Changes in the specificity of antibodies by site-specific mutagenesis followed by random mutagenesis.
The specificity for 11-deoxycortisol (11-DOC) of a monoclonal antibody (mAb), designated SCET, was changed to specificity for cortisol (CS) by site-specific mutagenesis followed by random mutagenesis. The Fab form of SCET was expressed on the surface of a phage. During the first step, mutations were introduced at 14 amino acid positions in three complementarity-determining regions (CDRs) of the VH domain that seemed likely to form the steroid-binding pocket. A clone, DcC16, was isolated from the resultant library with multiple mutations and this clone was shown to have CS-binding activity but also to retain high 11-DOC-binding activity. During the second step, mutations were introduced randomly into the entire VH-coding region of the DcC16 clone by an error-prone polymerase chain reaction, and CS-specific mutant antibodies were selected in the presence of 11-DOC as a competitor. Three representative clones were analyzed with the BIAcore instrument, and each revealed a large increase in the binding constant for CS and a decrease in that for 11-DOC. Structural models, constructed by computer simulation, indicated the probable molecular basis for these changes in specificity.
Miyazaki
C
C
Toyota Central R&D Laboratories Inc., Nagakute, Aichi 480-1192, Japan.
Iba
Y
Y
Yamada
Y
Y
Takahashi
H
H
Sawada
J
J
Kurosawa
Y
Y
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000918:Antibody Specificity / Q000235:genetics*; D001483:Base Sequence; D003198:Computer Simulation; D003350:Cortodoxone / Q000276:immunology; D017931:DNA Primers / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D006854:Hydrocortisone / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics* / Q000276:immunology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016133:Polymerase Chain Reaction; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007135:Immunoglobulin Variable Region; D003350:Cortodoxone; D006854:Hydrocortisone