PT  - JOURNAL ARTICLE
AU  - Alon Wellner
AU  - Conor McMahon
AU  - Morgan S. A. Gilman
AU  - Jonathan R. Clements
AU  - Sarah Clark
AU  - Kianna M. Nguyen
AU  - Ming H. Ho
AU  - Jung-Eun Shin
AU  - Jared Feldman
AU  - Blake M. Hauser
AU  - Timothy M. Caradonna
AU  - Laura M. Wingler
AU  - Aaron G. Schmidt
AU  - Debora S. Marks
AU  - Jonathan Abraham
AU  - Andrew C. Kruse
AU  - Chang C. Liu
TI  - Rapid generation of potent antibodies by autonomous hypermutation in yeast
AID  - 10.1101/2020.11.11.378778
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.11.11.378778
4099  - http://biorxiv.org/content/early/2020/11/11/2020.11.11.378778.short
4100  - http://biorxiv.org/content/early/2020/11/11/2020.11.11.378778.full
AB  - 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 somatic hypermutation. However, animal immunization is inherently slow, has poor compatibility with certain antigens (e.g., integral membrane proteins), and suffers from self-tolerance and immunodominance, which limit the functional spectrum of antibodies that can be obtained. Here, we describe Autonomous Hypermutation yEast surfAce Display (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside 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 as little as 2 weeks. We applied AHEAD to generate nanobodies against the SARS-CoV-2 S glycoprotein, a GPCR, and other targets. The SARS-CoV-2 nanobodies, concurrently evolved from an open-source naïve nanobody library in 8 independent experiments, 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.Competing Interest StatementProvisional patents have been filed on this work, with A.W., C.M., A.C.K., and C.C.L. as co-inventors. A.C.K. is a co-founder and advisor of Tectonic Therapeutic, Inc., and of the Institute for Protein Innovation. C.C.L. is a co-founder of K2 Biotechnologies, Inc., which focuses on the use of continuous evolution technologies applied to antibody engineering.