Development of a specific protein-based masking domain for EGFR binding.

Engineering and evaluation of a protease activated EGFR-specific affibody molecule.

Generation of an anti-idiotypic masking domain using directed evolution by bacterial display.

Conditional activation of engineered affinity proteins by proteolytic processing is an interesting approach for a wide range of applications. We have generated an anti-idiotypic masking domain with specificity for the binding surface of an EGFR-targeting affibody molecule using an in-house developed staphylococcal display method. The masking domain could specifically abrogate EGFR-binding on cancer cells when fused to the EGFR-targeting affibody molecule via a linker comprising a protease cleavage site. EGFR-binding was restored by proteolytic cleavage of the linker region resulting in release of the masking domain. A saturation mutagenesis study provided detailed information on the interaction between the EGFR-targeting affibody molecule and the masking domain. Introducing an anti-idiotypic masking affibody domain is a viable approach for blocking EGFR-binding and allows for conditional activation by proteolytic processing. The results warrant further studies evaluating the therapeutic and diagnostic applicability both in vitro and in vivo.

Albumin binding protein

Epidermal growth factor receptor

fluorescence-assisted cell sorting

magnetic activated cell sorting

Surface Plasmon Resonance

Affibody molecule

Conditional activation

Epidermal growth factor receptor

Staphylococcal display

© 2022 The Author(s). Published by Elsevier B.V.