when, in experiments using the antibody of the present invention, the induction of MuSK dimerization is the same or approximately the same (less than 20%, less than 10%, or the same, or more than 10%, or more than 20%) compared to the same experimental setup without a positive control antibody. Such MuSK dimerization can be assessed without agrin. The positive control in the assessment of MuSK dimerization is agrin.

The MuSK-induced signal by the anti-MuSK antibody of the present invention can be MuSK tyrosine phosphorylation and the induction of such phosphorylation, which can be assessed by Western blotting using an antibody specific for tyrosine phosphorylation. In the context of the present invention, in experiments using the antibody of the present invention, the agonistic activity of MuSK can be assessed when the induction of MuSK tyrosine phosphorylation is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, or more compared to the same experimental setting without any antibody. Alternatively, in the context of the present invention, in experiments using the antibody of the present invention, the agonistic activity of the MuSK antibody can be assessed when the induction of MuSK tyrosine phosphorylation is the same or approximately the same (20% lower, 10% lower, the same, or 10% higher, or 20% higher) compared to the same experimental setting without a positive control antibody. Such MuSK tyrosine phosphorylation can be assessed in the absence of synaptic proteoglycans. The positive control in the MuSK tyrosine phosphorylation assessment was synaptic glycan.

The MuSK-induced signal by the anti-MuSK antibody of the present invention can be the induction of acetylcholine receptor (AChR) aggregation at the NMJ, and such aggregation can be assessed by staining the AChR with an antibody that specifically binds to the AChR and visualizing such staining under a fluorescence microscope using techniques known to those skilled in the art. Alternatively, aggregation can be assessed in vitro in myotube AChR plaques. Preferred antibodies for visualizing AChR aggregation are antibodies specific to AChR. More preferred antibodies are AlexaFluor488-conjugated α-clade krait venom (B13422, ThermoFisher). Typically, the region to be analyzed is immobilized in paraformaldehyde and incubated at room temperature with the relevant antibody of the present invention or with a positive or negative control, and then each region is washed with PBS and observed under an epifluorescence microscope. In the context of this invention, in experiments using the antibody of this invention, the agonistic activity of the MuSK antibody can be assessed by comparing it with the same experimental setup without any antibody, when the induction of AChR aggregation at the NMJ is the same or approximately the same (i.e., 20% lower, 10% lower, the same, 10% higher, or 20% higher) or increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (i.e., significantly lower, lower, or the same, higher, or higher, or higher, or higher) or increased. Such AChR aggregation can be assessed in the absence of synaptic glycan. The positive control in the AChR aggregation assessment is synaptic glycan.

In a preferred embodiment, the anti-MuSK antibody of the present invention exhibits induction or enhancement of acetylcholine receptor aggregation at the NMJ, and such aggregation can be assessed by visualizing or enhancing AchR staining at the mouse diaphragm NMJ compared to staining obtained without the MuSK agonist antibody. In one embodiment, this induction or enhancement of AchR aggregation at the NMJ results in a more normal/physiological NMJ morphology that maintains synaptic innervation and/or presynaptic and postsynaptic alignment.

The MuSK-induced signal in myocytes at NMJs via the anti-MuSK antibody of the present invention can be an increase in the number or percentage of fully innervated NMJs, a decrease in the increase in the number or percentage of fully denervated NMJs, maintenance of the number or percentage of fully innervated NMJs (disease stabilization/disease progression stabilization), improved reliability of synaptic transmission, prevention/stabilization or even reduction/decrease of motor neuron death. Each of these characteristics can be assessed using techniques known to those skilled in the art, such as staining AchR with the α-cladosnipus venom antibody previously defined herein, presynaptic labeling and quantification of innervation by fluorescence confocal microscopy, EMG single-fiber EMG, electrophysiology of individual synapses, and staining of motor neuron cell bodies in bone marrow-specific regions. All of these assays have been described in Cantor et al. 2018 (Elife, 2018; 7:e34375).

Anti-MuSK antibodies or antigen-binding fragments can improve motor performance and/or grip strength in treated subjects. In experiments using the anti-MuSK antibodies of the present invention, motor performance and grip strength of the treated subject can be considered improved when such improvement is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the same experimental setting without any antibody. Motor performance (or grip strength) of the treated subject can be assessed using assays known to those skilled in the art. Some exemplary methods are disclosed in the experimental section.

Anti-MuSK antibodies or antigen-binding fragments can improve the contractile properties of muscles at the NMJ in treated subjects. In experiments using the anti-MuSK antibodies of the present invention, the contractile properties of the treated subject's muscles can be considered improved when, compared to the same experimental setup without any antibody, the contractile properties of such muscles are improved by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The contractile properties of the muscles (at the NMJ) in treated subjects can be evaluated using assays known to those skilled in the art. Some exemplary methods are disclosed in the experimental section. In this case, the subject can be an animal.

Anti-MuSK antibodies or antigen-binding fragments can improve the fatigue resistance of muscles at the NMJ in treated subjects. In experiments using the anti-MuSK antibodies of the present invention, the fatigue resistance of the treated subject's muscles can be considered improved when the fatigue properties of such muscles are improved by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the same experimental setup without any antibodies. The fatigue properties of the treated subject's (NMJ) muscles can be assessed using assays known to those skilled in the art. Some exemplary methods are disclosed in the experimental section. In this case, the subject can be an animal.

Anti-MuSK antibodies or antigen-binding fragments can induce an increase in muscle mass at the NMJ in treated subjects. In experiments using the anti-MuSK antibodies of the present invention, an improvement in muscle mass can be considered achieved when such an increase is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the same experimental setup without any antibody. Some exemplary methods are disclosed in the experimental section. In this case, the subjects can be animals.

The characteristic of the MuSK-induced signaling or effect of the anti-MuSK antibody of the present invention lies in the delayed occurrence of improvement in the quality of life or deterioration in the quality of life of the treated subject. Quality of life can be quantified by the subject's weight. The delay in the occurrence of improvement in quality of life or deterioration can be at least 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or longer. This is assessed by comparison with the expected quality of life (or expected deterioration) of a subject with the same condition who has not been treated with the antibody of the present invention. In this case, the subject can be an animal.

The characteristic of the MuSK-induced signaling or effect of the anti-MuSK antibody of the present invention lies in the lifespan of the treated subject. The extension can be at least 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, or longer. This is assessed by comparison with the expected lifespan of a subject suffering from the same condition who has not been treated with the antibody of the present invention. In this case, the subject can be an animal.

The properties of the anti-MuSK antibodies described herein can be measured according to the assays described herein. The activating activity of the MuSK agonist antibody can be measured relative to a control (e.g., a negative control antibody that does not bind to MuSK, such as an isotype control). A preferred control antibody that does not bind to MuSK is mavitizumab targeting RSV (Review, MAbs, 1(5), 439-442, Sept-Octo 2009, DOI: 10.4161/mabs.1.5.9496). A preferred positive control MuSK agonist antibody is mAb#13 from Genentech. Another preferred positive control molecule for demonstrating MuSK activation activity is synaptic glycan (rat synaptic glycan from the R&D system, 550-AG).

In another embodiment, an anti-MuSK antibody or its antigen-binding fragment (or polynucleotide, expression vector, host cell, composition) in combination with an anticholinergic compound previously defined herein exhibits therapeutic activity in the treated human subjects defined herein. In a preferred embodiment, the use of both compounds elicits additional and more preferably synergistic therapeutic effects compared to using either the anti-MuSK antibody or the antigen-binding fragment (or polynucleotide, expression vector, host cell, composition) as a standalone treatment.

Another therapeutic effect may be a reduction (“inhibition”) of muscarinic activity in perisynaptic Schwann cells (PSCs) and NMJ repair. Such an additional therapeutic effect could be a specific reduction (“inhibition”) of muscarinic activity in PSCs. Such an additional therapeutic effect could be a reduction (“inhibition”) of excessive excitation of PSCs in neuromuscular disorders. The compounds or combinations of the present invention specifically act on muscarinic receptors. The compounds or combinations of the present invention appear to have no effect on purified receptors expressed on PSCs.

NMJ repair can be achieved through the induction or enhancement of nerve sprouting and/or an increase in NMJ innervation. Each of these effects can be assessed using techniques known to the art. Furthermore, inhibition of the muscarinic activity of the PSC may help maintain NMJ innervation.

In the context of this invention, in experiments using anticholinergic compounds, the induction or enhancement of neurogenesis (or innervation of the NMJ) can be assessed when, compared to the same experimental setup without said compound, the induction of neurogenesis (or innervation of the NMJ) is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Immunohistochemistry of neuromuscular preparations can be used to assess neurogenesis or innervation. The experimental section discloses how to obtain such neuromuscular preparations.

In the context of this invention, in experiments using anticholinergic compounds, the reduction in muscarinic activity (or muscarinic hyperexcitability or overexcitability) of PSCs can be assessed when, compared to the same experimental setup without said compound, the reduction in muscarinic activity (or muscarinic hyperexcitability or overexcitability) of PSCs is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

Therefore, in one embodiment, the use of anti-MuSK antibodies or antigen-binding fragments (or polynucleotides, expression vectors, host cells, compositions), preferably in combination with anticholinergic compounds previously defined herein, exhibits one or more of the following therapeutic effects:

- An increase in the number or percentage of fully innervated NMJs in the subject; maintenance of the number or percentage of fully innervated NMJs in the subject; a decrease in the number or percentage of fully denervated NMJs in the subject; improved reliability of synaptic transmission; prevention, stabilization, or reduction of motor neuron death in the subject; and/or

- Improvement in the subject's motor performance and/or grip strength; and/or

- Improvement of the contractile properties of the muscle at the NMJ of the object; and/or

- Improvement of fatigue resistance in the muscles at the NMJ site of the object; and/or

- Inducing an increase in muscle mass at the NMJ of the subject; and/or

- The delay in the occurrence of improvement or deterioration in the quality of life of the subject; and/or

- Decreased muscarinic activity in perisynaptic Schwann cells (PSCs) of the subject (or decreased muscarinic hyperexcitability or overexcitability) or NMJ repair in the subject.

As demonstrated in the experimental section, a synergistic therapeutic effect was achieved when both compounds were used. These synergistic effects included improvements/enhancements in the following parameters/symptoms: motor function and grip strength, contractile properties of muscles at the NMJ, resistance to muscle fatigue, muscle mass, and effects on general life conditions (e.g., body weight).

The term "effective amount" for an antibody-based molecule refers to an amount sufficient, within the necessary dose and time period, to achieve the intended biological effect or desired therapeutic outcome (including, but not limited to, clinical outcomes). When applied to the antibody-based molecules of the present invention, the phrase "therapeutic effective amount" is intended to represent an amount of antibody sufficient to improve, alleviate, stabilize, reverse, slow, or delay the progression of a condition or disease state or the progression of its symptoms. In one embodiment, the method of the present invention provides the administration of an antibody-based molecule in combination with other compounds. In such a case, "effective amount" is the amount of the combination sufficient to cause the intended biological effect.

In another aspect, methods are provided for the prevention and/or treatment of neuromuscular diseases and/or disorders and/or conditions, comprising administering to a subject in need an anti-MuSK antibody or an antigen-binding fragment thereof (a polynucleotide, expression vector, host cell, or composition, as previously defined herein), and preferably an anticholinergic compound. All features of this method have been previously defined herein.

In another aspect, the use of anti-MuSK antibodies or antigen-binding fragments thereof (polynucleotides, expression vectors, host cells, or compositions, as previously defined herein) and preferably anticholinergic compounds is provided for the preparation of medicaments for the prevention and/or treatment of neuromuscular diseases and/or disorders and/or conditions. All characteristics of this use have been previously defined herein.

All documents referenced in this specification are incorporated herein by reference in their entirety. Unless otherwise defined, all terms used to disclose this invention (including technical and scientific terms) have the meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance, including terminology definitions, is provided to better understand the teachings of this invention. Unless otherwise indicated, each embodiment described herein may be combined with any other embodiment described herein.

The present invention is further described through the following embodiments, which should not be construed as limiting the scope of the invention.

References

1.L.R.Fischer et al., Amyotrophic lateral sclerosis is a distalaxonopathy: evidence in mice and man. Exp Neurol185, 232-240 (2004).

2.D.Frey et al., Early and selective loss of neuromuscular synapsesubtypes with low sprouting competence in motoneuron diseases.J Neurosci 20, 2534-2542(2000).

3. E. Martineau, A. Di Polo, C. Vande Velde, R. Robitaille, Dynamic neuromuscula r remodeling precedes motor-unit loss in a mouse model ofALS.Elife7, (2018).

4.S.Pun, A.F.Santos, S.Saxena, L.Xu, P.Caroni, Selective Vulnerability and pruning of phasic motoneuron axons in motoneuron disease alleviated by CNTF. Nat Neurosci 9, 408-419 (2006).

5. E. Tremblay, E. Martineau, R. Robitaille, Opposite Synaptic Alterations at the Neuromuscular Junction in an ALS Mouse Model: When Motor Units Matter. JNeurosci 37, 8901-8918 (2017).

6. C. Yang et al., Mutant PFN1 causes ALS phenotypes and progressive motor neuron degeneration in mice by a gain of toxicity. Proc Natl Acad Sci U SA 113, E6209-E6218 (2016).

7. D.Arbour, E.Tremblay, E.Martineau, J.P.Julien, R.Robitaille, Early and persistent abnormal decoding by glial cells at the neuromuscular junction inan ALS model.J Neurosci 35, 688-706 (2015).

8. D. Arbour, C. Vande Velde, R. Robitaille, New perspectives onamyotrophic lateral sclerosis: the role of glial cells at the neuromuscular junction. J Physiol 595, 647-661 (2017).

9. C.P.Ko, R.Robitaille, Perisynaptic Schwann Cells at the NeuromuscularSynapse: Adaptable, Multitasking Glial Cells.Cold Spring Harb Perspect Biol 7, a020503 (2015).

10. R.Robitaille, B.S.Jahromi, M.P.Charlton, Muscarinic Ca2+responsesresistant to muscarinic antagonists at perisynaptic Schwann cells of the frogneuromuscula r junction.J Physiol 504(Pt 2), 337-347(1997).

11.D.Rochon, L.Rousse, R.Robitaille, Synapse-glia interactions at themammalian neuromuscular junction.J Neurosci 21, 3819-3829(2001).

12. M.C.Wright et al., Distinct musca rinic acetylcholine receptorsubtypes contribute to stability and growth, but not compensatory plasticity, of neuromuscular synap ses.J Neurosci 29, 14942-14955(2009).13.J.Georgiou, R.Robitaille, M.P.Charlton, Muscarinic control of cytoskeleton in perisynapticglia.J Neurosci 19, 3836-3846(1999).

14. J. Georgiou, R. Robitaille, W. S. Trimble, M. P. Charlton, Synaptic regulation of glial protein expression in vivo. Neuron 12, 443-455 (1994).

15. J.P. O'Malley, M.T. Waran, R.J. Balice-Gordon, ln vivo observation of terminal Schwann cells at normal, denervated, and reinnervated mouseneuromuscular junctions. J Neurobiol 38, 270-286 (1999).

16. P.A. Perez-Gonzalez, Provost, F., Rousse, L., Benzina, O., Piovesana, R., Darabid, H., Lamoureux, B., Wang, Y-S, Arbour, D. and Robitaille, R. ., Functional adaptation of glial cells at neuromuscular junctions in response to injury.(ln revision to GLIA(GLIA-00440-202)).

17.M.L.Reynolds, C.J.Woolf, Terminal Schwann cells elaborate extensive processes following denervation of the motor endplate.J Neurocytol 21, 50-66 (1992).

18.Y.J.Son, W.J.Thompson, Nerve sprouting in muscle is induced and guided by processes extended by Schwann cells. Neuron 14, 133-141 (1995).

19. Y. J. Son, W. J. Thompson, Schwann cell processes guide regeneration of peripheral axons. Neuron 14, 125-132 (1995).

20. E.Martineau, D.Arbour, J.Vallee, R.Robitaille, Properties of GlialCell at the Neuromuscular Junction Are Incompatible with Synaptic Repair in the SOD1(G37R)ALS Mouse Model.J Neurosci 40, 7759-7777(2020).

21.S.J.Burden, The formation of neuromuscular synapses.Genes Dev 12, 133-148 (1998).

22.S.Cantor et al., Preserving neuromuscula r synapses in ALS bystimulating MuSK with a therapeutic agonist antibody. Elife 7, (2018).

23.M.J.Perez-Garcia, S.J.Burden, Increasing MuSK activity delays and improves motor function in ALS mice.Cell Rep 2, 497-502 (2012).

24.J.oury et al., Mechanism of disease and therapeutic rescue of Dok7 congenital myasthenia. Nature595, 404-408 (2021).

25.C.R.Chapple, Darifenacin: a novel M3muscarinic selective receptorantagonist for the treatment of overactive bladder. Expert Opinion lnvestigDrugs13, 1493-1500 (2004).

26. F. Haab, Darifenacin in the treatment of overactive bladder. Drugs Today (Ba rc) 41, 441-452 (2005).

27. C. Chapple et al., A pooled analysis of three phase III studies to investigate the efficacy, tolerability and safety of darifenacin, a muscarinicM3 selective receptor antagonist, in the treatment of overactive bladder. BJUInt95, 993-1001 (2005).

28. F. Haab, L. Stewart, P. Dwyer, Darifenacin, an M3 selective receptorantagonist, is an effective and well-tolerated once-daily treatment for overactive bladder. Eur Urol 45, 420-429; discussion 429 (2004).

29. E.Callegari et al.-A comprehensive non-clinical evaluation of the CNS penetration potential of antimuscarinic agents for the treatment of overactive bladder.Br J Clin Pharmacol 72, 235-246 (2011).

30.G.G.Kay, U.Ebinger, Preserving cognitive function for patients with overactive bladder: evidence for a differential effect with darifenacin.lnt JClin Pract 62, 1792-1800 (2008).

31. M. Filali, R. Lalonde, S. Rivest, Sensorimotor and cognitive functions in a SOD1(G37R) transgenic mouse model of amyotrophic lateral sclerosis. BehavBrain Res 225, 215-221 (2011).

32.P.C.Wong et al., An adverse property of a familial ALS-linked SOD1mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria. Neuron 14, 1105-1116 (1995).

33. A.C. Ludolph et al., Guidelines for preclinical animal research in ALS/MND: A consensus meeting. Amyotroph Lateral Scler 11, 38-45 (2010).

34.S.Boillee et al., Onset and progression in inherited ALS determined by motor neurons and microglia.Science 312, 1389-1392(2006).34.Knippenberg , S., et al., Significance of behavioral tests in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). Behav Brain Res, 2010.213(1): p.82-7.

35. Rizzuto, E., et al., Measuring Neuromuscular Junction Functionality in the SOD1(G93A) Animal Model of Amyotrophic Lateral Sclerosis. Ann Biomed Eng, 2015.43(9): p.2196-206.

36.Dibaj, P., E.D.Schomburg, and H.Steffens, Contractile characteristics of gastrocnemius-soleus muscle in the SOD1 G93A ALS mouse model. Neurol Res, 2015.37(8): p.693-702.

37.Kanning, K.C., A.Kaplan, and C.E.Henderson, Motor neuron diversity indevelopment and disease.Annu Rev Neurosci, 2010.33: p.409-40.

38.Atkin, J.D., et al., Properties of slow-and fast-twitch muscle fibersin a mouse model of amyotrophic lateral sclerosis. Neuromuscul Disord, 2005.15(5): p.377-88.

Example

Example 1: Method

animal

Mice, strain 29, expressing the human mutant SOD1 ^G37R transgene, were obtained from the Jackson Laboratory and housed in a C57BL/6 background at the University of Montreal's animal facility. This mouse model is a late-onset, slowly progressive ALS model that reproduces the human phenotype of the disease. Characterization of this strain's phenotype has been previously published in several ALS studies (5, 7, 20, 31, 32). All experiments were conducted in accordance with guidelines from the Canadian Commission on Animal Welfare and the Animal Ethics Committee of the University of Montreal.

Preclinical trial design

The preclinical trial was designed according to the preclinical animal research guidelines in ALS/MND (33). This study was conducted in a double-blind manner. Fifteen male mice from the SOD1 ^G37R background were randomly assigned to three groups.

1. ARGX-119 (3B2g2m1-hIgG1LALAdelk: full-length heavy chain with reduced effector function SEQ ID NO:268 and full-length light chain with reduced effector function SEQ ID:269): and dafinara

2. ARGX-119 (3B2g2m1-hIgG1LALAdelk) and its loading agent (dafinacin control)

3. Homotype control mAb + carrier (dafinacin control)

ARGX-119 treatment was initiated before an attack (before the onset of symptoms or in the absence of symptoms), and dafinaxine treatment was initiated during an attack (when symptoms appeared) and continued throughout the course of the disease. A set of neurological scores (grades 1 to 5, Appendix 1) was used to determine the onset of symptoms and the progression and severity of symptoms during disease progression. Onset of disease was assessed by the onset of weight loss (34) and the appearance of tremor, corresponding to a neurological score of 1, while the endpoint of this study was in the late symptomatic phase, corresponding to a neurological score of 3 to 5.

Mice were administered either ARGX-119MuSK antibody (3B2g2m1-hlgG1LALAdelk) or placebo (movizumab-hlgG1LALAdelk: full-length heavy chain SEQ ID NO:272 with reduced effector function and full-length light chain SEQ ID:273 with reduced effector function) intraperitoneally at an initial dose of 20 mg/kg at P400, followed by weekly intraperitoneal injections at a dose of 10 mg/kg until sacrifice. At disease onset (approximately P425), dafinarax (10 mg/kg, diluted in DMSO, 5 days/week) was initiated orally. The placebo of dafinarax was DMSO alone. Mice received both treatments for approximately 4 months until approximately 520 days of age, which is the median age at which they typically reach the severe disease endpoint.

Movitizumab-hlgG1LALAdelk: SEQ ID NO:272 is derived from SEQ ID NO:274 and SEQ ID:273 is derived from SEQ ID:275.

Treatment, behavioral monitoring, experiments, and outcome analysis were conducted blinded. Standard ALS behavioral measurements were performed weekly to measure disease progression in different study groups. These included rotarod testing, grip strength measurement, weight measurement, and tail suspension testing to assess hindlimb extension reflexes. At euthanasia, the extensor digitorum longus (EDL) and soleus (SOL) muscles, along with their innervations, were dissected and placed in the physiology chamber. Two sets of measurements were obtained. First, force sensors were used to determine the functional properties of the muscles (strength and fatigue). Second, the muscles were immobilized and muscle mass was determined.

Rotary bar speed-up solution

Motor coordination, strength, and balance were assessed using a rotarod apparatus (TSE rotarod apparatus, TSE system GmbH, Germany). Animals were placed on the rotarod at an initial speed of 4 rpm, increasing to 40 rpm over 300 seconds. During the acceleration program, mice had two attempts per block and two blocks per session (with rest periods between sessions) to remain on the rotarod apparatus, and the average of the two longest drop times was taken.

grip strength

To measure the overall limb strength of the mice, a grip strength meter was used (Figure 1C). The mice had nine attempts per limb (3 groups of 3 attempts per group; with a 1-minute rest period between groups), and the best three values from each group were averaged.

Neuromuscular specimen (preparation)

Specimens of EDL and SOL muscles and their innervating nerves were dissected in an oxygenated Ree's solution (in mM) containing: 110 NaCl, 5 KCl, ₁ MgCl₂, ₂₅ NaHCO₃, ₂ CaCl₂, 11 glucose, 0.3 glutamate, 0.4 glutamine, 5 BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonate sodium salt), 0.036 choline chloride, and 4.34 × ^10⁻⁷ cocarboxylase. Following dissection, the neuromuscular specimens were continuously perfused with an oxygenated Ree's solution (95% _O₂ , 5% _CO₂ ).

Measurement of neuromuscular properties

The EDL and SOL neuromuscular samples were vertically attached to a fixed force sensor (model 402A-500mN, Aurora Scientific Inc.) using surgical sutures. The samples were attached to a tactile sensor at one end at the tendon level and to an adaptive hook at the other end (Fig. 2A). A platinum reference electrode was then placed juxtaposed with the muscle, positioned near the muscle distal to the tendon. To stimulate the muscle, a second platinum electrode was placed juxtaposed at the other end of the muscle. To elicit muscle contraction and neuromuscular activity from the motor nerve, the tibial nerve (SOL) or deep peroneal nerve (EDL) was aspirated into an electrode made of PE tubing and filled with physiological solution. Thus, the system was designed to elicit muscle contraction from both muscle and/or nerve stimulation. A basic force response to neuromuscular contraction was induced by a single ultra-large square wave with a 500mV, 0.1ms pulse applied to the motor nerve. A basic force response to muscle contraction was induced by a 15V, 1ms square pulse stimulation. The optimal muscle length was determined by gradually stretching the muscle until maximum contractile force output was achieved.

Force-Frequency Curve: Neural and muscle stimulation was performed to generate a standard force-frequency curve. Alternating neural and muscle stimulation was performed for 500 ms at multiple frequencies (5 Hz, 10 Hz, 20 Hz, 30 Hz, 40 Hz, 50 Hz, 60 Hz, 70 Hz, 80 Hz, 90 Hz, 100 Hz, 120 Hz, 140 Hz, 160 Hz, 180 Hz, 200 Hz, 250 Hz, and 300 Hz), and the generated force was monitored. A 2-minute rest period was provided between each stimulation. The proportion of muscle capacity used by the neuromuscular system during neural stimulation is expressed as the contractile capacity ratio, and the following calculations were performed for each frequency:

Maximum force: At frequencies of 50 Hz and 80 Hz, the maximum force generated by alternating nerve and muscle stimulation is obtained for 2 seconds, with intervals of 2 (SOL) or 5 (EDL) minutes.

Muscle fatigue: The fatigue protocol is shown in Figure 4A. The fatigue protocol is applicable to each muscle due to differences in its intrinsic characteristics. For EDL, fatigue is tested using a cycle of 180 neural stimulations induced at 120 Hz for 300 ms. The rest period between each stimulation is 700 ms, for a total protocol duration of 3 minutes. Neural stimulation is superimposed on muscle stimulation every 10 stimulations (18 simultaneous neuromuscular stimulations) to evaluate muscle reserve. The fatigue protocol for SOL consists of the following: a cycle of 300 neural stimulations at 50 Hz for 500 ms, with a rest period of 600 ms between stimulations, for a total duration of 5 minutes and 30 seconds. Neural stimulation is superimposed on muscle stimulation every 10 stimulations (30 simultaneous neuromuscular stimulations).

Muscle recovery: A 30-minute recovery period was provided after each fatigue program, during which neuromuscular contractility and neuromuscular + muscle contractility (120Hz-300ms for EDL and 50Hz-500ms for SOL) were measured at 5s, 10s, 15s, 30s, 45s, 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 5 minutes, 10 minutes, 20 minutes, and 30 minutes after the fatigue program.

Muscle weight

After each experiment, the muscle was fixed (10 minutes, PFA) and washed (3 washes, 5 minutes each, PBS1X). Then, the two tendons were cut and the muscle was weighed and stored at 4°C for further processing.

statistics

Results are expressed as mean ± SEM, where the number of animals is defined as N (number of replicates) and the number of muscles is expressed as n (number of observations). In most cases comparing three or four different groups, one-way ANOVA, the Kruskal-Wallis test, and multiple t-tests were used. Repeated one-way ANOVA and post-hoc Bonferroni multiple comparisons were used to compare values obtained from the same animals in different groups at multiple frequencies or over time. The confidence level used in the studies was 95% (α = 0.05). All analyses were performed using GraphPad 8 software (Prism).

Example 2: Results

The new combination of ARGX-119 antibody with dafina improves motor function and grip strength.

The animals were tested for motor function and overall strength to investigate whether combination therapy with ARGX-119 antibody and dafinarax could improve muscle function.

First, motor performance, balance, and coordination were measured using a standard accelerated protocol on a rotarod (Fig. 1A), which is known to reveal motor deficits in ALS as the disease progresses (34). Fig. 1B shows the gradual decline in motor performance in mice treated with ARGX-119 alone, dafinarax alone, or placebo, as revealed by shorter waiting times for the rotarod to fall, indicating the expected progression of the ALS motor phenotype. However, the motor performance of mice treated with the combination of ARGX-119 antibody and dafinarax was less pronounced compared to mice treated with ARGX-119+DMSO (p<0.001), PBS+dafinarax (p<0.001), and placebo (p<0.001), resulting in a significant improvement in motor performance (Fig. 1B; ARGX-119+DMSO N=5, ARGX-119+dafinarax N=4, PBS+dafinarax N=5; placebo N=5, one-way ANOVA, Kruskal-Wallis’s test, and multiple t-tests). This was particularly evident near the endpoint, where mice treated with the combination therapy showed significantly higher scores at ages P475 to P525 compared to the placebo group. Indeed, at this late symptom stage, most placebo mice were no longer able to run on the rotarod, while more than half of the mice in the dafina treatment group were still able to run. Interestingly, mice treated with ARGX-119+DMSO showed a trend toward improved motor behavior on the rotarod from ages P510 to P525 (p = 0.07, p = 0.06, and p = 0.053, respectively). These results suggest that the combination therapy has a beneficial effect compared to other single treatments and the placebo group.

Next, grip strength was measured to assess whether the combination treatments improved the overall strength of the animals (Figure 1C). Mice in all groups started the trial with similar grip strength. However, mice in the combination treatments performed better than mice in the placebo group, as evidenced by the greater grip strength produced at P460, until the end of the preclinical trial (Figure 1D; ARGX-119+DMSO N=5, ARGX-119+dafinarax N=4, PBS+dafinarax N=5, placebo N=5, one-way ANOVA, p<0.05, Tukey’s test and multiple t-tests). Interestingly, at P507, mice treated with PBS+dafinarax showed a significant increase in grip strength compared to the placebo group.

Third, the study tested whether the combination treatment with ARGX-119 antibody and dafinarax had any effect on the general condition of mice. For this purpose, changes in animal body weight, a direct indicator of disease progression and survival, were monitored, with animals exhibiting significant gradual weight loss after symptom onset. However, no differences were observed between the two groups (ARGX-119+DMSO N=5, ARGX-119+dafinarax N=4, PBS+dafinarax N=5, placebo N=5, p>0.05, one-way ANOVA, Tukey’s test, and multiple t-tests).

Example 2.1: Combined treatment improves neuromuscular contractile strength and NMJ efficacy

Improved contractile muscle properties are a powerful indicator that muscle and NMJ function should also be improved through combined treatment. Two types of muscles with different properties and disease resistance were investigated. EDL was used as a disease-sensitive, fast-twitching, fatigue-prone muscle, and SOL was used as a disease-resistant, slow-twitching, fatigue-prone muscle. Muscle force sensors were used to measure the force generated by muscles when stimulated by motor nerves and/or direct muscle stimulation (see Figure 2A). Using this system, stimulation of motor nerves at multiple frequencies elicited muscle contraction via NMJ efficacy, reflecting only the strength of contractile fibers associated with the innervated NMJ. Conversely, muscle stimulation depolarized all muscle fibers and reflected the maximum twitching force of all muscles, regardless of innervation state. This approach is particularly suitable for characterizing diseases exhibiting NMJ and muscle defects, such as ALS (35, 36).

Fast-acting, easily fatigued EDL muscles

First, a standard stimulation protocol was executed to generate force-frequency curves (5 Hz to 300 Hz) to characterize the NMJ potency following chronic treatment with ARGX-119 and dafinarax. During the protocol, muscle force generated by contractions induced by motor nerve stimulation and NMJ activation was significantly higher than the EDL in the placebo group (p < 0.001) or the ARGX-119+DMSO group (p < 0.05) (Figure 2B; ARGX-119+DMSO N = 5, ARGX-119+dafinarax N = 4, placebo N = 5; repeated one-way ANOVA, Bonferroni post-hoc test). Indeed, the combined treatment produced a twitching force of 66.3 ± 15.7 mN, compared to 47.2 ± 12.4 mN in the placebo group and 53.6 ± 14.9 mN in the ARGX-119+DMSO group. There was no significant difference in the ARGX-119 antibody group compared to the placebo-treated mice.

For direct muscle stimulation, significant differences were observed between the combined-treatment mice and other groups at higher frequencies (Fig. 2C). The combined-treatment groups showed significantly higher peak force (mN) compared to the ARGX-119 and DMSO groups (p<0.05) and the placebo group (p<0.001). This indicates preservation of the rapid twitching properties of EDL (37, 38) (Fig. 2C, ARGX-119+DMSO N=5, ARGX-119+dafinarax N=4, placebo N=5, repeated one-way ANOVA, Bonferroni post-hoc test, and multiple t-tests). Interestingly, as shown in Fig. 2E, increased contractile force and better preservation of EDL muscle weight were also observed in the combined-treatment animals compared to the placebo group.

Next, the proportion of muscle capacity used by the neuromuscular system during neural stimulation was determined. This is expressed as the contractile capacity ratio. In WT mice, this ratio was 100%, indicating that the neuronal control of the muscle recruited 100% of its contractile capacity. Therefore, if the treatment improved NMJ innervation, resulting in increased force, it was assumed that this proportion in the EDL muscle of the combined-treated animals should be higher compared to other groups. As shown in Figure 2D, the EDL proportion in ARGX-119+DMSO mice was significantly higher at 61.1±1.0% compared to 52.3±1.8% (p<0.001) in the combined-treated mice and 53.3±3.6% (p<0.0001) in the placebo group (ARGX-119+DMSO N=5, ARGX-119+dafinarax N=4, placebo N=5, one-way ANOVA, Kruskal-Wallis’s test).

Slow twitching SOL muscles

Then, the same protocol was performed, but targeting the SOL muscles (Figure 3). During the neural stimulation protocol, the combination-treated group showed significantly higher twitching force compared to the ARGX-119+DMSO group (p<0.0001) and the placebo group (p<0.0001) (Figure 3A; ARGX-119+DMSO N=5, ARGX-119+dafinarax N=4, placebo N=5, repeated one-way ANOVA and multiple t-tests). However, compared to placebo, the ARGX-119+DMSO group produced smaller contractile force (p<0.05). The twitching force produced by the combination treatment was 114.5±3.5 mN, compared to 83.1±4.5 mN in the placebo group and 64.6±5.1 mN in the ARGX-119+DMSO group. Under direct muscle stimulation, during the neural stimulation protocol, the combined treatment group again showed significantly higher twitching force (140.6±2.1 mM) compared to the ARGX-119+DMSO group (98.3±2.1 mN; p<0.01) and the placebo group (119.5±1.6 mN; p<0.001) (Figure 3B; ARGX-119+DMSO N=5, ARGX-119+dafinarax N=4, placebo N=5, repeated one-way ANOVA). There was also a significant difference between the ARGX-119+DMSO group and the placebo group (p<0.05).

Compared with the ARGX-119-treated group (68.4 ± 7.8%, p < 0.0001) and the control mice (56.1 ± ), the contractile strength ratio of the combined-treatment mice (Figure 3C) was significantly higher, at 82.0 ± 2.8% (p < 0.0001; ARGX-119 + DMSO N = 5, ARGX-119 + dafinarax N = 4, placebo N = 5, repeated one-way ANOVA). Interestingly, improvements in contractile force and contractile strength ratio were observed in the treated animals while maintaining good SOL muscle mass. In fact, compared with the placebo group, mice treated with ARGX-119 + dafinarax had better muscle mass retention (p < 0.05; ARGX-119 + DMSO N = 5, ARGX-119 + dafinarax N = 4, placebo N = 5, one-way ANOVA). Interestingly, there was also a significant difference between the ARGX-119+DMSO treatment group and the placebo group (p<0.001), where the SOL from ARGX-119 produced a better contractile capacity ratio than the SOL from the placebo group.

Overall, these results indicate that the combined treatment improved muscle and neuromuscular contractile force and muscle weight in both muscles, but only in the ARGX-119+DMSO group was the contractile capacity of EDL and SOL increased.

Example 2.2: The combination of ARGX-119 antibody and dafinarax maintains muscle fatigue properties

In addition to the force generated, muscles are also characterized by their resistance to fatigue. For example, fast-twitching muscles (such as EDL), which are mainly composed of fast, easily fatigued motor units, exhibit higher fatigue resistance compared to slow-twitching muscles (such as SOL) (37). In ALS, alterations in innervation type (from fast to slow twitching) and changes in the properties of the muscles themselves can alter fatigue characteristics, making them more resistant. Since treatment with the ARGX-119 + dafina significantly preserved muscle strength, the fatigue resistance of EDL and SOL muscles was then investigated.

The fatigue stimulation protocol was followed by a 30-minute recovery period (see Figure 4A). EDL muscles from the placebo-treated group exhibited atypical resistance to fatigue when the nerves were directly stimulated. However, as revealed by the delayed recovery, EDL muscles from mice treated with the ARGX-119 + dafinarax combination showed a more typical level of fatigue for this type of rapid muscle twitching (Figure 4B; ARGX-119 + dafinarax N=4, ARGX-119 + DMSO N=5, placebo N=5; p<0.05, one-way ANOVA, Kruskal-Wallis’s test, and multiple t-tests). Interestingly, a significant difference existed between the ARGX-119-treated and placebo groups during recovery (p<0.05), with muscles treated with ARGX-119 + DMSO exhibiting more typical fatigue. This suggests that the use of combination treatments, as well as single treatments such as the ARGX-119 antibody, improves muscle properties.

However, no difference in fatigue and recovery rates was found between neuromuscular stimulation and other methods when all muscle fibers were recruited (Fig. 4C; ARGX-119+Dalfinavidin N=4, ARGX-119+DMSO N=5, placebo N=5; p>0.05; one-way ANOVA, Kruskal-Wallis’s test).

SOL is a chronically twitching and resistant muscle, and compared to fast-twitching muscle EDL, it is expected to be more resistant to denervation observed in degenerative diseases and recover more quickly. Regarding neural stimulation of SOL muscles, the placebo group showed more pronounced fatigue compared to the ARGX-119+ dafinarax treatment group, as revealed by slower fatigue recovery, which is atypical for this fatigue-resistant muscle. Therefore, the combined treatment restored the fatigue-resistant properties of SOL muscles. (Figures 4D to E; ARGX-119+ dafinarax N=4, ARGX-119+ DMSO N=5, placebo N=5; p<0.01; one-way ANOVA, Kruskal-Wallis’s test and multiple t-tests). For neuromuscular stimulation, there were significant differences between the ARGX-119+ dafinarax and ARGX-119+ DMSO groups (p<0.001), and between the ARGX-119+ DMSO and placebo groups (p<0.0001).

Claims (45)

1. Anti-MuSK antibody or its antigen-binding fragment, used in human subjects to treat neuromuscular disorders. 2. The anti-MuSK antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment binds to the MuSK coil (Fz)-like domain sequence of SEQ ID NO:129. 3. The anti-MuSK antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a constant Fc region of wild-type human IgG, and the constant Fc region of wild-type human IgG contains at least 80% sequence identity with SEQ ID NO:266 or SEQ ID NO:267. 4. The anti-MuSK antibody or its antigen-binding fragment for use according to any of the preceding claims, wherein it is an agonist MuSK antibody and/or the effector function has been reduced or eliminated. 5. Preferably, the anti-MuSK antibody or its antigen-binding fragment according to any of the preceding claims, wherein the reduction or elimination of effector function is obtained by introducing one or more of the following mutations (all numbered according to the EU numbering system) into the constant region of the antibody-based molecule SEQ ID NO:266 or SEQ ID NO:267: N297A substitution; N297Q substitution; L234A substitution; L234D substitution; L234E substitution; L234G substitution; L234H substitution; L234F substitution; L234K substitution; L234Q substitution; L234R substitution; L234S substitution; L234T substitution; L235A substitution; L235D substitution; L235E substitution; L235F substitution; L235G substitution; L235V substitution; L235H substitution; L235I substitution; L235K substitution; L235R substitution; L235 Replace with S; Replace with L235T; Replace with L235Q; Replace with L237A; Replace with S239D; Replace with E233P; Replace with L234V; C236 is missing; Replace with G236E; Replace with G236R; Replace with G236K; Replace with G237A; Replace with P238A; Replace with F243L; Replace with D265A; Replace with S267E; Replace with H268A; Replace with R292P; Replace with Y300L; Replace with K322A; K Replacement with 322Q; A327Q; L328F; L328R; P329A; P329G; A330L; A330S; P331S; I332E; P396L; or each of the combinations of mutations described above in the fourth embodiment of this application, preferably the mutation is L234A or L235A, more preferably the mutation is L234A and L235A. 6. Preferably, an anti-MuSK antibody or antigen-binding fragment thereof for the application according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a constant Fc region of wild-type human IgG SEQ ID NO:266 or SEQ ID NO:267, and wherein the L234A and L235A mutations numbered according to the EU numbering system are introduced into the Fc region. 7. An anti-MuSK antibody or antigen-binding fragment thereof for any of the preceding claims, wherein the antibody or antigen-binding fragment comprises: a) A heavy chain variable domain (VH) comprising an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234, and b) A light chain variable domain (VL) containing an amino acid sequence that has at least 80% identity or similarity to SEQ ID NO:235. 8. An anti-MuSK antibody or antigen-binding fragment thereof for any of the preceding claims, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain (VH) and a light chain variable domain (VL): The VH contains: - Contains a CDR-H1 amino acid sequence that is SEQ ID NO:147 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:147. - Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and - Contains a CDR-H3 amino acid sequence (3B2g2m1) that is SEQ ID NO:156 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:156 and The VL contains: - Contains a CDR-L1 amino acid sequence that is SEQ ID NO:159 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159. - Contains a CDR-L2 amino acid sequence that is SEQ ID NO:172 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and - Contains a CDR-L3 amino acid sequence (3B2g2m1) that is SEQ ID NO:195 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195. 9. An anti-MuSK antibody or antigen-binding fragment thereof for any of the preceding claims, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain (VH) and a light chain variable domain (VL): - wherein the VH comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234, and the VL comprises an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:235, and - wherein the VH contains: The sequence contains SEQ ID NO:147 or a CDR-H1 amino acid sequence having 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:147. ο Contains a CDR-H2 amino acid sequence that is SEQ ID NO:153 or has 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:153, and The sequence contains SEQ ID NO:156 or a CDR-H3 amino acid sequence (3B2g2m1) with 1, 2, 3, 4, or 5 amino acid changes relative to SEQ ID NO:156. -The VL contained therein includes: The sequence contains SEQ ID NO:159 or a CDR-L1 amino acid sequence having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:159. ο Contains SEQ ID NO:172 or a CDR-L2 amino acid sequence having 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:172, and ο Contains SEQ ID NO:195 or a CDR-L3 amino acid sequence (3B2g2m1) with 1, 2, 3, 4 or 5 amino acid changes relative to SEQ ID NO:195. 10. An anti-MuSK antibody or antigen-binding fragment thereof for any of the preceding claims, wherein the antibody or antigen-binding fragment comprises: - Heavy chain variable structural domain (VH), which includes SEQ ID NO:234, and - Light chain variable structural domain (VL), which contains SEQ ID NO:235. 11. An anti-MuSK antibody or an antigen-binding fragment thereof for use in treating neuromuscular disorders in human subjects, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) as defined in Table 3 and/or a CDR as defined in Table 1 or 2. 12. An anti-MuSK antibody or an antigen-binding fragment thereof, preferably used in human subjects to treat neuromuscular disorders, wherein said antibody or antigen-binding fragment comprises: - A full-length heavy chain containing an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:270 and a full-length light chain containing an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:271, or - A full-length heavy chain containing an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:268 and a full-length light chain containing an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:269. - One or more of the following mutations (all numbered according to the EU numbering system) have been introduced into the full-length heavy chain: N297A substitution; N297Q substitution; L234A substitution; L234D substitution; L234E substitution; L234G substitution; L234H substitution; L234F substitution; L234K substitution; L234Q substitution; L234R substitution; L234S substitution; L234T substitution; L235A substitution; L235D substitution; L235E substitution; L235F substitution; L235G substitution; L235V substitution; L235H substitution; L235I substitution; L235K substitution; L235R substitution; L235S substitution; L235T substitution; L235Q substitution; L237A substitution; S239D substitution; E233P substitution ; L234V replacement; C236 deletion; G236E replacement; G236R replacement; G236K replacement; G237A replacement; P238A replacement; F243L replacement; D265A replacement; S267E replacement; H268A replacement; R292P replacement; Y300L replacement; K322A replacement; K322Q replacement; A327Q replacement; L328F replacement; L328R replacement; P329A replacement; P329G replacement; A330L replacement; A330S replacement; P331S replacement; I332E replacement; P396L replacement; or each of the combinations of mutations described above in the fourth embodiment of this application, preferably the mutation is L234A or L235A, more preferably the mutation is L234A and L235A. 13. An anti-MuSK antibody or an antigen-binding fragment thereof, preferably used in the application according to claim 12, wherein said antibody or antigen-binding fragment comprises: - Full-length heavy chain, which contains SEQ ID NO:270, and - A full-length light chain containing SEQ ID NO:271, and -The full-length heavy chain contains L234A and L235A mutations numbered according to the EU numbering system. 14. An anti-MuSK antibody or an antigen-binding fragment thereof, preferably used in the application according to claim 12, wherein said antibody or antigen-binding fragment comprises: a) A full-length heavy chain, comprising SEQ ID NO:268, and b) A full-length light chain containing SEQ ID NO:269, and c) The full-length heavy chain contains L234A and L235A mutations numbered according to the EU numbering system. 15. A polynucleotide for treating neuromuscular disorders in human subjects, said polynucleotide comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof or its VH or VL or CDR as described in any one of claims 1 to 14. 16. An expression vector for treating neuromuscular disorders in human subjects, comprising the polynucleotide of claim 15, preferably operatively linked to a regulatory region that allows expression of an antibody or its antigen-binding fragment or its VH or VL or CDR in a host cell or cell-free expression system. 17. A host cell or cell-free expression system for treating neuromuscular disorders in human subjects, comprising the expression vector of claim 16. 18. A composition for treating neuromuscular disorders in human subjects, comprising an antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 14, a polynucleotide as defined in claim 15, an expression vector as defined in claim 16, or a host cell or cell-free expression system as defined in claim 17. 19. The composition of claim 18, used for treating neuromuscular disorders in human subjects, is a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier or excipient. 20. An anti-MuSK antibody or its antigen-binding fragment, polynucleotide, expression vector, host cell, cell-free expression system or composition thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment, polynucleotide, expression vector, host cell, cell-free expression system or composition is administered in combination with an anticholinergic compound. 21. The anti-MuSK antibody or its antigen-binding fragment, polynucleotide, expression vector, host cell, cell-free expression system or composition according to claim 20, wherein the anticholinergic compound is administered alone, sequentially or simultaneously. 22. The anti-MuSK antibody or its antigen-binding fragment, polynucleotide, expression vector, host cell, cell-free expression system or composition according to any one of claims 20 or 21, wherein the anticholinergic compound is a muscarinic receptor antagonist, preferably a muscarinic receptor antagonist selective for muscarinic receptor M1 and/or muscarinic receptor M3 and/or muscarinic receptor M5. 23. The anti-MuSK antibody or its antigen-binding fragment, polynucleotide, expression vector, host cell, cell-free expression system or composition according to any one of claims 22, wherein the muscarinic receptor antagonist is selective for muscarinic receptor M3, preferably wherein the anticholinergic compound is dafinacin, ipratropium bromide, tiotropium bromide or troxodium chloride. 24. An anti-MuSK antibody or its antigen-binding fragment, polynucleotide, expression vector, host cell, cell-free expression system or composition thereof according to any of the preceding claims, wherein the neuromuscular disease is characterized by impaired neuromuscular transmission and/or denervation at the NMJ (neuromuscular junction). 25. An anti-MuSK antibody or its antigen-binding fragment, polynucleotide, expression vector, host cell, cell-free expression system, or composition according to any one of the preceding claims, wherein the neuromuscular disease is characterized by at least one of the following: a. Excessive stimulation by muscarinic substances, b. Motor neuron death, c. Neuromuscular junction (NMJ) denervation and d. Impaired synaptic transmission. 26. An anti-MuSK antibody or its antigen-binding fragment, polynucleotide, expression vector, host cell, cell-free expression system, or composition according to any one of the preceding claims, wherein the neuromuscular disease is selected from the following: amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), myasthenia gravis (MG), congenital myasthenia gravis, Lambert-Eton myasthenic syndrome (LEMS), Lyme disease, poliomyelitis, post-poliomyelitis, heavy metal poisoning, Kennedy syndrome, adult-onset Ty Sachs disease, hereditary spastic paraplegia, multifocal neuropathy, cervical spondylosis, extramedullary tumors with compressive radiculopathy and myelopathy, inclusion body myositis, progressive bulbar palsy, progressive muscular atrophy, motor neuron syndrome, and thyrotoxic myopathy. 27. An anti-MuSK antibody or antigen-binding fragment for the application of any one of claims 1 to 14, or, when referencing any one of claims 1 to 14, any one of claims 15 to 26, wherein the condition is ALS. 28. An anti-MuSK antibody or an antigen-binding fragment thereof for the treatment of ALS in human subjects, wherein the antibody or antigen-binding fragment is administered to asymptomatic human subjects, preferably within 1, 2, 3, 4, 5 or 6 months prior to the onset of disease. 29. The anti-MuSK antibody or its antigen-binding fragment according to claim 28, wherein the asymptomatic subject is diagnosed as being prone to neuromuscular disorders or diseases. 30. The anti-MuSK antibody or antigen-binding fragment thereof according to claim 28 or 29, wherein the antibody or antigen-binding fragment binds to the MuSK coil (Fz)-like domain sequence of SEQ ID NO:129. 31. The anti-MuSK antibody or antigen-binding fragment thereof for the application according to claims 28 to 30, wherein the antibody or antigen-binding fragment comprises: c) A heavy chain variable domain (VH) comprising an amino acid sequence having at least 80% identity or similarity to SEQ ID NO:234, and d) Light chain variable domain (VL) containing an amino acid sequence that has at least 80% identity or similarity to SEQ ID NO:235. 32. The anti-MuSK antibody or antigen-binding fragment thereof for any one of claims 28 to 31, wherein said antibody or antigen-binding fragment comprises: a) A full-length heavy chain, comprising SEQ ID NO:268, and b) A full-length light chain containing SEQ ID NO:269, and c) The full-length heavy chain contains L234A and L235A mutations numbered according to the EU numbering system. 33. The anti-MuSK antibody or its antigen-binding fragment, polynucleotide, expression vector, host cell, cell-free expression system or composition according to any one of claims 20 to 27, wherein the anticholinergic compound is administered at the onset of disease or within 1, 2, 3, 4, 5, 6 or 7 weeks after the onset of disease. 34. A combination comprising an anti-MuSK antibody or an antigen-binding fragment thereof and an anticholinergic compound, preferably for use in treating ALS in human subjects. 35. The combination of claim 34, wherein the antibody or antigen-binding fragment is administered to an asymptomatic subject, preferably within 1, 2, 3, 4, 5 or 6 months prior to the onset of disease, and/or wherein the anticholinergic compound is administered at the onset of disease or within 1, 2, 3, 4, 5, 6 or 7 weeks after the onset of disease. 36. The combination of claim 35, wherein the asymptomatic human subject treated with the antibody has been pre-diagnosed as being prone to neuromuscular disorders or diseases. 37. The combination according to claims 34 to 36, wherein the anti-MuSK antibody or its antigen-binding fragment binds to the MuSK coil (Fz)-like domain sequence of SEQ ID NO: 129. 38. The combination of any one of claims 34 to 37, wherein the anti-MuSK antibody or its antigen-binding fragment comprises: The heavy chain variable domain (VH) contains an amino acid sequence that has at least 80% identity or similarity to SEQ ID NO:234, and The light chain variable domain (VL) contains an amino acid sequence that has at least 80% identity or similarity to SEQ ID NO:235. 39. The combination of any one of claims 34 to 38, wherein the antibody or antigen-binding fragment comprises: a) A full-length heavy chain, comprising SEQ ID NO:268, and b) A full-length light chain containing SEQ ID NO:269, and c) The full-length heavy chain contains L234A and L235A mutations numbered according to the EU numbering system. 40. The anti-MuSK antibody or its antigen-binding fragment, combination, polynucleotide, expression vector, host cell, cell-free expression system, or composition according to any one of claims 1 to 39, wherein the anti-MuSK antibody or its antigen-binding fragment, combination, polynucleotide, expression vector, host cell, cell-free expression system, or composition is administered to an asymptomatic human subject at the onset of disease, preferably within 1, 2, 3, 4, 5, or 6 months prior to the onset of disease. 41. The anti-MuSK antibody or antigen-binding fragment, combination, polynucleotide, expression vector, host cell, cell-free expression system or composition thereof as described in claim 40, wherein the disease attack includes at least one symptom selected from the following: muscle twitching, muscle spasm, cramps, muscle weakness, slurred speech and/or nasal speech, dysphagia or dysphagia, dysphagia, dysarthria and dyspnea. 42. An anti-MuSK antibody or antigen-binding fragment, combination, polynucleotide, expression vector, host cell, cell-free expression system or composition thereof used according to any of the preceding claims, wherein neuromuscular disorders are analyzed by electrophysiological or pharmacodynamic evaluation in the following: neurofilaments (e.g., neurofilament light chains (NFL)) in serum, plasma and/or cerebrospinal fluid (CSF); or NMJ biopsy. 43. An anti-MuSK antibody or antigen-binding fragment thereof, combination thereof, polynucleotide, expression vector, host cell, cell-free expression system or composition thereof, according to any one of the preceding claims, wherein administration of the anti-MuSK antibody or antigen-binding fragment thereof, the combination thereof, polynucleotide, expression vector, host cell, cell-free expression system or composition to the human subject results in one or more of the following therapeutic effects: - An increase in the number or percentage of fully innervated NMJs in the subject; maintenance of the number or percentage of fully innervated NMJs in the subject; a decrease in the number or percentage of fully denervated NMJs in the subject; improved reliability of synaptic transmission; prevention, stabilization, or reduction of motor neuron death in the subject; and/or - Improvement in the subject's motor performance and/or grip strength; and/or - Improvement of the contractile properties of the muscle at the NMJ of the object; and/or - Improvement of fatigue resistance in the muscles at the NMJ site of the object; and/or - Inducing an increase in muscle mass at the NMJ of the subject; and/or - The delay in the occurrence of improvement or deterioration in the quality of life of the subject; and/or - Decreased muscarinic activity in perisynaptic Schwann cells (PSCs) of the subject (or decreased muscarinic hyperexcitability) or NMJ repair in the subject. 44. An anti-MuSK antibody or antigen-binding fragment, combination, polynucleotide, expression vector, host cell, cell-free expression system or composition thereof used according to any of the preceding claims, wherein the treatment results in stabilization of the condition. 45. An anti-MuSK antibody or antigen-binding fragment, combination, polynucleotide, expression vector, host cell, cell-free expression system, or composition thereof for use according to any of the preceding claims, wherein, relative to a human subject not treated with said anti-MuSK antibody or antigen-binding fragment, polynucleotide, expression vector, host cell, cell-free expression system, or composition thereof, treatment of the neuromuscular disease results in improvement in the treated human subject as assessed by electrophysiological or pharmacodynamic evaluation; improvement in neurofilaments (e.g., neurofilament light chains (NFL)) in serum, plasma, and/or cerebrospinal fluid (CSF); or improvement in NMJ biopsy.