Structural differences among monoclonal antibodies with distinct fine specificities and kinetic properties

The mAbs HyHEL-8, HyHEL-26 (HH8, and HH26, respectively) recognize epitopes on hen egg-white lysozyme (HEL) highly overlapping with the structurally d efined HH10 epitope, while the structurally related XRPC-25 is specific for DNP and does not bind HEL. All four Abs appear to use the same V(k)23 germ line gene, and all but HH8 use the same V(H)36-60 germ line gene. Of the t hree anti-MEL Abs, the sequences of HH26 variable regions are closest to th ose encoded by the respective germ line sequences. HH8 utilizes a different member of the V(H)36-60 gene family. Thus, the same V-k and V-H genes, com bined with somatically derived sequence differences, are used to recognize the unrelated Ags HEL and DNP. In contrast, different V(H)36-60 germ line g enes are used to bind the same antigen (e.g. HH8 vs HM10 and HH26). While t he affinities of HH10, HH8, and HH26 for HEL vary by less than 10-fold, the ir affinities for mutated Ag vary over several orders of magnitude. Analyse s of Fab binding kinetics with natural species variants and site-directed m utants of lysozyme indicate that these cross-reactivity differences reflect the relative sensitivities of both the association and dissociation rates to antigenic mutation: HH8 has relatively mutation-insensitive association and dissociation rates, HH10 has a relatively mutation-sensitive associatio n rate but more variable dissociation rates, and HH26 has variable associat ion and dissociation rates. Only a few amino acid differences among the ant ibodies produce the observed differences in the robustness of the associati on and dissociation rates. Our results suggest that association and dissoci ation rates and mutation sensitivity of these rates may be independently mo dulated during antibody repertoire development. (C) 2000 Published by Elsev ier Science Ltd. All rights reserved.