A novel multivalent (99m)Tc-labeled EG2-C4bpα antibody for targeting the epidermal growth factor receptor in tumor xenografts

Abstract

Introduction: The C4b binding protein (C4bp) α/β-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-C4bpα. We radiolabeled EG2-C4bpα with (99m)Tc and evaluated its targeting efficiency and pharmacokinetics in tumor xenografts.

Methods: EGFR expression and EGFR-EG2-C4bpα binding was evaluated in A431 and OCM-1 cells by Western blotting and flow cytometry, respectively. EG2-C4bpα was radiolabeled with [(99m)Tc(CO)3(OH2)3](+) using a tricarbonyl vial followed by purification on a PD-10 column. In vitro studies with (99m)Tc-EG2-C4bpα were performed in A431 and/or OCM-1 cells. Single photon emission computed tomography (SPECT) imaging and biodistribution studies were carried out in (99m)Tc-EG2-C4bpα-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-C4bpα bound more A431 cells than OCM-1 cells. (99m)Tc-EG2-C4bpα was successfully prepared with radiochemical yields of 30.3-50.4%. The binding affinity of (99m)Tc-EG2-C4bpα to A431 cells was approximately 20 nM. (99m)Tc-EG2-C4bpα specifically bound A431 cells and this binding was blocked by 41% in the presence of 50 nM excess unlabeled EG2-C4bpα. In vivo radioactivity uptake in A431 tumors was detected 2h after (99m)Tc-EG2-C4bpα administration and sustained up to 18h. The highest ratio of A431 tumor-to-muscle and tumor-to-blood was 3.69 ± 0.48 at 10h and 0.77 ± 0.14 at 20 h, respectively. Excess unlabeled EG2-C4bpα blocked radioactivity uptake in A431 tumors by 55% at 10h. (99m)Tc-EG2-C4bpα was barely detectable in OCM-1 tumors, and biodistribution analysis confirmed that radioactivity uptake was significantly lower than in A431 tumors.

Conclusions: (99m)Tc-EG2-C4bpα specifically and efficiently targets EGFR over-expressing tumors suggesting that EG2-C4bpα 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-to-blood should be noticed and improved.

Keywords: Biodistribution; C4b binding protein; Epidermal growth factor receptor; Multimerization; Single photon emission computed tomography; Single-domain antibody.