In vitro assay development to assess successful removal of T and B cell epitopes through Seismic’s IMPACT platform

Abstract Proteases derived from human pathogens can specifically cleave IgG into F(ab’)2 and Fc fragments. IdeS, an cleaving enzyme Streptococcus pyogenes, has shown clinical proof of concept, is approved for use before kidney transplantation. Due to the immunogenic nature these proteases, dosing limited by high prevalence pre-existing antibodies induction anti-drug after dosing. Therefore, mitigate impact immune system on our enzyme, we identify remove putative T B cell epitopes using Seismic’s IMPACT platform leveraging machine learning. To experimentally validate in silicopredictions, developed two complementary vitroassays intravenous immunoglobulin (IVIg) assess removal epitopes. First, how mutating wild type (WT) protease sequence impacts a competitive binding assay followed evaluating global protease. Additionally, vitroPBMC proliferation determine success epitope removal. Using flow cytometry, quantitively CD4 via bromodeoxyuridine (BrdU) incorporation. We demonstrate examples molecules designed through reduce both The assays show decrease competition as compared WT while achieving reduction IVIg binding. These data suggest that be used screen successful invisibilization interest.