Selection of the optimal chelator for labeling of DARPin Ec1 with gallium-68 for PET imaging of EpCAM expression.

Abstract

Background: Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein, which is overexpressed in several types of malignancies. Designed ankyrin repeat protein (DARPin) Ec1 is a 19 kDa engineered scaffold protein that binds with high affinity to EpCAM. Radiolabelled Ec1 might be used as a companion diagnostic for the selection of patients for personalized therapy. This study aimed to investigate the influence of different radiometal-chelator complexes on the biodistribution and imaging contrast of ⁶⁸Ga-labelled Ec1. To investigate this, two macrocyclic chelators, 1,4,7-triazacyclononane-N,N,N-triacetic acid (NOTA) and 1-(1,3-carboxypropyl)-1,4,7-triazacyclononane-4,7-diacetic acid (NODAGA) were conjugated to the C-terminus of the Ec1. The previously developed DARPin Ec1 conjugated to 1,4,7,10-tetraazacylododecane-1,4,7,10-tetraacetic acid (DOTA) was used as a comparator.

Results: All Ec1 variants were successfully labelled with ⁶⁸Ga. The use of NOTA and NODAGA provided twice higher radiochemical yield and improved label stability compared to DOTA. All labelled Ec1 variants bound to the EpCAM-expressing cells with nanomolar affinity and preserved targeting specificity in vitro and in vivo. Biodistribution studies in mice bearing EpCAM-expressing SKOV-3 xenografts showed that [⁶⁸Ga]Ga-Ec1-NOTA had lower uptake in most normal organs while maintaining tumor uptake. Among all variants, [⁶⁸Ga]Ga-Ec1-NOTA showed the lowest liver uptake, with no significant differences in tumor uptake. Additionally, [⁶⁸Ga]Ga-Ec1-NOTA provided the highest tumor-to-blood ratio compared to [⁶⁸Ga]Ga-Ec1-DOTA and [⁶⁸Ga]Ga-Ec1-NODAGA.

Conclusion: [⁶⁸Ga]Ga-Ec1-NOTA is the preferred radioconjugate for PET imaging of EpCAM expression.