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.

Our experience suggests that there is often a significant additive or synergistic effect on functional activity when combining beneficial single point mutations (Gray et al., 2001; Palackal et al., 2004) . In addition, we wanted the best antibodies to have humanized frameworks. Thus, a new library was constructed to place all combinations of the positive or neutral single point mutations into the backbone of the framework reassembly clones. The two framework clones used as the backbone for the library were 61G4 and 61H4. Fig. 3 . Binding of the spike protein-antibody mixture to Vero E6 cells. Vero E6 cells were analyzed by flow cytometry using a bandpass filter of 580/30 with collection of 10 000 cells. (A) Cells incubated with streptavidin-phycoerythrin only, (B) cells incubated with 4 nM spike protein and a bacterial lysate from cells expressing an irrelevant antibody, (C) same as (B) but with an anti-spike antibody that does not block binding, (D -E) same as (B) but with three unique anti-spike antibodies that block binding of spike to its receptor. All antibodies were added at a 12 nM concentration with the exception of (D) which was at 8 nM. Listed % represents the % of cells following in the defined gate. These clones had unique light chains but were paired with the same heavy chain framework clone. Four different point mutations (51E7, 52G3, 51H8, 51E2) were chosen from the light chain and a single mutation from the heavy chain (49B9) ( Table II ). The total library size was 64 possible combinations and it was screened on the same two ELISAs as described above. The top 10 performers all had $1.75-fold or better binding activity when compared to the WT chimera (Fig. 5) .