W138 - In Vitro Assay Development to Assess Successful Removal of T and B Cell Epitopes Through Seismic’s IMPACT Platform

Jordan Anderson – Protein Sciences – Seismic Therapeutic; Nathan higginson-Scott – Seismic Therapeutic; Melissa Hollfelder – Scientist, Protein Sciences, Seismic Therapeutic; Ivan Mascanfroni – Seismic Therapeutic; Kevin Otipoby – Seismic Therapeutic; Ryan Peckner – Seismic Therapeutic; Alex Pellerin, PhD – Principal Scientist, Immunology, Seismic Therapeutic; Nathan Rollins, PhD – Sr. Scientist, Machine learning, Seismic Therapeutic

Abstract Text: Proteases derived from human pathogens can specifically cleave IgG into F(ab’)2 and Fc fragments. IdeS, an IgG cleaving enzyme derived from Streptococcus pyogenes, has shown clinical proof of concept, and is approved for use before kidney transplantation. Due to the immunogenic nature of these proteases, dosing is limited by high prevalence of pre-existing antibodies and the induction of anti-drug antibodies after dosing. Therefore, to mitigate the impact of the immune system on our enzyme, we identify and remove putative T and B cell epitopes using Seismic’s IMPACT platform leveraging machine learning.

To experimentally validate in silico predictions, we developed two complementary in vitro assays using intravenous immunoglobulin (IVIg) to assess removal of B cell epitopes. First, we assess how mutating the wild type (WT) protease sequence impacts pre-existing antibodies in a competitive binding assay followed by evaluating global binding of pre-existing antibodies to our protease. Additionally, we developed an in vitro PBMC proliferation assay to determine the success of T cell epitope removal. Using flow cytometry, we quantitively assess CD4 proliferation via bromodeoxyuridine (BrdU) incorporation.

We demonstrate examples of molecules designed through our IMPACT platform to reduce both T and B cell epitopes. The B cell epitope assays show a decrease in competition as compared to the WT protease while achieving a reduction in IVIg binding. These data suggest that these in vitro assays can be used to screen for successful invisibilization of molecules of interest.