Functional monovalency amplifies the pathogenicity of anti-MuSK IgG4 in myasthenia gravis.

Human immunoglobulin (Ig) G4 usually displays antiinflammatory activity, and observations of IgG4 autoantibodies causing severe autoimmune disorders are therefore poorly understood. In blood, IgG4 naturally engages in a stochastic process termed "Fab-arm exchange" in which unrelated IgG4s exchange half-molecules continuously. The resulting IgG4 antibodies are composed of two different binding sites, thereby acquiring monovalent binding and inability to cross-link for each antigen recognized. Here, we demonstrate that this process amplifies autoantibody pathogenicity in a classic IgG4-mediated autoimmune disease: muscle-specific kinase (MuSK) myasthenia gravis. In mice, monovalent anti-MuSK IgG4s caused rapid and severe myasthenic muscle weakness, whereas the same antibodies in their parental bivalent form were less potent or did not induce a phenotype. Mechanistically this could be explained by opposing effects on MuSK signaling. Isotype switching to IgG4 in an autoimmune response thereby may be a critical step in the development of disease. Our study establishes functional monovalency as a pathogenic mechanism in IgG4-mediated autoimmune disease and potentially other disorders.

MuSK antibodies, lessons learned from poly- and monoclonality.

Muscle-specific kinase (MuSK) plays a critical role in establishing and maintaining neuromuscular synapses. Antibodies derived from immunizing animals with MuSK were important tools to help detect MuSK and its activity. The role of antibodies in MuSK-related research got an extra dimension when autoantibodies to MuSK were found to cause myasthenia gravis (MG) in 2001. Active immunization with MuSK or passive transfer of polyclonal purified IgG(4) fractions from patients reproduced myasthenic muscle weakness in a range of animal models. Polyclonal patient-purified autoantibodies were furthermore found to block agrin-Lrp4-MuSK signaling, explaining the synaptic disassembly, failure of neuromuscular transmission and ultimately muscle fatigue observed in vivo. MuSK autoantibodies are predominantly of the IgG4 subclass. Low levels of other subclass MuSK antibodies coexist, but their role in the pathogenesis is unclear. Patient-derived monoclonal antibodies revealed that MuSK antibody subclass and valency alters their functional effects and possibly their pathogenicity. Interestingly, recombinant functional bivalent MuSK antibodies might even have therapeutic potential for a variety of neuromuscular disorders, due to their agonistic nature on the MuSK signaling cascade. Thus, MuSK antibodies have proven to be helpful tools to study neuromuscular junction physiology, contributed to our understanding of the pathophysiology of MuSK MG and might be used to treat neuromuscular disorders. The source of MuSK antibodies and consequently their (mixed) polyclonal or monoclonal nature were important confounding factors in these experiments. Here we review the variety of MuSK antibodies described thus far, the insights they have given us and their potential for the future.

ARGX-119 is an agonist antibody for human MuSK that reverses disease relapse in a mouse model of congenital myasthenic syndrome.

Muscle-specific kinase (MuSK) is essential for the formation, function, and preservation of neuromuscular synapses. Activation of MuSK by a MuSK agonist antibody may stabilize or improve the function of the neuromuscular junction (NMJ) in patients with disorders of the NMJ, such as congenital myasthenia (CM). Here, we generated and characterized ARGX-119, a first-in-class humanized agonist monoclonal antibody specific for MuSK, that is being developed for treatment of patients with neuromuscular diseases. We performed in vitro ligand-binding assays to show that ARGX-119 binds with high affinity to the Frizzled-like domain of human, nonhuman primate, rat, and mouse MuSK, without off-target binding, making it suitable for clinical development. Within the Fc region, ARGX-119 harbors L234A and L235A mutations to diminish potential immune-activating effector functions. Its mode of action is to activate MuSK, without interfering with its natural ligand neural Agrin, and cluster acetylcholine receptors in a dose-dependent manner, thereby stabilizing neuromuscular function. In a mouse model of DOK7 CM, ARGX-119 prevented early postnatal lethality and reversed disease relapse in adult Dok7 CM mice by restoring neuromuscular function and reducing muscle weakness and fatigability in a dose-dependent manner. Pharmacokinetic studies in nonhuman primates, rats, and mice revealed a nonlinear PK behavior of ARGX-119, indicative of target-mediated drug disposition and in vivo target engagement. On the basis of this proof-of-concept study, ARGX-119 has the potential to alleviate neuromuscular diseases hallmarked by impaired neuromuscular synaptic function, warranting further clinical development.

Development and characterization of agonistic antibodies targeting the Ig-like 1 domain of MuSK.

Muscle-specific kinase (MuSK) is crucial for acetylcholine receptor (AChR) clustering and thereby neuromuscular junction (NMJ) function. NMJ dysfunction is a hallmark of several neuromuscular diseases, including MuSK myasthenia gravis. Aiming to restore NMJ function, we generated several agonist monoclonal antibodies targeting the MuSK Ig-like 1 domain. These activated MuSK and induced AChR clustering in cultured myotubes. The most potent agonists partially rescued myasthenic effects of MuSK myasthenia gravis patient IgG autoantibodies in vitro. In an IgG4 passive transfer MuSK myasthenia model in NOD/SCID mice, MuSK agonists caused accelerated weight loss and no rescue of myasthenic features. The MuSK Ig-like 1 domain agonists unexpectedly caused sudden death in a large proportion of male C57BL/6 mice (but not female or NOD/SCID mice), likely caused by a urologic syndrome. In conclusion, these agonists rescued pathogenic effects in myasthenia models in vitro, but not in vivo. The sudden death in male mice of one of the tested mouse strains revealed an unexpected and unexplained role for MuSK outside skeletal muscle, thereby hampering further (pre-) clinical development of these clones. Future research should investigate whether other Ig-like 1 domain MuSK antibodies, binding different epitopes, do hold a safe therapeutic promise.