Abstract
Radionuclide molecular imaging of epidermal growth factor receptor (EGFR) expression might permit the selection of patients for EGFR-targeting therapies. Designed ankyrin repeat protein (DARPin) E01 with a high affinity to the ectodomain III of the EGFR is a possible EGFR imaging probe. The goal of this study was to evaluate the potential of radiolabeled DARPin E01 for in vivo imaging of EGFR. DARPin E01 containing the (HE)(3)-tag was site-specifically labeled with a residualizing (99m)Tc (using (99m)Tc]Tc(CO)(3)). Two methods providing non-residualizing (123)I labels, direct electrophilic radioiodination and indirect radioiodination using [(123)I]I-para-iodobenzoate (PIB), were tested. [(99m)Tc]Tc-(HE)(3)-E01 and [(123)I]I-(HE)(3)-E01-PIB preserved specific binding to EGFR-expressing cells and affinity in the single-digit nanomolar range. Direct labeling with (123)I resulted in a substantial loss of binding. In vitro cellular processing studies showed that both [(99m)Tc]Tc-(HE)(3)-E01 and [(123)I]I-(HE)(3)-E01-PIB had rapid binding and relatively slow internalization. Evaluation of [(99m)Tc]Tc-(HE)(3)-E01 biodistribution in normal CD1 mice showed that its hepatic uptake was non-saturable, suggesting that this tracer does not bind to murine EGFR. A side-by-side comparison of biodistribution and tumor targeting of [(99m)Tc]Tc-(HE)(3)-E01 and [(123)I]I-(HE)(3)-E01-PIB was performed in Nu/j mice bearing EGFR-positive A-431 and EGFR-negative Ramos human cancer xenografts. Both radiolabeled DARPins demonstrated EGFR-specific tumor uptake. However, [(123)I]I-(HE)(3)-E01-PIB had appreciably lower uptake in normal organs compared to [(99m)Tc]Tc-(HE)(3)-E01, which provided significantly (p < 0.05) higher tumor-to-organ ratios. Gamma-camera imaging confirmed that [(123)I]I-(HE)(3)-E01-PIB demonstrated a higher imaging contrast in preclinical models than [(99m)Tc]Tc-(HE)(3)-E01. In conclusion, DARPin (HE)(3)-E01 labeled using a non-residualizing [(123)I]I-para-iodobenzoate (PIB) label is the preferred radiotracer for in vivo imaging of EGFR expression in cancer.