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 al-lows for conditional activation by proteolytic processing. The results warrant further studies evaluating the therapeutic and diagnostic applicability both in vitro and in vivo.

Automated summary

Conditional activation of engineered affinity proteins can be achieved by proteolytic processing. In this study, an anti-idiotypic masking domain was generated to block EGFR-binding on cancer cells when fused to an affibody molecule via a protease cleavage site linker. The results demonstrate the potential therapeutic and diagnostic applications of this approach and suggest further studies are warranted.