The Use of Glycine-Containing Peptide-Based Chelators for Labeling with ^99m Tc Improves the Imaging Properties of EpCAM-Targeting Designed Ankyrin Repeat Ec1

Noninvasive radionuclide imaging of epithelial cell adhesion molecule (EpCAM) expression in lung, ovarian, breast, kidney, and other cancers can stratify patients for EpCAM-targeted therapy. The constructed scaffold proteins, designed ankyrin repeat proteins (DARPins), are highly specific high-affinity probes for radionuclide imaging. A clinical study demonstrated that the anti-EpCAM DARPin [^99mTc]Tc-(HE)₃-Ec1 showed precise EpCAM imaging at 2, 4, and 6 h after injection in patients with nonsmall cell lung cancer. However, a noticeable accumulation in healthy organs has prompted the development of new Ec1-based agents with improved biodistribution properties. In addition, it would be desirable to substitute a labor-intensive labeling procedure. The purpose of this study was to test the hypothesis that the use of Gly-Gly-Gly-Cys (G₃C) or Glu-Glu-Glu-Cys (E₃C) peptide chelators placed at the C-terminus of DARPin for labeling with ^99mTc (V) could improve the image contrast and biodistribution of Ec1. The radiochemical yield of the new variants exceeded 95%. The labeled proteins specifically bound to human EpCAM-expressing cancer cell lines with affinities of 8-10 nM. The biodistribution of [^99mTc]Tc-Ec1-G₃C and [^99mTc]Tc-Ec1-E₃C in mice was compared with the biodistribution of clinically tested [^99mTc]Tc-(HE)₃-Ec1 in a Nu/j mouse model with SKOV-3 xenografts. The new variants specifically accumulate in human xenografts with EpCAM expression. The accumulation of new variants in healthy organs (liver, salivary glands, spleen, and stomach) was reduced compared to [^99mTc]Tc-(HE)₃-Ec1. [^99mTc]Tc-Ec1-G₃C provided the best imaging contrast and is suitable for clinical testing.