AUTHOR=Sivelle Coline , Sierocki Raphael , Lesparre Youen , Lomet Aurore , Quintilio Wagner , Dubois Steven , Correia Evelyne , Moro Ana Maria , Maillère Bernard , Nozach Hervé TITLE=Combining deep mutational scanning to heatmap of HLA class II binding of immunogenic sequences to preserve functionality and mitigate predicted immunogenicity JOURNAL=Frontiers in Immunology VOLUME=14 YEAR=2023 URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2023.1197919 DOI=10.3389/fimmu.2023.1197919 ISSN=1664-3224 ABSTRACT=

Removal of CD4 T cell epitopes from therapeutic antibody sequences is expected to mitigate their potential immunogenicity, but its application is complicated by the location of their T cell epitopes, which mainly overlap with complementarity-determining regions. We therefore evaluated the flexibility of antibody sequences to reduce the predicted affinity of corresponding peptides for HLA II molecules and to maintain antibody binding to its target in order to guide antibody engineering for mitigation of predicted immunogenicity. Permissive substitutions to reduce affinity of peptides for HLA II molecules were identified by establishing a heatmap of HLA class II binding using T-cell epitope prediction tools, while permissive substitutions preserving binding to the target were identified by means of deep mutational scanning and yeast surface display. Combinatorial libraries were then designed to identify active clones. Applied to adalimumab, an anti-TNFα human antibody, this approach identified 200 mutants with a lower HLA binding score than adalimumab. Three mutants were produced as full-length antibodies and showed a higher affinity for TNFα and neutralization ability than adalimumab. This study also sheds light on the permissiveness of antibody sequences with regard to functionality and predicted T cell epitope content.