Abstract
Here we applied ribosome display to in vitro selection and evolution of single-chain antibody fragments (scFvs) from a large synthetic library (Human Combinatorial Antibody Library; HuCAL) against bovine insulin. In three independent ribosome display experiments different clusters of closely related scFvs were selected, all of which bound the antigen with high affinity and specificity. All selected scFvs had affinity-matured up to 40-fold compared to their HuCAL progenitors, by accumulating point mutations during the ribosome display cycles. The dissociation constants of the isolated scFvs were as low as 82 pM, which validates the design of the naïve library and the power of this evolutionary method. We have thus mimicked the process of antibody generation and affinity maturation with a synthetic library in a cell-free system in just a few days, obtaining molecules with higher affinities than most natural antibodies.
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Acknowledgements
This work was supported by the Schweizerischer Nationalfonds grant 31-46624.96. C.S. is supported by a predoctoral Kékule fellowship from the Fonds der Chemischen Industrie (Germany). The authors would like to acknowledge Annemarie Honegger, Lutz Jermutus, and Stephen Marino for help, advice, and discussion, and MorphoSys AG for the constructive collaboration on HuCAL.
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Hanes, J., Schaffitzel, C., Knappik, A. et al. Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display. Nat Biotechnol 18, 1287–1292 (2000). https://doi.org/10.1038/82407
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DOI: https://doi.org/10.1038/82407
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