Integrating Fab Phage and Yeast Display through Seamless Transfer of Paired Heavy and Light Chain Libraries for Efficient Antibody Discovery

Abstract Background In vitro display technologies, especially phage and yeast display, are revolutionary tools in antibody discovery, enabling high-throughput screening and engineering. Previous efforts to integrate phage and yeast display for synergistic antibody discovery have predominantly focused on scFvs and sdAbs. Methods Here, we developed an integrated phage-yeast display platform for efficient Fab antibody discovery. We transferred paired Fab libraries from phage output into yeast display vectors while preserving VH-VL pairings with the engineered compatible Fab display vectors that we developed for both phage and yeast. Specifically, we identified restriction enzyme sites that are rare in antibody variable regions and designed novel signal peptides compatible with these sites for Fab display. These signal peptides performed at least as well as commonly used benchmarks (such as PelB or StII for phage display, and AGA2 or MFAL1 for yeast display) in bio-panning experiments. Our modular vector architecture supports straightforward library shuttling across mouse-derived, fully human, and naïve Fab libraries. Results Using a mouse immune Fab library, we applied our integrated platform to isolate antibodies against a soluble protein. Following two rounds of liquid-phase phage display and four rounds of FACS combined with a yeast-based receptor-blocking screening method developed herein, we successfully isolated antigen-specific Fabs with sub-nanomolar affinities and potent receptor-blocking activity. Conclusions Our integrated approach leverages the capacity of phage display to screen large Fab libraries while harnessing the strength of yeast display for functional screening, exemplified here by the successful identification of receptor-blocking antibodies, thereby streamlining the discovery of high-quality Fab candidates.