A yeast mating-based platform enables the generation and screening of ultra large antibody libraries

Yeast display is a powerful platform for antibody engineering, offering eukaryotic protein folding and compatibility with fluorescence-activated sorting (FACS). However, conventional yeast display faces limitations in terms of library size and display heterogeneity due to plasmid-based expression. Here, we present a yeast display platform that combines genomic library integration and high-efficiency mating to generate ultra-diverse antibody libraries approaching the diversity of phage-libraries (>1011). We engineered two yeast strains, LFYa and LFYalpha, featuring genomically integrated landing pads for high-efficiency library insertion. To enable compatibility with deep sequencing, we integrated the recombinase BxB1, which links heavy (HC) and light chain (LC) information onto a single chromosome. Using our platform, we constructed synthetic antibody libraries with HC and LC diversities of 4.1×107 and 1.7×107 variants, respectively and with an improved mating protocol, we achieved a combinatorial library diversity exceeding 1011. We demonstrated that genomic integration yields uniform surface display. Screening this antibody library against multiple antigens resulted in the discovery of binders with affinities in the single-digit nanomolar to picomolar range, demonstrating the platform’s utility for the discovery of antibodies with therapeutically relevant affinities. This work establishes a robust and deep sequencing-compatible yeast display system that overcomes key limitations of previous mating-based platforms.