Project Summary. Myasthenia gravis (MG) is an autoimmune disorder of neuromuscular transmission. The most common subtype is characterized by autoantibodies (autoAbs) targeting the acetylcholine receptor (AChR), a membrane channel expressed on the muscle end-plate at the neuromuscular junction. The pathology is directly due to the AChR autoAbs. The AChR autoAbs in the serum of patients are broadly heterogeneous in their specificity as it includes autoAbs that recognize each of the four different subunits (a, b, e or d) of AChR. Furthermore, AChR autoAbs can use three distinct mechanisms to effect pathology: (i) complement-directed tissue damage, (ii) blocking the binding site for acetylcholine and (iii) modulation (internalization) of the AChR. Although AChR autoAbs are pathogenic, titers vary widely between different patients and within individuals during the course of their disease; importantly, titer neither correlates well with clinical severity nor predicts treatment outcome. Furthermore, new biological therapeutic strategies that specifically target AChR autoAb complement activation or reduce circulating titers have shown wide variability in response including patients that do not respond. The disassociation between titer and disease severity, and the heterogeneous response to autoantibody targeting treatments are not well understood. This gap in our knowledge exists first because AChR autoAbs are often studied with patient serum, which are confounded by the complex mixture of specificities. Second, existing assays only measure AChR autoAb binding, but not their pathogenic capacity or mechanism. We will address these gaps in our knowledge by (i) generating rare human monoclonal AChR autoAbs, (ii) using novel approaches to measure the different mechanisms of autoAb pathogenicity, and (iii) associating the autoAb properties with pathogenic capacity. To this end, we applied an unbiased high-throughput approach for producing AChR-specific human mAbs by cloning B cells, which circumvented technical challenges associated with cloning mAbs targeting this multi-subunit membrane channel. This will afford production of an AChR specific mAb library, which includes mAbs that recognize different subunits and epitopes within those subunits. New approaches to study the different effector mechanisms of AChR autoAb pathogenicity have also been developed. We will measure AChR autoAb-mediated acetylcholine blocking and AChR modulating functions of the AChR mAbs. Importantly, an assay for measuring complement activation, in which the complement inhibitors (CD46, CD55 and CD59) are knocked out, will be used to test AChR autoAb-dependent complement activity. These experiments will define how the specificity and molecular properties of AChR autoAb are associated with pathogenic effector function. Overall, this investigation will (i) provide a set of well-characterized mAbs which will serve as tools for more accurate modeling of AChR autoAb pathology; (ii) provide...