Poster Presentation The 42nd Lorne Conference on Protein Structure and Function 2017

Engineering a non-antibody scaffold for binding to therapeutic targets (#119)

Peter G Chandler 1 , David E Hoke 1 , Blake T Riley 1 , Sebastian S Broendum 1 , Paul J Conroy 1 , Ashley M Buckle 1
  1. Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Victoria 3800, Australia

As a non-antibody scaffold, the fibronectin type III (FN3) domain overcomes antibody size and complexity while maintaining analogous binding loops [1]. We have previously produced FN3Con, an ultra-stable FN3 scaffold with significant potential for engineering for therapeutic purposes [2]. Previously a wild-type FN3 domain, FNfn10, has undergone evolution for targeted binding to the therapeutic targets VEGFR2 [3] and PCSK9 [4], but such function is gained at the expense of large losses in stability. In order to overcome this stability-function trade-off, we grafted binding loops from FNfn10-α-VEGFR2 and FNfn10-α-PCSK9 onto the FN3Con scaffold to produce domains with binding to therapeutic targets and to further develop the FN3Con scaffold for evolution of function. These FN3Con grafts were expressed solubly in bacteria with little aggregation. The FN3Con scaffold was robust to loop grafting, maintaining characteristically high thermostability. Binding affinity to the targets was also transferred to varying degrees. The consensus design of FN3Con resulted in a fibronectin domain that is robust to engineering for function. Loop grafting studies examine the ability of FN3Con to mimic the targeted binding of antibodies and identify components of the scaffold that are disruptive of binding. This study widens our knowledge of the FN3Con scaffold’s ability to be engineered for function, providing insights for further redesign of the scaffold towards therapeutic application.

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