Generation of an anti-idiotypic affibody-based masking domain for conditional activation of EGFR-targeting.

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.
Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: SS is a minority shareholder of Affibody AB. The other authors declare no conflict of interest.
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