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.

Gene site saturation mutagenesis (GSSM TM ) was performed as described previously (Kretz et al., 2004) using 32-fold degenerate oligonucleotides to randomize each codon in the CDR regions so that all possible amino acids would be encoded. The mutated DNA pool was transformed into XL1-Blue cells and all possible variants for each site were identified by sequence analysis. After sequencing two 96-well plates (188 clones), we found that at least 16 (usually greater) of the possible 20 amino acids were represented at most positions and decided this diversity was sufficient for the screen. Approximately 13% of the residues required an extra 96-well plate of variants sequenced in order to achieve at least 16 amino acid changes.