Flores-Mora, F. E.; Brodsky, J.; Cerna, G. M.; Tse, A.; Hoover, R. L.; Bartelle, B. B.

Despite >50 years of methods development, specific antibodies are still generated at low throughput and remain in high demand across biotechnology. Most biologics and immunoprobes are monoclonal antibodies, developed using a combination of inoculating animals with a target antigen, engineered candidate libraries, and multiple rounds of selection using phage or yeast display. Here we introduce a synthetic biology scheme to eliminate the need for nearly all of these steps, by combining Surface display on E. coli and Phage display with the microvirus {Phi}X174, Assisting Continuous Evolution (SurPhACE). Instead of building libraries for screening, SurPhACE runs a closed evolutionary program. A typical experiment can have 1011 mutant candidates under active selection, with complete turnover of the mutant population every 30min, or >5x1012 unique mutants per day, using less than 100mL of bacterial culture media. We demonstrate SurPhACE for optimizing a nanobody to a related epitope, and develop novel nanobodies for an arbitrary target using a minimal starting library to establish a proof of concept and identify best practices for this scalable method for generating protein binders.