EVALUATION OF RADIOIODINATED AND RADIOCOPPER LABELED MONOVALENT FRAGMENTS OF MONOCLONAL-ANTIBODY CHCE7 FOR TARGETING OF NEUROBLASTOMA

Monovalent fragments of antineuroblastoma antibody mAb chCE7 were eval uated for their in vitro and in vivo tumor cell binding properties, Si ngle chain fragments were constructed from the variable region genes c loned from hybridoma cells, expressed in E.coli and purified by metal chelate affinity chromatography, Radioiodinated CE7-scFv fragments wer e found to bind with high affinity (K-d similar to 10(-9)M) to target cells in vitro but formed aggregates at 37 degrees C, and bound to ser um proteins in vitro and in vivo. Circular Dichroism spectra revealed the protein to be in a conformationally altered form and no permanent ''refolding'' could be achieved. In contrast, chCE7-Fab fragments were found to bind to target tumor cells with similar affinity than the pa rent mAb chCE7 (K-d similar to 10(-10)M), showed no tendency to aggreg ate and were stable in serum both in vitro and in vivo. Kinetics of as sociation and dissociation of radioiodinated scFv and Fab fragments we re found to be rapid. Radioiodination with the Iodogen method led to i mpaired immunoreactivity which was found to further increase the off-r ates of radioiodinated fragments from tumor cells, Radioiodination wit h the Bolton Hunter reagent as well as labeling of chCE7-Fab fragments with Cu-67 via the macrocyclic CPTA ligand led to fully immunoreactiv e Fab fragments, Radioiodinated and radiocopper labeled monovalent CE7 fragments did not internalize into target tumor cells as the parent m Ab and its F(ab')(2) fragment, A comparison of the biodistribution in tumor bearing nude mice of the radiocopper labeled monovalent, non int ernalizing Fab fragments with the internalizing divalent F(ab')(2) fra gments showed in both cases high levels of radioactivity in the kidney s. Concerning tumor uptake, radioactivity from both internalizing and non internalizing fragments remained associated with tumor tissue for longer times than in case of the corresponding radioiodinated fragment s. When compared with the radioiodinated forms, tumor uptake of radioc opper-labeled Cu-67-chCE7 and its F(ab')(2) fragments was found to be higher. However in the case of the non internalizing Cu-67-chCE7-Fab f ragment no increase in the absolute amount of radioactivity in tumor t issue compared with the radioiodinated Fab was observed, indicating an advantage of using radiocopper labeling in conjunction with internali zing antibody fragments for delivering high doses of radioactivity to neuroblastoma. (C) 1997 Elsevier Science Inc.