(531d) Enriching Inhibitory Proteins Using a Tethered Yeast Surface Display System

Protein engineering for therapeutic applications is a field that has seen much growth and innovation in the past two decades, but challenges remain. Diseases, such as autoimmune diseases, infectious diseases and cancer can all be related to irregular biological activity stimulated by specific sets of proteins. A valid method of therapy is the identification and inhibition of proteins which play key roles in symptom expression and disease progression. One of the greatest deficiencies in current strategies is a means to efficiently tune screening of candidate proteins to recover active binders, those that bind and demonstrate function. This requires an appropriate selective pressure. The goal of this work is to create an efficient screening platform for inhibitory proteins which utilizes a concept known as tethering to perform this selective screening in a high-throughput manner using yeast surface display. We have designed a bivalent yeast surface displayed construct which aims to take advantage of high local concentrations provided by tethering a library of candidate inhibitory proteins to a target protein of interest (POI) via a flexible peptide linker. These high local concentrations will allow for library screening based on decrease of signal due to inhibitory function when the bivalent construct is assayed in the presence of a competitive binder to the POI. We have shown expression of these complex tethered constructs and functional inhibition in this context, wherein a Cetuximab single chain variable fragment (scFv) tethered to the extracellular domain of the epidermal growth factor receptor (EGFR) inhibits EGFR binding to the Cetuximab antibody when compared to a control where the Cetuximab scFv is replaced by a non-EGFR binding Bevacizumab scFv. This behavior was recapitulated in an atezolizumab-PD-L1 system as well. We have also shown our system is capable of robust enrichment of functional binders from mock libraries when sorted via flow cytometry at a range of affinities. The effect of linker length and structure on the titratability and efficiency of the system has also been investigated, revealing differential functional outcomes. Applications of this system to generate novel, active binding proteins will also be discussed.