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.

This work introduces a novel DNA display method for rapid screening of functional antibodies. As with other display technologies, this method bypasses the laborious task of hybridoma production and perhaps more importantly, allows access to the total diversity of antibodies found within the mice. In fact, as all heavy chains have the chance to interact with all light chains, this method can provide even greater diversity than found in mice. After screening an immunized mouse library for antibodies against the spike protein, three antibodies were isolated that inhibited infection of SARS-CoV in Vero E6 cells. This library was recovered from three immunized mice that had been paired with three additional mice immunized with the same virus on the same schedule. The other three mice had significantly higher serum antibody titers against SARS-CoV and were used to generate antibodies following the traditional hybridoma approach. Although the analysis of the hybridomas was conducted solely with the SARS virus rather than with the spike protein alone, it is interesting to note that no neutralizing Discovery and optimization of therapeutic antibodies antibodies were found using the hybridoma approach (data not shown) while multiple neutralizing antibodies were found using DNA display. There are many protein display systems described in the literature, generally falling into three categories: viral display, cell surface display and DNA/RNA display (Li, 2000) . Phage-based systems are among the more commonly used methods, typically based on fusion of the protein of interest to a coat protein expressed on the phage surface. Despite significant successes, there are some limitations to this approach. For example, fusion to phage proteins or other aspects of phage display may interfere with the binding properties of the protein of interest. As well, multi-subunit proteins are more difficult to screen by phage display. Finally, phage display proteins are transported to the cell surface, where the in vitro environment might not be suitable for some protein -protein interactions. For ribosome display, the limitation is that proteins are made entirely in vitro, and thus might not have the right chaperones, necessary cofactors, or appropriate redox state to fold properly.