In vitro Evolution of Antibody Affinity via Insertional Scanning Mutagenesis of an Entire Antibody Variable Region
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We report the first systematic combinatorial exploration of affinity enhancement of antibodies by insertions and deletions (InDels). Transposon-based introduction of InDels via the method TRIAD was used to generate large libraries with random in-frame InDels across the entire scFv gene that were further recombined and screened by ribosome display. Knowledge of potential insertion points from TRIAD libraries formed the basis of exploration of length and sequence diversity of novel insertions by insertional-scanning mutagenesis (InScaM). An overall 256-fold affinity improvement of an anti-IL-13 antibody BAK1 as a result of InDel mutagenesis and combination with known point mutations validates this approach and suggests that the results of this InDel approach and conventional exploration of point mutations can synergize to generate antibodies with higher affinity.
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Biotechnology and Biological Sciences Research Council (BB/L002469/1)
European Commission Horizon 2020 (H2020) Marie Sk?odowska-Curie actions (750772)