bioRxiv2018
Myasthenia gravis (MG) is a chronic autoimmune disorder characterized by muscle weakness and caused by autoantibodies that bind to functional membrane proteins at the neuromuscular junction. Most patients have autoantibodies to the acetylcholine receptor (AChR), but a subset of patients instead have autoantibodies to muscle specific tyrosine kinase (MuSK). MuSK is an essential component of the Agrin/Lipoprotein receptor-related protein 4(LRP4)/MuSK/downstream of tyrosine kinase 7 (DOK7) pathway that is responsible for synaptic differentiation, including clustering of AChRs at the neuromuscular junction, both during development and in adult muscle. Nerve-released Agrin binds to LRP4 which then binds to MuSK, stimulating autophosphorylation and recruitment of DOK7 to complete the membrane component of the pathway. Serum-derived IgG4 subclass MuSK autoantibodies prevent the binding of LRP4 to MuSK, subsequently impairing autophosphorylation, resulting in the loss of Agrin-induced AChR clustering in the mouse myotube-forming C2C12 line. Although this autoimmune mechanism appears well understood, MuSK autoantibodies from patients are polyclonal. Most are IgG4 but IgG1, 2 and 3 are also present and can achieve similar results on AChR clustering. In addition, most bind the first Ig-like domain in MuSK, however some patients harbor serum autoantibodies that recognize other domains in MuSK. We sought to establish individual MuSK IgG clones so that the disease mechanisms could be better understood. We isolated MuSK autoantibody-expressing B cells from MuSK MG patients with a fluorescent-tagged MuSK antigen multimer and generated a panel of human monoclonal autoantibodies (mAbs) from these cells. We produced 77 mAbs from single B cells collected from six MuSK MG patients. Here we focused on three highly specific mAbs that bound quantitatively to MuSK in solution, to MuSK-expressing HEK cells and at mouse neuromuscular junctions where they co-localized with AChRs. These three IgG isotype mAbs (two IgG4 and one IgG3 subclass) recognized the Ig-like domain 2 of MuSK. The three MuSK mAbs inhibited Agrin induced-AChR clustering in C2C12 myotubes, but intriguingly, they enhanced rather than inhibited MuSK phosphorylation. This approach for ex vivo isolation of MuSK MG autoantibody-producing cells and production of human recombinant mAbs has identified distinct autoantibody specificities and likely divergent effector mechanisms. Collectively, these findings will enable a better understanding of MuSK autoantibody-mediated pathology.