Background Granulomatosis with polyangiitis (GPA) is a disease characterized by inflammation in small blood vessels, leading to significant organ damage and ultimately great morbidity and mortality. The presence of anti-neutrophil cytoplasmic antibodies (ANCAs) is a hallmark of the disease, and thought to directly play a role in pathogenesis by activating primed neutrophils. The two major antigens that ANCAs are believed to recognize are myeloperoxidase (MPO) and proteinase 3 (PR3). Although anti-MPO antibodies are directly pathogenic when transferred or induced in animal models, the evidence is less clear for PR3. Clinically, it is debated whether levels of anti-PR3 correlate with disease activity. Therefore, we believe that anti-PR3 antibodies are not the primary drivers of disease in patients with PR3+ ANCA-vasculitis (i.e. GPA), and that novel autoantibodies may play a role in disease pathogenesis.
Objectives The objective of this work is to identify the human autoantigen targets of novel autoantibodies identified in GPA, and to test their association with disease. The goal is to discover novel autoantigens that will ultimately lead to new diagnostics and therapeutics.
Methods Cell-barcode-enabled antibody repertoire sequencing was performed on blood plasmablasts (antibody-producing cells formed during an immune response) from five PR3+ ANCA-vasculitis patients with GPA treated with rituximab in the RAVE trial. Phylogenetic trees were bioinformatically created in order to identify clonal families of plasmablasts. Antibodies representing clonal families of plasmablasts were recombinantly expressed and are being tested using human protein arrays and ELISAs to determine their antigen specificity.
Results All five PR3+ ANCA-vasculitis patients sequenced from the RAVE trial achieved complete remission but subsequently flared. Plasmablasts were isolated at the baseline flare, at remission and at the post-rituximab flare. Phylograms of the antibody repertoires revealed clonal families of affinity-matured antibodies that share heavy and light chain VJ usage. A total of 24 representative antibodies were selected for recombinant expression, including representative antibodies derived from clonal families: (1) shared across patients at baseline flare and/or post-rituximab flare (n=5), (2) present at baseline flare and post-rituximab flare (n=7) [persistent clone or same clone came back], (3) present at baseline flare or post-rituximab flare (n=6), (4) present in remission and post-rituximab flare (n=5) [achieves remission despite clone presence], (5) present in remission (but not flare) (n=1) [patient in remission despite clone presence]. None of the 24 antibodies bind PR3 in an ELISA and thus these antibodies do not represent ANCAs. Antigen discovery to determine the targets of these recombinant plasmablast antibodies is ongoing.
Conclusions Plasmablasts from patients with GPA in the RAVE trial were sequenced and antibody repertoires were generated. Clonal families of plasmablasts, including those expressing antibodies possessing shared CDR sequences across multiple patients, were identified and recombinantly expressed. None of the 24 antibodies bound to PR3, suggesting these are not ANCAs and are potentially novel autoantibodies. Identification of the antigen targets of these antibodies is ongoing.
Acknowledgements The Immune Tolerance Network
Training Program in Adult and Pediatric Rheumatology 2T32AR050942–11
Disclosure of Interest None declared