Changes in the specificity of antibodies by site-specific mutagenesis followed by random mutagenesis

The specificity for 11-deoxycortisol (11-DOC) of a monoclonalantibody (mAb), designated SCET, was changed to specificityfor cortisol (CS) by site-specific mutagenesis followed by randommutagenesis. The Fab form of SCET was expressed on the surfaceof a phage. During the first step, mutations were introducedat 14 amino acid positions in three complementarity-determiningregions (CDRs) of the V_H domain that seemed likely to form thesteroid-binding pocket. A clone, DcC16, was isolated from theresultant library with multiple mutations and this clone wasshown to have CS-binding activity but also to retain high 11-DOC-bindingactivity. During the second step, mutations were introducedrandomly into the entire V_H-coding region of the DcC16 cloneby an error-prone polymerase chain reaction, and CS-specificmutant antibodies were selected in the presence of 11-DOC asa competitor. Three representative clones were analyzed withthe BIAcore instrument, and each revealed a large increase inthe binding constant for CS and a decrease in that for 11-DOC.Structural models, constructed by computer simulation, indicatedthe probable molecular basis for these changes in specificity.