Identifying new antigens in human autoantibody-mediated disease - Project Summary. Myasthenia gravis (MG) is an autoimmune disorder affecting neuromuscular transmission. MG patients suffer from severe muscle weakness and increased muscle fatigability due to diminished neuromuscular signaling. MG is caused by autoantibodies that target components of the neuromuscular junction (NMJ). The most common subtype of autoantibody-mediated MG—comprising approximately 80% of the patient population—is characterized by pathogenic autoantibodies targeting the nicotinic acetylcholine receptor (AChR). There is a subset of MG patients that is termed seronegative MG (SNMG). SNMG is defined by the absence of detectable autoantibodies to the known NMJ targets. The mechanisms underlying the immunopathology SNMG are poorly understood. Consequently, the impact on these patients is considerable; treatment paradigms and outcomes are uncertain due to the lack of data associated with the disease mechanisms. In addition, SNMG patients are often excluded from clinical trials in which autoantibody positive MG patients participate. To address this knowledge gap, we will identify immune mechanisms driving SNMG. This will be achieved through the application of two high-throughput autoantibody-discovery technologies, both of which allow for the screening of exceptionally large human antigen libraries. The first (termed PhIP-seq) leverages the entire human protein catalog represented by a synthetic peptide library (peptidome), which includes all open reading frames in the human genome. The library is presented on the surface of bacteriophages as forty-nine amino acid peptides that “tile” or overlap each other. The second (termed REAP) affords screening of antigens displayed on cells as full-length domains of extracellular and membrane proteins, which are often the target of pathogenic autoantibodies. Because the antigens are expressed in live cells, protein folding into biologically relevant conformations and post-translational modifications are included. Together these approaches complement each other by collectively offering broad antigen coverage and presentation of biologically relevant epitope conformations. Carefully curated SNMG patient samples from the US, Italy and Eastern Europe will be screened. Validation of candidate antigens will be performed with independent assays used for measuring antigen-antibody binding. The second phase of this project focuses on defining the mechanisms these newly identified autoantibodies use to mediate SNMG pathology. The focus will be given to antibody characteristics including isotype and IgG subclass, followed by complement activation, receptor internalization, and interruption of receptor/channel function. We will apply in vitro assays, which we have developed, to measure these characteristics and mechanisms. Overall, this study is designed to provide detailed insights into the molecular mechanisms used by autoantibodies to facilitate the pathology of this poorly defined MG subtype. In addition to providing valuable diagnostic biomarkers, these newly defined immune mechanisms are expected to impact treatment outcomes by informing individualized and mechanism-specific applications of biological therapeutics.
