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 (>10 11 ). 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 4.1x10 7 and 1.7x10 7 variants, respectively and with an improved mating protocol, we achieved a combinatorial library diversity exceeding 10 11 . 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.