Restoring the potency of a neutralizing antibody via guided hypermutation with hyper-antibody editor HAE1

Somatic hypermutation (SHM) drives antibody affinity maturation in B cells. By mimicking this process, guided hypermutation (GHM) tools employing CRISPR systems and activation-induced cytidine deaminase (AID) have advanced antibody development. However, GHM-induced mutations in cultured cells exhibit mutation patterns distinct from those observed in natural antibody diversification following in vivo affinity selection. To address this, we engineer a hyper-antibody editor, HAE1, by integrating cytidine and adenine deaminases with a nicked, PAMless Cas9 variant, SpRY, to closely resemble the mutation spectrum of natural SHM. Moreover, to streamline mutation, selection, and validation within the same cells, we develop a dual-expression system in HEK293F cells that allows simultaneous expression of both transmembrane and secreted full-length antibodies. Using this system, we apply HAE1 to the SARS-CoV-2 neutralizing antibody CV07-209 and restore the antibody's binding affinity and neutralization potency against Omicron variants, specifically BA.1, including at least one mutation beyond the reach of current GHM tools. HAE1 thus provides a versatile, high-throughput strategy for expediting antibody evolution, presenting significant potential for therapeutic antibody development and protein engineering.