Affinity maturation of a cyclic peptide handle for therapeutic antibodies using deep mutational scanning

M. van Rosmalen, B.M.G. Janssen, N. Hendrikse, A.J. van der Linden, P.A. Pieters, D. Wanders, T.F.A. de Greef, M. Merkx

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Meditopes are cyclic peptides that bind in a specific pocket in the antigen-binding fragment of a therapeutic antibody such as cetuximab. Provided their moderate affinity can be enhanced, meditope peptides could be used as specific non-covalent and paratope-independent handles in targeted drug delivery, molecular imaging, and therapeutic drug monitoring. Here we show that the affinity of a recently reported meditope for cetuximab can be substantially enhanced using a combination of yeast display and deep mutational scanning. Deep sequencing was used to construct a fitness landscape of this protein-peptide interaction, and four mutations were identified that together improved the affinity for cetuximab 10-fold to 15 nM. Importantly, the increased affinity translated into enhanced cetuximab-mediated recruitment to EGF receptor-overexpressing cancer cells. Although in silico Rosetta simulations correctly identified positions that were tolerant to mutation, modeling did not accurately predict the affinity-enhancing mutations. The experimental approach reported here should be generally applicable and could be used to develop meditope peptides with low nanomolar affinity for other therapeutic antibodies.

Original languageEnglish
Pages (from-to)1477-1489
Number of pages13
JournalJournal of Biological Chemistry
Issue number4
Publication statusPublished - 27 Jan 2017


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