Development and Preclinical Evaluation of a Novel EpCAM-Targeted ¹¹¹In-Labeled Antibody for SPECT Imaging

Epithelial cell adhesion molecule (EpCAM) is overexpressed in various epithelial malignancies and represents an attractive target for molecular imaging and radionuclide therapy. This study aimed to develop and evaluate a novel EpCAM-targeted monoclonal antibody for single-photon emission computed tomography (SPECT) imaging. To this end, Ep4-1, a high-affinity anti-EpCAM monoclonal antibody, was conjugated to the bifunctional chelator p-SCN-Bn-diethylenetriaminepentaacetic acid (DTPA) and radiolabeled with indium-111 (¹¹¹In). In vitro EpCAM-binding affinity, plasma stability, and cellular internalization were examined using high-EpCAM-expressing DLD-1 cells. In vivo biodistribution, blocking studies, and SPECT/computed tomography (CT) imaging were performed in DLD-1 tumor-bearing mice. Results showed that [¹¹¹In]-In-DTPA-Ep4-1 retained high, EpCAM-specific binding after chelator conjugation and showed excellent plasma stability. The radiolabeled antibody exhibited pronounced cellular internalization; approximately 93% of cell-associated radioactivity was internalized in DLD-1 cells at 6 h, exceeding the reported internalization efficiency of previously described EpCAM-targeted antibodies. In vivo biodistribution indicated progressive and selective tumor uptake, reaching 15.1 ± 1.3% ID/g at 72 h after injection. Co-injection of excess unlabeled Ep4-1 reduced tumor accumulation, consistent with EpCAM-specific targeting. Uptake in the liver and spleen was consistent with the expected pharmacokinetics of intact IgG antibodies. SPECT/CT imaging enabled clear visualization of EpCAM-expressing tumors at 72 h after injection. In conclusion, Ep4-1 is a highly specific and strongly internalizing EpCAM-targeted monoclonal antibody that enables robust SPECT imaging of EpCAM-positive tumors. Given the favorable internalization properties of Ep4-1, it may serve as a promising imaging platform and a potential candidate for future EpCAM-directed radiotheranostics applications, including α-particle-based radionuclide therapy.