Synthetic human antibody libraries used by Lee and colleagues are based on phage-displayed bivalent F(ab′)2 or monovalent Fab with randomized CDRs. Starting out from bivalent F(ab′)2 libraries that use avidity to make up for weak affinities, a Fab lead with micromolar affinity to the murine antigen and even weaker cross-reactivity with the human antigen was selected. Iterative optimization strategies that involved both simultaneous and sequential selections of randomized CDRs resulted in a final Fab that bound both murine and human antigen with subnanomolar affinity. Compared with the lead Fab, the final Fab had acquired 13 mutations spread over 4 CDRs (bold). Illustration by Paulette Dennis.
