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

We have developed a strategy for making antibody fragments with high b inding affinities by harnessing the chelate effect. We create a bispec ific antibody fragment (Chelating Recombinant Antibody or CRAb) that r ecognizes adjacent and non-overlapping epitopes of the target antigen, and is flexible enough to bind to both epitopes simultaneously Here t he strategy is illustrated with two antibodies that form complexes of known three-dimensional structure against different epitopes of lysozy me. Computer graphic modelling indicated that two single-chain antibod y 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 s urface of lysozyme by a flexible and hydrophilic polypeptide between t he C terminus of one fragment and the N terminus of the other. The CRA b gene was assembled and the CRAb expressed by secretion from bacteria . The purified CRAb was shown to have a much higher affinity than eith er 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 quenc h (K-a > 1.3 x 10(10) M(-1)).