Rapid generation of potent antibodies by autonomous hypermutation in yeast

Antibodies are critical tools in all areas of the life sciences, ranging from basic research to diagnostics and therapeutics. Currently, the predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of B cell somatic hypermutation. However, animal immunization is inherently slow, expensive and has poor compatibility with certain antigens (e.g., integral membrane proteins). Here, we describe Autonomous Hypermutation yEast surfAce Display (AHEAD), a synthetic recombinant antibody generation technology that avoids the problems of animal immunization by imitating somatic hypermutation in engineered yeast. In AHEAD, antibody fragments are encoded on an error-prone orthogonal DNA replication system, resulting in Saccharomyces cerevisiae populations that continuously mutate surface-displayed antibody repertoires. Simple cycles of yeast culturing and enrichment for antigen binding drive the evolution of high-affinity antibody clones in a readily parallelizable process that takes only 1.5-3 weeks. Using AHEAD, we generated nanobodies against several targets including a GPCR and the receptor-binding domain of the SARS-CoV-2 S glycoprotein. The SARS-CoV-2 nanobodies, reached subnanomolar affinities through the sequential fixation of multiple mutations over 3-8 AHEAD cycles that saw ~580-fold and ~925-fold improvements in binding affinities and pseudovirus neutralization potencies, respectively. These experiments highlight the defining speed, parallelizability, and effectiveness of AHEAD and provide a template for streamlined antibody generation at large with salient utility in rapid response to current and future viral outbreaks.