Abstract 2357 Generation of target specific nanobodies

Nanobodies (Nb) are single chain antibodies that bind available protein epitopes with remarkable specificity making them valuable tools for molecular and structural biology studies. The goal of this work is to identify target specific nanobodies for downstream applications. We have established a pipeline for rapid generation of conformational specific Nb using a yeast phage display library that contains 108 different combinations of variable single chain Nb that differ only in their complementary determining regions that recognize and bind their targets. To select Nb against an antigen of interest, the Nb are tethered to the yeast cell wall. The yeast displaying the desired Nb bind to a fluor-labeled antigen of interest which allows for selective enrichment of yeast containing the desired Nb through magnetic-activated-cell-sorting (MACS) and florescence-activated-cell-sorting (FACS). After iterative rounds of isolation and amplification, Nb are sequenced and cloned into plasmids for expression using either the small-scale cell-free expression system or large-scale expression using bacterial systems. Here we present our pipeline as well as target specific Nb generated using this pipeline and their ability to bind to their target.