Characterization of PR3 Epitopes for the Precision Targeting of Autoreactive B cells in ANCA-Associated Vasculitis

About This Grant

PROJECT SUMMARY/ABSTRACT: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of systemic autoimmune dis- eases characterized by organ- and life-threatening inflammation of small and medium blood vessels. Half of AAV cases are driven by ANCAs targeting neutrophil cell-surface bound proteinase 3 (PR3), which leads to patho- genic neutrophil activation. However, total PR3-ANCA plasma levels correlate poorly with disease severity and relapse risk, and limited knowledge of the variable immunopathogenic potential of distinct PR3-ANCA subsets has challenged the advancement of more accurate diagnostic and prognostic tests. Moreover, severe infection caused by current, broadly immunosuppressive treatment strategies is the leading cause of mortality in AAV. To this end, we have developed PR3-chimeric autoantigen-T cell receptor (CATCR)-T cells, a precision cellular immunotherapy which uses engineered receptors (PR3 fused to T cell receptor subunits) to specifically engage and eliminate pathogenic ANCA+ B cells through their B cell receptor (BCR). Initial CATCR-T cell designs demonstrated potent killing of ANCA+ B cells, though moderate killing of normal B cells indicated off-target bind- ing of CATCRs to other surface antigens. The overall goal of this proposal is to characterize the pathogenic features of PR3-ANCA on the monoclonal antibody level, and to increase PR3-CATCR selectivity by identifying and disrupting off-target binding partners through targeted mutagenesis of the CATCR PR3 binding moiety. In preliminary work, we have synthesized and expressed 18 novel patient-derived monoclonal PR3-ANCAs to identify shared features of pathogenic autoantibodies in AAV (Aim 1). Standard kinetic and competitive surface plasmon resonance (SPR) and ELISA assay formats will be used to measure PR3-ANCA binding affinity, epitope specificity, and their ability to disrupt PR3 binding with its neutrophil surface receptor NB1. Individual PR3-ANCA monoclonal autoantibodies will then be assayed for their ability to trigger pathogenic neutrophil activation in vitro to identify common determinants of pathogenicity. To mitigate off-target PR3-CATCR-T cells cytotoxicity (Aim 2), we will use BioID-mass spectrometry to identify non-BCR binding partners of PR3-CATCRs. Rounds of tar- geted mutagenesis informed by in silico modeling of protein-protein interactions will be validated using in vitro binding assays. Optimized PR3-CATCR-T cells using mutagenized PR3 as a binding domain will be tested for their selective killing in model human ANCA+ B cell lines and primary human B cells from patients with AAV. Overall, this proposal aims to i) advance our understanding of PR3-AAV immunopathogenesis with the goal of improving diagnostic and prognostic clinical tests, and ii) enable the development of PR3-CATCR-T cells, a novel cellular immunotherapy modality for the treatment of AAV. This research plan provides valuable training in im- munological wet lab techniques, cell culture modeling of rheumatic disease, protein engineering, and the pro- cessing and assaying of clinical samples. Findings from this proposal will be of broad interest to the immuno- therapy field and will serve as preliminary data for a future NIH K99 Career Development Award.