title: Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning.
abstract: 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.
authors.affiliation: From the Laboratory of Chemical Biology and Institute for Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands.
authors.forename: Martijn
authors.initials: M
authors.lastname: van Rosmalen
authors.affiliation: From the Laboratory of Chemical Biology and Institute for Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands.
authors.forename: Brian M G
authors.initials: BM
authors.lastname: Janssen
authors.affiliation: From the Laboratory of Chemical Biology and Institute for Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands.
authors.forename: Natalie M
authors.initials: NM
authors.lastname: Hendrikse
authors.affiliation: From the Laboratory of Chemical Biology and Institute for Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands.
authors.forename: Ardjan J
authors.initials: AJ
authors.lastname: van der Linden
authors.affiliation: From the Laboratory of Chemical Biology and Institute for Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands.
authors.forename: Pascal A
authors.initials: PA
authors.lastname: Pieters
authors.affiliation: From the Laboratory of Chemical Biology and Institute for Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands.
authors.forename: Dave
authors.initials: D
authors.lastname: Wanders
authors.affiliation: From the Laboratory of Chemical Biology and Institute for Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands.
authors.forename: Tom F A
authors.initials: TF
authors.lastname: de Greef
authors.affiliation: From the Laboratory of Chemical Biology and Institute for Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands m.merkx@tue.nl.
authors.forename: Maarten
authors.initials: M
authors.lastname: Merkx
mesh_terms: D045744:Cell Line, Tumor; D000068818:Cetuximab / Q000737:chemistry*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae
chemical_list: D010456:Peptides, Cyclic; C512478:EGFR protein, human; D066246:ErbB Receptors; D000068818:Cetuximab
keywords: antibody; cyclic peptide; directed evolution; epidermal growth factor receptor (EGFR); protein engineering