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.

With the potential for these antibodies to be used as human therapeutics, our HuFR TM technology was used to convert the mouse variable region sequences of the 4049Fab14 antibody to human antibody sequences. The random combination of all the possible heavy and light chains results in 62 720 combinations (280 Â 224, respectively). To limit the size of the screen, it was divided into two parts with the heavy chain library screened first followed by the light chain library. The first screen used eight fixed light chains composed of the three CDRs of the mouse variable region (4049Fab14) together with consensus sequences from the selected pools of the four defined framework families in the variable region. These eight light chains were each paired with a heavy chain library composed of 176 unique variants arrayed in duplicate on four 96-well plates (88 clones per plate) for a total of 32 '96-well plates' (1408 clones). The supernatants derived from the transfected framework library were assayed both for functional activity (i.e. reactivity to the SARS spike protein) and for relative antibody expression levels (i.e. production of sufficient antibody). From this initial screen, 176 unique hits (12.5%) were chosen that had a specific activity of 0.7 or greater when normalized to the mouse-human chimera (i.e. 4049Fab14). Following confirmation of the hits (assayed in duplicate) using the same two ELISA assays, the top 10 performers were chosen based on normalized specific activity (i.e. the degree of binding activity to the purified spike protein normalized to IgG concentration and further normalized to the specific activity of the original mouse-human chimeric antibody). The 10 best performers had specific activities ranging from 0.9 to 1.381 (Fig. 4) . The second screen consisted of these 10 heavy chain candidates paired with the light chain library composed of 176 variants arrayed in duplicate on four 96-well plates for a total of 40 '96-well plates' (1760 clones). From this initial screen, 352 hits ($20%) were chosen with a specific activity .1.1 when compared to the original 4049Fab14 antibody. These hits derived from the primary screen were assayed again in duplicate using the same ELISA assays. The top 10 performers from this secondary screen exhibited specific activities ranging around 1.5-2-fold (with a high of 2.8 and a low of 1.12) greater than the mouse -human chimera (Fig. 4) . These high and low values were chosen due to the very high level of expression (1.12 value) and very low expression but relatively high functional activity (2.8 value). Five of the 10 heavy chains appeared in these top light chain candidates with one of the heavy chains (23F9) represented in five of the clones.