Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Engineering a non-antibody scaffold for binding to therapeutic targets  (#110)

Peter G Chandler , Blake T Riley , Sebastian S Broendum , James S Green , David E Hoke , Ashley M Buckle

As a non-antibody scaffold, the fibronectin type III (FN3) domain overcomes antibody size and complexity while maintaining analogous binding loops. Previously, We have 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, Adnectin, 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. In order to overcome this stability-function trade-off, we grafted binding loops from Adnectin-α-VEGFR2 and Adnectin-α-PCSK9 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. Grafts demonstrated varying degrees of binding affinity to the targets, and further structural analysis identified that regions of the scaffold disrupted binding.

These loop grafting 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.

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