title: High-affinity antigen binding by chelating recombinant antibodies (CRAbs).
abstract: We have developed a strategy for making antibody fragments with high binding affinities by harnessing the chelate effect. We create a bispecific antibody fragment (Chelating Recombinant Antibody or CRAb) that recognizes adjacent and non-overlapping epitopes of the target antigen, and is flexible enough to bind to both epitopes simultaneously. Here the strategy is illustrated with two antibodies that form complexes of known three-dimensional structure against different epitopes of lysozyme. Computer graphic modelling indicated that two single-chain antibody fragments (scFv) derived from antibodies D1.3 (Ka = 10(8) M-1) and mutant HyHEL-10 (Ka = 10(6) M-1) could be linked together on the surface of lysozyme by a flexible and hydrophilic polypeptide between the C terminus of one fragment and the N terminus of the other. The CRAb gene was assembled and the CRAb expressed by secretion from bacteria. The purified CRAb was shown to have a much higher affinity than either of the scFv fragments, as shown by competition ELISA (Kd > 10(9) M-1), bandshift on gels (Ka > 2 x 10(9) M-1) and fluorescence quench (Ka > 1.3 x 10(10) M-1).
authors.affiliation: Cambridge Centre for Protein Engineering, MRC Centre, England.
authors.forename: D
authors.initials: D
authors.lastname: Neri
authors.forename: M
authors.initials: M
authors.lastname: Momo
authors.forename: T
authors.initials: T
authors.lastname: Prospero
authors.forename: G
authors.initials: G
authors.lastname: Winter
mesh_terms: D000595:Amino Acid Sequence; D018033:Antibodies, Bispecific / Q000235:genetics / Q000276:immunology*; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D017931:DNA Primers; D000939:Epitopes / Q000276:immunology; D008969:Molecular Sequence Data; D015202:Protein Engineering
chemical_list: D018033:Antibodies, Bispecific; D017931:DNA Primers; D000939:Epitopes