Surface Display For Phage Assisted Continuous Evolution: A Platform For Evolving / Screening Nanobodies In Prokaryote Systems

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 ΦX174, Assisting Continuous Evolution (SurPhACE). Instead of building libraries for screening, SurPhACE runs a closed evolutionary program. A typical experiment can have 10 ¹¹ mutant candidates under active selection, with complete turnover of the mutant population every 30min, or >5×10 ¹² 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.