Effects on interaction kinetics of mutations at the VH-VL interface of Fabs depend on the structural context

The influence of framework residues belonging to VH and VL modules of antib ody molecules on antigen binding remains poorly understood. To investigate the functional role of such residues, we have performed semi-conservative a mino acid replacements at the VH-VL interface. This work was carried out wi th (i) variants of the same antibody and (ii) with antibodies of different specificities (Fab fragments 145P and 1F1h), in order to check if functiona l effects are additive and/or similar for the two antibodies. Interaction k inetics of Fab mutants with peptide and protein antigens were measured usin g a BIACORE(R) instrument. The substitutions introduced at the VH-VL interf ace had no significant effects on k(a) but showed small, significant effect s on k(d), Mutations in the VH module affected k(d) not only for the two di fferent antibodies but also for variants of the same antibody, These effect s varied both in direction and in magnitude. In the VL module, the double m utation (FL)-L-L37-Q(L38)L, alone or in combination with other mutations, c onsistently decreased kd about two-fold in Fab 145P. Other mutations in the VL module had no effect on kd in 145P, but always decreased kd in 1F1h, Mo reover, in both systems, small-magnitude non-additive effects on k(d) were observed, but affinity variations seemed to be limited by a threshold, When comparing functional effects in antibodies of different specificity, no ge neral rules could be established. In addition, no clear relationship could be pointed out between the nature of the amino acid change and the observed functional effect. Our results show that binding kinetics are affected by alteration of framework residues remote from the binding site, although the se effects are unpredictable for most of the studied changes. Copyright (C) 2000 John Wiley & Sons, Ltd.