NEXT-GENERATION SEQUENCING AND MOTIF GRAFTING APPLICATIONS IN SYNTHETIC ANTIBODY DISCOVERY

Abstract

The overall objective of this PhD project was to develop and validate methods for advancing the applications of two techniques, next-generation sequencing (NGS) and motif grafting, in synthetic antibody discovery. In the first part of this project, we developed an NGS-assisted antibody discovery platform by integrating phage-displayed single-framework synthetic antigen- binding fragment (Fab) libraries with Ion Torrent sequencing. We constructed a new single- framework synthetic Fab library containing 8.5 billion unique Fab clones, and validated its functionality by generating high affinity Fabs against Notch and Jagged receptors. We developed a rapid and simple method to link and sequence all diversified complementarity-determining regions (CDRs) in phage Fab pools without losing the CDR pairing information. We identified and reconstructed low-frequency rare Fab clones from NGS information in a reliable and high- throughput manner. In some cases, reconstructed rare clones (frequency ~0.1%) showed higher affinity and better specificity than high-frequency top clones isolated by Sanger sequencing, highlighting the importance of NGS in synthetic antibody discovery. In the second part of this project, we employed motif grafting to semi-rationally design phage-displayed synthetic Fab libraries that are biased towards interacting with a specific site on a receptor. We used structural information on the epidermal growth factor receptor (EGFR) homo-dimerization interaction to design a structure-guided Fab library that was biased towards interacting with domain II of EGFR. We used this structure-guided Fab library to obtain Fabs against the EGFR extracellular domain. For comparison, we used a naïve synthetic Fab library to generate an anti-EGFR Fab whose binding overlapped with the Fab isolated from the structure-guided Fab library. Both Fabs possessed low-nM binding values for recombinant and cell-surface EGFR and inhibited EGF- mediated EGFR activation. Epitope mapping showed that domain II is partially responsible for the interaction of Fabs with EGFR. Further, both Fabs target unique epitopes that are different from previously validated epitopes on EGFR. In total, this PhD project resulted in novel methods for discovering synthetic antibodies using NGS and motif grafting techniques, three functional Fab libraries and numerous high-affinity Fabs against Notch, Jagged and EGF receptors.