Radiolabeling of fab and f(ab′)2antibody fragments with 99mTc(I) tricarbonyl core using a new bifunctional tridentate ligand

The objective of this study was to design and evaluate a new histidine-modified tridentate chelator for labeling antimesothelin fab and f(ab′)2antibody fragments with the 99mTc(I) tricarbonyl (99mTc(CO)3) core. N-(ortho-phenol)-histidine chelator was synthesized by modifying the single amino acid L-histidine, a natural amino acid, with an additional phenol group to obtain the bifunctional tridentate ligand after reductive amination. Bioconjugation was based on the carbodiimide activation of the carboxylate of chelator and on further reaction with the amine groups present on the antibody fragments. Radiolabeling was accomplished by replacing the three aqua ligands of the complex precursor 99mTc(CO)3(H2O)3with the tridentate chelator. The antibody fragments radiolabeled with 99mTc(CO)3core were tested for stability by the cysteine challenge test. The immunoreactivity and binding affinity of the radiolabeled fragments were studied using in-vitro cell-binding assays. Radiochemical yields achieved for 99mTc(CO)3core labeling of fab and f(ab′)2were 91.6±9.1 and 80.7±8.5, respectively. Stability studies of radiolabeled antibody fragments showed that the 99mTc label was stable to transchelation by cysteine. Both 99mTc-fab and 99mTc-f(ab′)2retained their reactivity and affinity to the mesothelin antigen. From our studies, it can be concluded that the newly synthesized N-(ortho-phenol)-histidine chelator is a promising candidate for 99mTc(CO)3labeling of biomolecules and for developing other novel 99mTc radiopharmaceuticals.