Characterization of pathogenic monoclonal autoantibodies derived from muscle-specific kinase myasthenia gravis patients.

Kazushiro Takata,Leslie Jacobson,Erik S Benotti,Richard J Nowak,Michelangelo Cao,Mario Losen,Patrick Waters,Angela Vincent,David Beeson,Marina Mané-Damas,Sarosh R Irani,Pilar Martinez-Martinez,Miriam L Fichtner,Kevin C O’Connor,Panos Stathopoulos

doi:10.1172/jci.insight.127167

Abstract

Myasthenia gravis (MG) is a chronic autoimmune disorder characterized by muscle weakness and caused by pathogenic autoantibodies that bind to membrane proteins at the neuromuscular junction. Most patients have autoantibodies against the acetylcholine receptor (AChR), but a subset of patients have autoantibodies against muscle-specific tyrosine kinase (MuSK) instead. MuSK is an essential component of the pathway responsible for synaptic differentiation, which is activated by nerve-released agrin. Through binding MuSK, serum-derived autoantibodies inhibit agrin-induced MuSK autophosphorylation, impair clustering of AChRs, and block neuromuscular transmission. We sought to establish individual MuSK autoantibody clones so that the autoimmune mechanisms could be better understood. We isolated MuSK autoantibody-expressing B cells from 6 MuSK MG patients using a fluorescently tagged MuSK antigen multimer, then generated a panel of human monoclonal autoantibodies (mAbs) from these cells. Here we focused on 3 highly specific mAbs that bound quantitatively to MuSK in solution, to MuSK-expressing HEK cells, and at mouse neuromuscular junctions, where they colocalized with AChRs. These 3 IgG isotype mAbs (2 IgG4 and 1 IgG3 subclass) recognized the Ig-like domain 2 of MuSK. The mAbs inhibited AChR clustering, but intriguingly, they enhanced rather than inhibited MuSK phosphorylation, which suggests an alternative mechanism for inhibiting AChR clustering.

Highlights

Patients with myasthenia gravis (MG) experience skeletal muscle weakness, worsened by activity
Myasthenia gravis (MG) is a chronic autoimmune disorder characterized by muscle weakness and caused by pathogenic autoantibodies that bind to membrane proteins at the neuromuscular junction
Patients (n = 6, all female; mean age 44 ± 12 years, range 37–63; consistent with reported demographics of muscle-specific tyrosine kinase (MuSK) MG in refs. 34–36) with laboratory and clinically confirmed MuSK autoantibody– positive MG were selected for study

Summary

Introduction

Patients with myasthenia gravis (MG) experience skeletal muscle weakness, worsened by activity. They present with ocular muscle weakness, which generalizes to involve limb muscles and bulbar and respiratory muscles in particular [1, 2]. The molecular immunopathology of MG is directly attributed to the presence of circulating autoantibodies targeting extracellular domains of postsynaptic membrane proteins at the neuromuscular junction Autoantibodies against the acetylcholine receptor (AChR), which are present in around 85% of patients, are mainly IgG1 and cause loss of AChRs via divalent binding, which leads to internalization of AChRs and complement-mediated damage to the NMJ [1, 8].

Methods

Results

Conclusion