Isolation of potent human Fab fragments against a novel highly immunogenic region on human muscle acetylcholine receptor which protect the receptor from myasthenic autoantibodies.

In the autoimmune disease myasthenia gravis (MG), antibodies against the muscle nicotinic acetylcholine receptor (AChR) cause loss of functional AChR in the neuromuscular junction. To isolate AChR-specific human antibody fragments (Fab), a phage-display library was constructed from an MG patient's thymic B lymphocytes. The first Fab isolated had a low affinity for human AChR, but two sequential antibody chain shufflings using the MG donor heavy and light chain gene repertoires resulted in isolating two new Fab with an approximately 30-fold higher binding ability. The selected Fab contained extensively mutated heavy and light chains and probably represent intraclonal variants of a common progenitor having diverged in vivo by somatic hypermutation. Interestingly, the isolated Fab bound to an extracellular highly immunogenic region located either on an alpha-subunit site affected by the gamma/epsilon-subunits or on the interface between alpha- and gamma/epsilon-subunits. This region is not the previously described "main immunogenic region" (MIR), although it seems to be close to it, as one improved Fab and an anti-MIR mAb competed for AChR binding with distinctly different subpopulations of MG sera. Furthermore, this Fab protected surface AChR in cell cultures against MG autoantibody-induced antigenic modulation, suggesting a potential therapeutic use in MG, especially in combination with a human anti-MIR Fab.