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

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 VH domain combined with only 36 VL domain variants yielding 3 × 1010 unique members in the phage-displayed library. After three rounds of panning the enriched VH genes from the primary library selections against lysozyme were incorporated into a ready-made circular single-stranded affinity maturation library containing 7 × 108 VL gene variants. Several unique antibodies with 0.8–10 nM (Kd, dissociation constant) affinities against lysozyme were found after panning from the affinity maturation library, contrasted by only one anti-lysozyme scFv clone with Kd