As a non-antibody scaffold, the fibronectin type III (FN3) domain overcomes antibody size and complexity while maintaining analogous binding loops. We have previously produced FN3Con, an ultra-stable FN3 scaffold with significant potential in engineering for therapeutic purposes. We then aimed to examine if the scaffold could take on antibody-like binding to therapeutic targets.
In an earlier study, a wild-type FN3 domain was subjected to directed evolution for targeted binding to the therapeutic targets VEGFR2 and PCSK9, but such function was gained at the expense of large losses in thermostability and increased aggregation. To overcome this stability-function trade-off, we grafted anti-VEGFR2 and anti-PCSK9 binding loops onto the FN3Con scaffold to produce domains that successfully bound to therapeutic targets. The resulting FN3Con grafts were expressed solubly in bacteria with little aggregation, and maintained characteristically high thermostability. Further, we developed a library of FN3Con domains which can be screened for binding to novel targets through Yeast Surface Display. Variants of FN3Con were selected from a screen for binding against Lysozyme, which show evolved affinity for the target.
These loop grafting and directed evolution studies examine the ability of FN3Con to mimic the targeted binding of antibodies, and support the assertion that consensus design of FN3Con resulted in a fibronectin domain that is robust to engineering for function. Future work will focus on expanding the repertoire of binding targets and the ability of the FN3Con scaffold to tolerate further functionalization with an aim towards therapeutic or diagnostic applications.