A Sortase A Programmable Phage Display Format for Improved Panning of Fab Antibody Libraries

Phage display of combinatorial antibody libraries is a versatile tool in the field of antibody engineering, with diverse applications including monoclonal antibody (mAb) discovery, affinity maturation, and humanization. To improve the selection efficiency of antibody libraries, we developed a new phagemid display system that addresses the complication of bald phage propagation. The phagemid facilitates the biotinylation of fragment of antigen binding (Fab) antibody fragments displayed on phage via Sortase A catalysis and the subsequent enrichment of Fab-displaying phage during selections. In multiple contexts, this selection approach improved the enrichment of target-reactive mAbs by depleting background phage. Panels of cancer cell line-reactive mAbs with high diversity and specificity were isolated from a naïve chimeric rabbit/human Fab library using this approach, highlighting its potential to accelerate antibody engineering efforts and to empower concerted antibody drug and target discovery. [Display omitted] •A phagemid display system for site-specific biotinylation of Fabs displayed on phage was developed.•Based on this phagemid display system, a Fab-phage biotinylation and capture (FBC) approach, which depletes phage that do not display Fabs, was established.•A naïve rabbit antibody library that leverages FBC was generated and selected against whole cells.•FBC improves target-reactive mAb enrichment.