HIGH-AFFINITY ANTIGEN-BINDING BY CHELATING-RECOMBINANT-ANTIBODIES (CRABS)

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 (K-a= 10(8) M(-1)) and mutant HyHEL-10 (K-a = 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 (K-a > 10(9) M(-1)), bandshift on gels(K-d > 2 x 10(9) M(-1)) and fluorescence quench (K-a > 1.3 x 10(10) M(-1)).