Feasibility of Imaging EpCAM Expression in Ovarian Cancer Using Radiolabeled DARPin Ec1

Abstract

Epithelial cell adhesion molecule (EpCAM) is overexpressed in 55%–75% of ovarian carcinomas (OC). EpCAM might be used as a target for a treatment of disseminated OC. Designed ankyrin repeats protein (DARPin) Ec1 is a small (18 kDa) protein, which binds to EpCAM with subnanomolar affinity. We tested a hypothesis that Ec1 labeled with a non-residualizing label might serve as a companion imaging diagnostic for stratification of patients for EpCAM-targeting therapy. Ec1 was labeled with 125I using N-succinimidyl-para-iodobenzoate. Binding affinity, specificity, and cellular processing of [125I]I-PIB-Ec1 were evaluated using SKOV-3 and OVCAR-3 ovarian carcinoma cell lines. Biodistribution and tumor-targeting properties of [125I]I-PIB-Ec1 were studied in Balb/c nu/nu mice bearing SKOV-3 and OVCAR-3 xenografts. EpCAM-negative Ramos lymphoma xenografts served as specificity control. Binding of [125I]I-PIB-Ec1 to ovarian carcinoma cell lines was highly specific and had affinity in picomolar range. Slow internalization of [125I]I-PIB-Ec1 by OC cells confirmed utility of non-residualizing label for in vivo imaging. [125I]I-PIB-Ec1 provided 6 h after injection tumor-to-blood ratios of 30 ± 11 and 48 ± 12 for OVCAR-3 and SKOV-3 xenografts, respectively, and high contrast to other organs. Tumor targeting was highly specific. Saturation of tumor uptake at a high dose of Ec1 in SKOV-3 model provided a rationale for dose selection in further studies using therapeutic conjugates of Ec1 for targeted therapy. In conclusion, [125I]I-PIB-Ec1 is a promising agent for visualizing EpCAM expression in OC.

Radionuclide Molecular Imaging of EpCAM Expression in Triple-Negative Breast Cancer Using the Scaffold Protein DARPin Ec1

“…Three different variants of non-residualizing radioiodine labels were evaluated for DARPins: A product of so-called direct electrophilic radioiodination, when the radioiodine is incorporated into the phenolic ring of tyrosine [ 22 , 23 , 30 ], a conjugate with [ 125 I]-PIB [ 30 , 31 ], and a site-specific conjugate of [ 125 I]I-iodo-[(4-hydroxyphenyl)ethyl]-maleimide (HPEM) to a unique cysteine engineered to a C-terminus of DARPin G3 [ 24 ]. It turned out that the use of the HPEM label results in a high level of hepatobiliary excretion and accumulation of activity in the content of gastrointestinal tract.…”

“…Apparently, slow internalization of Ec1 after binding to TNBC cells is critical for the use of a non-residualizing label for imaging in this cancer. Such a slow internalization of radiolabeled Ec1 was observed in the case of binding to pancreatic and ovarian cell lines [ 30 , 31 ], which was an indication that the cellular processing pattern would be similar for TNBC as well. However, our previous observations with ESP suggest that an internalization rate might depend on origin of cancer cells.…”

“…Site-specific radiolabeling of DARPin Ec1-H 6 bearing a C-terminal His 6 -tag with tricarbonyl technetium-99m was performed as described earlier [ 31 ]. The solution eluted from the technetium generator (500 µL) containing 3–4 GBq of [ 99m Tc]Tc pertechnetate was added to the CRS kit, and the mixture was incubated at 100 °C for 30 min.…”

“…Binding specificity to EpCAM was evaluated as described previously [ 30 , 31 ]. To saturate EpCAM receptors, 100-fold excess of nonlabeled Ec1 DARPin (200 nM) in cell culture medium was added to one group of cells and an equal volume of media only was added to the second group.…”

“…In this case, tumor retention of activity depends mainly on the high affinity of a probe to its molecular target. A successful use of non-residualizing radiohalogen labels was demonstrated earlier for ESPs such as HER2-binding Albumin-binding domain (ABD)-Derived Affinity ProTein 6 (ADAPT6) [ 29 ] and HER2-binding DARPins 9_29 and G3 [ 22 , 23 ], as well as for Ec1 for imaging of EpCAM expression in pancreatic and ovarian cancer models [ 30 , 31 ]. Importantly, good retention in tumors was accompanied by a rapid clearance from normal tissues, which increased tumor-to-organ ratios.…”