The contribution of contact and non-contact residues of antibody in the affinity of binding to antigen. The interaction of mutant D1.3 antibodies with lysozyme.
We have analysed the contribution of residues of the D1.3 Fv fragment to binding of hen egg lysozyme. We altered residues at the contact interface by site-directed mutagenesis, and determined the affinity of the mutant Fv fragments for lysozyme by fluorescence quench titration. We found that a band of residues at the centre of the contact interface were much more important for binding affinity than those at the periphery. We also subjected the seFv fragment to random mutagenesis to simulate somatic mutation and affinity maturation. By display of the mutants on the surface of filamentous phages, and selection of the phages with biotinylated lysozyme, we were able to select mutants with modest improvements in binding affinity to lysozyme. By combining the mutations we obtained a scFv fragment with a fivefold improved affinity (Kd approximately 0.6 nM compared to wild-type Kd = 3.3 nM). However, none of the altered residues leading to improved affinity was located in the contact interface. This indicates that the interactions of a few residues at the centre of the contact interface are responsible for the binding affinity to antigen, but that these interactions can be modulated by alterations of residues outside the binding site. This may represent a typical mechanism for the affinity maturation of antibodies.
Hawkins
R E
RE
MRC Laboratory of Molecular biology, Cambridge, U.K.
Russell
S J
SJ
Baier
M
M
Winter
G
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000915:Antibody Affinity; D001483:Base Sequence; D002645:Chickens; D017931:DNA Primers / Q000737:chemistry; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D046911:Macromolecular Substances; D009113:Muramidase