Rapid discovery and optimization of therapeutic antibodies against emerging infectious diseases

Rogers, J.; Schoepp, R.J.; Schröder, O.; Clements, T.L.; Holland, T.F.; Li, J.Q.; Li, J.; Lewis, L.M.; Dirmeier, R.P.; Frey, G.J.; Tan, X.; Wong, K.; Woodnutt, G.; Keller, M.; Reed, D.S.; Kimmel, B.E.; Tozer, E.C.

The DNA display method described here combines the in vivo production of proteins and an in vitro screen for desired protein characteristics. The in vitro screen allows for a highthroughput analysis of complex libraries with great diversity. For in vivo production, we made functional Fabs in specific E. coli redox strains that allowed the proper intracellular environment for Fab production at sufficient concentrations to perform a screen. The Fab was produced as a fusion of a zinc finger to the Fab heavy chain. The opposite construction with the light chain fused to the zinc finger resulted in a lower level of enrichment (data not shown). The heavy chain alone is known to be insoluble (Davies and Riechmann, 1994) and thus, we postulate that the fusion with the zinc finger, in combination with the proper oxidizing environment, could make the heavy chain soluble long enough to pair with the light chain (the light/heavy chain binding results in a soluble Fab). Due to the intracellular production of the targeted protein during DNA display, broad applications for a variety of protein-based interactions are possible. One could use the method to probe any protein -protein or protein -nucleic acid interactions in vivo, unlike a phage display screen. As well, because the proteins are produced in vivo, they could have access to chaperones that allow proper folding. Furthermore, DNA display is simpler than phage display in that there is no need to make a phage preparation and the entire library can be screened in about half the time ($1.5 days compared to three days).