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.

Unlike a rational approach, GSSM TM does not require any knowledge of the structure of the protein. In contrast to the other random approaches such as error-prone PCR, chemical mutagenesis, and site-directed mutagenesis by sequence overlap extension PCR, GSSM TM is comprehensive and thus allows access to substitutions missed by these other methods. Using this method for an enzymatic protein, it was observed that the thermostability of a haloalkane dehalogenase was increased by 30 000-fold when combining eight single-point mutations discovered by GSSM TM (Gray et al., 2001) . Of the eight mutations, three required multiple base pair changes at each codon, thus the chance of finding these mutations using error-prone PCR would be exceedingly rare (Kretz et al., 2004) . Similarly, for the optimized antibodies presented here, three of the five amino acid positions incorporated into the reassembly screen required multiple base pair changes at each position (Table II) . In this study, the best single point mutation (i.e. 51E7) resulted in .8-fold improvement in functional activity of the antibody. It is possible that if mutagenesis of the entire variable region (instead of simply the CDRs) had been done, a further improvement in neutralization would have been observed.