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.

Clones were sequenced to identify appropriate amino acid changes and arrayed in duplicate into 96-well plates with two amino acid positions per plate ($20 amino acids per position for a total of 40 clones). Thus, the total number of plates screened for the 67 amino acids found in the CDR regions was 34 '96-well plates' ($1350 clones). These antibody variants were screened in the same functional and quantitative ELISAs as the framework reassembly screen. From this initial screen, 330 clones ($24%) were chosen which exhibited a specific activity when normalized to the mouse -human chimera !1.0. These clones derived from the primary screen were assayed in duplicate again in the same ELISA assays and the top 10 performers chosen. These 10 antibodies, when normalized to the mouse -human chimera, showed specific activities ranging from 1.16 to 1.92 (Fig. 5) . The purified antibodies were subsequently analyzed in PRNTs with two of the GSSM TM variants achieving 80% neutralization at $3.5-fold (52G3) and .8-fold (51E7) lower concentrations than that of the WT chimeric antibody. For the 51E7 variant, the neutralization activity was .80% at the lowest titer used (i.e. 0.195 mg/ml). Clones not shown had 80% neutralization capabilities at higher concentrations than the WT chimera, ranging from 3.12 to 12.5 mg/ml.