Affinity improvement of CB1. (A) Flow chart of CB1 affinity maturation. Combinations of CDR loops were diversified in different libraries by soft randomization (see “Materials and methods”) or restricted randomization mimicking natural antibody diversities (underlined). The CDR combinations shown in bold text indicate the libraries from which improved binders were isolated. (B) Amino acid sequences of CB1 CDRs as deduced from DNA sequencing are aligned with the template of the synthetic antibody libraries, h4D5. The positions randomized in the libraries are in bold. Alterations in the affinity-improved variants are shown with their affinities measured as Fab-phage IC₅₀ values in competition ELISA, as Fab K_D values measured with SPR technology (BIAcore), or as IgG protein IC₅₀ values competing with BAFF for binding to mBR3-(BHK) or hBR3-(BJAB) expressing cells, as described in “Materials and methods.”