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

Somatic hypermutation (SHM) drives antibody affinity maturation in B cells, and mimicking this process has advanced therapeutic antibody development. Unlike conventional base editing, antibody diversification requires diverse mutations within the antibody complementary determining region (CDR) loops. Here, we engineered a hyper-antibody editor, HAE1, by integrating cytosine and adenine deaminases with a nicked, PAMless Cas9 variant, SpRY. HAE1 enables broad-spectrum mutagenesis with unrestricted targeting, fully covering mutation types observed in nature. Moreover, we developed a dual-expression system in mammalian cells that allows simultaneous expression of both transmembrane and secreted full-length antibodies, streamlining mutation, selection, and validation within the same cells. Using this platform, we rejuvenated the SARS-CoV-2 antibody CV07-209, restoring potency against Omicron variants. These results demonstrate the effectiveness of HAE1 as a powerful tool for antibody engineering, offering a practical strategy for evolving antibodies to address rapidly mutating pathogens and other therapeutic challenges. Overall design: We introduced Amplicon sequencing to detect the editing products of different antibody editor. eGFP gene was targeted by different sgRNAs and then cloned with specific primers for high-throughput sequencing.