Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning

Abstract

Edited by Luke O'NeillMeditopes 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.

Nonproteinogenic deep mutational scanning of linear and cyclic peptides

“…E scores alone are sufficient for analysis of deep mutational scanning data ( 2 – 9 ). However, to aid interpretation of the mutant data and any structure–activity relationships that follow, we chose to calibrate log 2 E against a handful of experimentally validated mutants and calculate true thermodynamic parameters, ∆∆ G / K D , for each mutant.…”

“…Deep mutational scanning methods, in particular, allow for the analysis of many thousands of mutants ( 2 ): Mutant proteins or peptides are each coupled to their encoding DNA, libraries of pooled mutants are sorted for activity, mutants are counted via deep sequencing, and each mutant is scored. The throughput of these experiments is sufficient for exhaustive saturation mutagenesis; that is, testing all proteinogenic amino acids at all positions in a sequence and returning all effects on folding ( 3 – 5 ), binding ( 2 , 5 – 8 ), or function ( 9 ). Extensive structure–activity maps are produced, but these methods are currently limited to the chemistry accessible within the universal genetic code—the 20 proteinogenic amino acids.…”

Dual-Color Bioluminescent Sensor Proteins for Therapeutic Drug Monitoring of Antitumor Antibodies

“… 27 In a recent study using deep mutational scanning, we identified three mutations that together increased the affinity of the meditope peptide for cetuximab further to 60 nM (CVFDLGTRRLRC, meditope 2). 40 Two LUMABS variants were cloned and expressed that contained either meditope 1 or meditope 2. To assess the importance of peptide cyclization, we also included a third variant, (CTX-LUMABS-3) that contains a linear version of meditope 2 in which the cysteines were replaced by alanines (AVFDLGTRRLRA, meditope 3).…”

Lasso-grafting of macrocyclic peptide pharmacophores yields multi-functional proteins

“…However, the rst-generation binders obtained from such partially randomized protein libraries often show modest level of a nity due to the limited size of the library diversity, and it is often necessary to conduct additional selection campaigns for identifying improved binding clones worthy of the practical utility 27 . This second step usually involves directed evolution or saturation mutagenesis 28,29 , and leads to a signi cant extension of development time and increase the uncertainty of the success.…”