Engineering conditional binding for enhanced protein therapeutics

QC 2024-11-21

Abstract

Protein therapeutics hold great potential in cancer treatment as they combine specificity with effective delivery to tumor sites, but traditional antibodies face limitations related to size, production cost, and stability. As an alternative, smaller protein scaffolds present a promising approach, offering reduced costs due to production in bacterial hosts, greater stability, and versatile engineering potential for diverse functionalities.

This thesis aims to contribute to the field of engineered scaffold proteins, optimizing the discovery workflow, introducing a new conditional binding scaffold, and evaluating its applicability as protein-drug conjugates for targeted cancer therapies.

The first part of this thesis aims to streamline the discovery pipeline for protein scaffolds. Study I presents an optimized high-throughput phage display system incorporating automated selection and sequencing techniques to discover protein binders efficiently, as demonstrated with the albumin binding domain-derived affinity protein (ADAPT) and Calcium-regulated affinity (CaRA) libraries. This semi-automated system reduces hands-on time and increases robustness, making the discovery process more accessible for labs without high-cost equipment. Results show the possibility of generating high-affinity binders with broad applications in diagnostics and therapeutics. Following the foundation laid by the workflow optimization, Study II introduces the CaRA library more deeply. The library is engineered to provide calcium-dependent and pH-dependent binding capabilities. The library’s design enables conditional interactions, where calcium levels modulate binding. This feature is particularly beneficial for therapeutic applications requiring precise targeting and controlled binding release. The CaRA scaffold demonstrated stability, nanomolar affinities, and calcium-dependent binding across diverse targets, with potential in both therapeutic and biotechnological settings. Study III introduces an accelerated maturation process for conditional binders to further enhance the therapeutic potential of small scaffold proteins. Utilizing deep sequencing data from initial selections with the CaRA library and E. coli display screening, a high-affinity binder for HER3 with pH-dependent binding, CaRA_HER3, was developed. This characteristic allows for rapid release in acidic environments, mimicking endosomal conditions, which could be advantageous for intracellular drug delivery. The final paper, Study IV, focuses on applying CaRA binders in developing protein-drug conjugates for targeted cancer treatment. Specifically, a CaRA-based EGFR binder (CaRA_EGFR) was engineered to bind EGFR conditionally, depending on calcium levels. This calciumregulated binding allows the protein to dissociate in the low-calcium environment of endosomes, potentially enhancing cytotoxic drug delivery directly to tumor cells. Confocal microscopy confirmed that the CaRA_EGFR binder effectively internalizes and trafficks to lysosomes, achieving targeted cytotoxicity in EGFR-expressing cells. This approach highlights the value of conditional affinity in challenges related to the biological fate of receptors, paving the way for more effective, receptor-specific drug delivery systems. This thesis advances protein engineering for small scaffold therapeutics through new discovery workflows and calcium- and pH-dependent binding mechanisms. By advancing new ways to engineer these scaffolds, the findings contribute to developing safer, more effective proteinbased therapies for cancer treatment.

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