A novel multivalent 99mTc-labeled EG2-C4bpα antibody for targeting the epidermal growth factor receptor in tumor xenografts

Introduction: The C4b binding protein (C4bp) alpha/beta-chain C-terminal effectively induces polymerization during protein synthesis. Using this fragment and the single-domain antibody EG2, which targets the epidermal growth factor receptor (EGFR), we generated the novel multimeric antibody EG2-C4bp alpha. We radiolabeled EG2-C4bp alpha with Tc-99m and evaluated its targeting efficiency and pharmacokinetics in tumor xenografts. Methods: EGFR expression and EGFR-EG2-C4bp alpha binding was evaluated in A431 and OCM-1 cells by Western blotting and flow cytometry, respectively. EG2-C4bp alpha was radiolabeled with [Tc-99m(CO)(3)(OH2)(3)](+) using a tricarbonyl vial followed by purification on a PD-10 column. In vitro studies with Tc-99m-EG2-C4bp alpha were performed in A431 and/or OCM-1 cells. Single photon emission computed tomography (SPEC') imaging and biodistribution studies were carried out in Tc-99m-EG2-C4bp alpha-injected mice bearing A431- and OCM-1-derived tumors. EGFR immunofluorescent staining in A431 and OCM-1 tumors was performed. Results: A431 cells showed higher EGFR expression levels than OCM-1 cells, and flow cytometry confirmed EG2-C4bp alpha bound more A431 cells than OCM-1 cells. Tc-99m-EG2-C4bp alpha was successfully prepared with radiochemical yields of 303-50.4%. The binding affinity of Tc-99m-EG2-C4bp alpha to A431 cells was approximately 20 nM. Tc-99m-EG2-C4bp alpha specifically bound A431 cells and this binding was blocked by 41% in the presence of 50 nM excess unlabeled EG2-C4bp alpha. In vivo radioactivity uptake in A431 tumors was detected 2 h after Tc-99m-EG2-C4bp alpha. administration and sustained up to 18 h. The highest ratio of A431 tumor-to-muscle and tumor-to-blood was 3.69 +/- 0.48 at 10 hand 0.77 +/- 0.14 at 20 h, respectively. Excess unlabeled EG2-C4bp alpha blocked radioactivity uptake in A431 tumors by 55% at 10 h. Tc-99m-EG2-C4bp alpha was barely detectable in OCM-1 tumors, and biodistribution analysis confirmed that radioactivity uptake was significantly lower than in A431 tumors. Conclusions: Tc-99m-EG2-C4bp alpha specifically and efficiently targets EGFR over-expressing tumors suggesting that EG2-C4bp alpha may be a promising antibody alternative for future diagnostic application and potential radioimmunotherapy. However, the high activity in the blood and liver, and the relative low ratio of tumor-toblood should be noticed and improved. (C) 2015 Elsevier Inc. All rights reserved.