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

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 ^99mTc 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 [^99mTc(CO)₃(OH₂)₃]⁺ using a tricarbonyl vial followed by purification on a PD-10 column. In vitro studies with ^99mTc-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 ^99mTc-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. ^99mTc-EG2-C4bpα was successfully prepared with radiochemical yields of 30.3–50.4%. The binding affinity of ^99mTc-EG2-C4bpα to A431 cells was approximately 20 nM. ^99mTc-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 2 h after ^99mTc-EG2-C4bpα 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 h and 0.77 ± 0.14 at 20 h, respectively. Excess unlabeled EG2-C4bpα blocked radioactivity uptake in A431 tumors by 55% at 10 h. ^99mTc-EG2-C4bpα was barely detectable in OCM-1 tumors, and biodistribution analysis confirmed that radioactivity uptake was significantly lower than in A431 tumors. Conclusions: ^99mTc-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