Isolation and characterization of nanobodies against epithelial cell adhesion molecule as novel theranostic agents for cancer therapy

•Four consecutive rounds of biopanning were performed on immoblilized EpCAM.•Selected high affinity nanobodies significantly inhibited proliferation of MCF-7 cells in vitro .•The in vivo study revealed that after 14 days monitoring a significant reduction in tumor size occurred.•Our data revealed that Nb4 and Nb5 could be considered as theranostic agents for EpCAM overexpressing cancers. Epithelial cell adhesion molecule (EpCAM) plays an important role in tumorigenesis. Camelids produce functional antibodies composed of heavy chains only that bind to their antigens via a single domain variable fragment known as nanobody. Nanobodies show multiple advantages over traditional monoclonal antibodies. Isolation of functional anti-EpCAM nanobodies (Nbs) was the main aim of this study. An immune nanobody library containing 108 members was constructed previously. Anti -EpCAM nanobodies were isolated from camel immune library using phage display. Four consecutive rounds of biopanning were performed on immobilized EpCAM. Four nanobodies (Nb4, Nb5, Nb22, and Nb23) with highest signal intensity in monoclonal phage ELISA were selected. Affinity of these selected nanobodies for EpCAM was in the nanomolar range. Selected nanobodies significantly inhibited proliferation of MCF-7 cells. The in vivo study revealed that a significant reduction in tumor size occurred when treated with nanobodies Nb4 and Nb5, after 14 days monitoring. Our data revealed that nanobodies Nb4 and Nb5 could be considered as attractive theranostic agents for EpCAM overexpressing cancers.