ISOMERIZATION OF AN ASPARTIC-ACID RESIDUE IN THE COMPLEMENTARITY-DETERMINING REGIONS OF A RECOMBINANT ANTIBODY TO HUMAN IGE - IDENTIFICATION AND EFFECT ON BINDING-AFFINITY

This report describes the effect on antigen binding of an isomerized a spartate residue located in the complementarity-determining regions (C DRs) of a recombinant monoclonal antibody. The antibody, which binds h uman IgE, contains two Asp-Gly sequences within its CDRs, but only one site was found to be labile to isomerization. Isolation and character ization of antibody fragments differing in the labile sequence were fa cilitated by using a technique involving hydrophobic interaction chrom atography (HIC) that separates aspartyl, isoaspartyl, and cyclic imide variants to the residue located in CDR-L1. The variants were isolated for structural characterization and for determination of their relati ve antigen binding affinities. Mutants were constructed with altered r esidues to obviate the effects of isomerization and were evaluated for their ability to bind to IgE. Inspection of published crystal structu res of CDRs of antibodies indicated that hydrogen binding of the Asp s ide chain of the unreactive residue may be the constraint that prevent s isomerization. The strategy outlined here may prove to be of general utility in the biochemical and immunochemical characterization of rec ombinant antibodies.