RADIOIODINATION OF INTERNALIZING MONOCLONAL-ANTIBODIES USING N-SUCCINIMIDYL 5-IODO-3-PYRIDINECARBOXYLATE

Monoclonal antibodies (mAbs) that internalize following binding to cel l-surface receptors require radiolabeling approaches that minimize los s of radioactivity from the cell after intracellular processing. One c lass of internalizing mAbs of great interest fur imaging and radioimmu notherapy are those specific for EGFRvIII, a truncated form of the epi dermal growth factor receptor found on gliomas, non-small cell lung ca rcinomas, breast carcinomas, and ovarian carcinomas. Because lysosomes are known to retain positively charged compounds, N-succinimidyl 5-io do-3-pyridine-carboxylate (SIPC) might he ideal for radioiodination of these mAbs because of the positive charge on its pyridine ring. To in vestigate this hypothesis, the anti-EGFRvIII mAb L8A4 was labeled usin g SIPC, and internalization assays were performed using the EGFRvIII-p ositive cell lines HC2 20 d2 and NR6M. Compared with L8A4 labeled usin g Iodogen or N-succinimidyl 3-iodobenzoate, SIPC increased intracellul ar retention of activity by up to 65%. Reverse-phase high-performance liquid chromatography analyses indicated that a significantly higher f raction of the low molecular weight catabolites from mAbs labeled via SIPC were retained within cells (SIPC, 28.1%; Iodogen, 7.6% at 1 h). W ith SIPC, the primary labeled species in cell lysates was the 5-iodoni cotinic acid (INA)-lysine conjugate, whereas in the supernatant, both INA-lysine and INA were seen. A 3-4-fold higher percentage of these ca tabolites were charged at lysosomal pH in comparison with those from m Ab labeled using N-succinimidyl 3-iodobenzoate, in concert with the di fferences in cellular retention observed between these two labeling me thods. In mice bearing HC2 20 d2 xenografts, a significant improvement in tumor retention of radioiodine and tumor:normal tissue ratios mas seen when L8A4 was labeled using SIPC instead of the Iodogen method. T hese results suggest that SIPC is a promising reagent for the radioiod ination of anti-EGFRvIII L8A4 and, possibly, other internalizing mAbs.