Synthetic single-framework antibody library integrated with rapid affinity maturation by VL shuffling

Abstract

Affinity maturation is often applied to improve the properties of antibodies isolated from universal antibody libraries in vitro. A synthetic human scFv antibody library was constructed in single immunoglobulin framework to enable rapid affinity maturation by updated Kunkel's mutagenesis. The initial diversity was generated predominantly in the V_H domain combined with only 36 V_L domain variants yielding 3 × 10¹⁰ unique members in the phage-displayed library. After three rounds of panning the enriched V_H genes from the primary library selections against lysozyme were incorporated into a ready-made circular single-stranded affinity maturation library containing 7 × 10⁸ V_L gene variants. Several unique antibodies with 0.8–10 nM (K_d, dissociation constant) affinities against lysozyme were found after panning from the affinity maturation library, contrasted by only one anti-lysozyme scFv clone with K_d H domain diversity to affinity maturation without bimolecular ligation leading to a diverse set of antibodies with binding affinities in the low nanomolar range.