Identification of Important Residues in Metal−Chelate Recognition by Monoclonal Antibodies

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Abstract
The molecular characterization of antibodies directed against metal−chelate complexes will provide important insights into the design and development of radiotherapeutic and radioimaging reagents. In this study, two monoclonal antibodies directed against different metal−chelate complexes were expressed as recombinant Fab fragments. Covalent modification and site-directed mutagenesis were employed to ascertain those residues important in antigen recognition. Antibody 5B2 was raised to a Pb(II)-loaded isothiocyanatobenzyl-diethylenetriamine pentaacetic acid (DTPA)−protein conjugate. The native antibody bound to complexes of Pb(II)−p-aminobenzyl-DTPA with an affinity of 4.6 × 10-9 M. A monovalent Fab fragment prepared from the native protein and a bivalent recombinant fragment exhibited comparable affinities for the same Pb(II)−chelate complex, approximately 6-fold lower than that of the intact antibody. Covalent modification and molecular modeling predicted that Lys58 in the heavy chain contacted the Pb(II)-chelate ligand. Mutational analysis supported a role for Lys58 in ion pair or hydrogen bond formation with the carboxylate groups on the chelate. Antibody E5 was directed toward an isothiocyanatobenzyl-ethylenediamine tetraacetic acid (EDTA)−protein conjugate loaded with ionic Cd(II). The native immunoglobulin recognized Cd(II)−p-aminobenzyl-EDTA with an affinity of 8.2 × 10-12 M. A proteolytically derived fragment and a bivalent recombinant fragment bound to the same Cd(II)−chelate complex with affinities that were comparable to that of the native antibody. Homology modeling and mutagenesis identified three residues (Trp52 and His96 in the heavy chain and Arg96 in the light chain) that were important for Cd(II)−chelate recognition. His96 likely mediates a direct ligation to the Cd(II) ion and Trp52 appears to be involved in hydrophobic stacking with the benzyl moiety of the chelator. Arg96 appeared to mediate an electrostatic or hydrogen bond to the chelate portion of the complex. These studies demonstrate that antibody recognition of metal−chelate haptens occurs through a limited number of molecular contacts and that these molecular interactions involve both direct ligation between the antibody and the metal ion and interactions between the antibody and the chelator.