ImmunoPET imaging of EpCAM in solid tumours with nanobody tracers: a preclinical study.

Abstract

Epithelial cell adhesion molecule (EpCAM) is a potential therapeutic target and anchoring molecule for circulating and disseminated tumour cells (CTC/DTC) in liquid biopsy. In this study, we aimed to construct EpCAM-specific immuno-positron emission tomography (immunoPET) imaging probes and assess the diagnostic abilities in preclinical cancer models. By engineering six single-domain antibodies (e.g., EPCD1 - 6) targeting EpCAM of different binding properties and labelling with ⁶⁸Ga (T_1/2 = 1.1 h) and ¹⁸F (T_1/2 = 110 min), we developed a series of EpCAM-targeted immunoPET imaging probes. The probes' pharmacokinetics and diagnostic accuracies were investigated in cell-derived human colorectal (LS174T) and esophageal cancer (OE19) tumour models. Based on in vitro binding affinities and in vivo pharmacokinetics of the first three tracers ([⁶⁸Ga]Ga-NOTA-EPCD1, [⁶⁸Ga]Ga-NOTA-EPCD2, and [⁶⁸Ga]Ga-NOTA-EPCD3), we selected [⁶⁸Ga]Ga-NOTA-EPCD3 for tumour imaging which showed an average tumour uptake of 2.06 ± 0.124%ID/g (n = 3) in LS174T cell-derived tumour model. Development and characterisation of [¹⁸F]AIF-RESCA-EPCD3 showed comparable tumour uptake of 1.73 ± 0.0471%ID/g (n = 3) in the same tumour model. Further validation of [⁶⁸Ga]Ga-NOTA-EPCD3 in OE19 cell-derived tumour model showed an average tumour uptake of 4.27 ± 1.16%ID/g and liver uptake of 13.5 ± 1.30%ID/g (n = 3). Near-infrared fluorescence imaging with Cy7-EPCD3 confirmed the in vivo pharmacokinetics and relatively high liver accumulation. We further synthesized another three ¹⁸F-labeled nanobody tracers ([¹⁸F]AIF-RESCA-EPCD4, [¹⁸F]AIF-RESCA-EPCD5, and [¹⁸F]AIF-RESCA-EPCD6) and found that [¹⁸F]AIF-RESCA-EPCD6 had the best pharmacokinetics with low background. [¹⁸F]AIF-RESCA-EPCD6 showed explicit uptake in the subcutaneously inoculated OE19 tumour model with an average uptake of 4.70 ± 0.26%ID/g (n = 3). In comparison, the corresponding tumour uptake (0.17 ± 0.25%ID/g, n = 3) in the EPCD6 blocking group was substantially lower (P < 0.001), indicating the targeting specificity of the tracer. We developed a series of ⁶⁸Ga/¹⁸F-labeled nanobody tracers targeting human EpCAM. ImmunoPET imaging with [¹⁸F]AIF-RESCA-EPCD6 may facilitate better use of EpCAM-targeted therapeutics by noninvasively displaying the target's expression dynamics.