Development of specific peptide reagents for serologic monitoring of Exostosin autoantibodies in membranous lupus nephritis

Project SummaryUp to half of patients with systemic lupus erythematosus (SLE) will develop lupus nephritis (LN), and nearly a third of LN patients will develop kidney failure requiring dialysis or transplantation. One type of LN, membranous lupus nephritis (MLN) exhibits similar clinical hallmarks and patterns of injury as compared to non- lupus associated membranous nephropathy (MN). MN and MLN are largely diagnosed through histopathological analysis of kidney biopsies. Moreover, autoantigens have been identified in MN, with M-type phospholipase A2 receptor (PLA2R) autoantibodies present in nearly 70% of cases and thrombospondin type-1 domain containing 7A (THSD7A) autoantibodies present in approximately 2% of cases. Given the strong connection between these autoantibodies and MN, there is significant movement toward eliminating kidney biopsies in patients showing serological positivity for autoantibodies against PLA2R and THSD7A. Moreover, serologic tests specific for the inciting autoantibody (anti-PLA2R or anti-THSD7A) have proven to be highly valued by nephrologists for monitoring response to therapy and guiding patient care. Reducing the requirement for renal biopsy and progressing towards a precision medicine approach represent dramatic benefits to patients and the US healthcare system, alike. For SLE patients at risk for MLN however, predictive autoantigens are only just now becoming available, with a need for subsequent serological tests. Arkana Laboratories, a leading provider of histopathological and molecular nephrology diagnostics, evaluated a broad range of MLN biopsy samples in an effort to identify autoantigens for MLN that may have the same impact as PLA2R and THSD7A have had on non- lupus MN diagnostics. Using laser capture microdissection, immune complex elution, mass spectrometry, and bioinformatic proteomic analysis, two proteins, Exostosin 1 and Exostosin 2 (EXT1/2), emerged as candidate autoantigens for a subset of MLN. Recently published reports from the Mayo Clinic confirm EXT1/2 as likely autoantigens in MLN. Autoantigenicity was confirmed by staining MLN biopsies for EXT1/2, which exhibited tight co-localization with IgG in the glomerular membrane. EXT1/2 also co-immunoprecipitated with IgG in extracts from MLN kidney biopsy tissue. After evaluating over 80 SLE samples, more than 30% were found to be positive for EXT1/2, with only 1% and 4% exhibiting PLA2R or THSD7A signal, respectively. Interestingly, EXT1/2 circulating autoantibodies have not yet been detected in serological analysis indicating that traditional ELISA- based assays using recombinant proteins to detect autoantibodies may not support an EXT1/2 diagnostic test for MLN. This Phase I program will therefore employ phage display biopanning, an unbiased screening method used to identify high affinity peptide binders, against MLN patient sera and immune complexes from patients with exostosin-positive MLN that likely contain EXT1/2 autoantibodies. Peptides which bind EXT1/2 autoantibodies will be further refined to maximize affinity and specificity for future Phase II testing as diagnostic reagents.