FUNCTIONAL MAPPING OF CONSERVED RESIDUES LOCATED AT THE VL AND VH DOMAIN INTERFACE OF A FAB

The interface between the VL and VH domains of antibodies is highly co nserved. To investigate the influence of conserved interface residues on Fab function, 13 interface residues were subjected to codon-based c ombinatorial alanine scanning mutagenesis in Fab 57P, specific for pep tide 134 to 151 of the coat protein of tobacco mosaic virus. The 13 si ngle mutants were analysed by Western blot to determine the effect of interface modifications on Fab expression. The kinetic rate constants of peptide-Fab mutant interactions were measured using the biosensor t echnology. Alanine replacements did not prevent assembly of the mutate d Fabs and led to a modification of their binding properties in every case. Twelve of the 13 target residues correspond to homologous positi ons in the VL and VH domains, which have similar folds. Mutation at ho mologous positions mostly had different effects on antigen binding aff inity. The replacement of bulky side-chains had the most drastic effec t on binding. When smaller side-chains were replaced by alanine, the b inding properties of Fab mutants differed slightly (by less than a fac tor of two), but significantly from that of Fab 57P. Modification of s ome of these residues, which are located 9 to 12 Angstrom away from th e base of CDR loops, is unlikely to alter loop conformation. They may affect antigen binding indirectly by influencing the relative position of the VL and VH domains. Our results demonstrate that residues situa ted at the VL-VH interface and which are remote from the paratope are able to influence the antigen binding properties of antibodies. (C) 19 96 Academic Press Limited