SPECT imaging with 99mTc-labeled EGFR-specific nanobody for in vivo monitoring of EGFR expression

Purpose: Overexpression of the epidermal growth factor receptor (EGFR) occurs with high incidence in various carcinomas. The oncogenic expression of the receptor has been exploited for immunoglobulin-based diagnostics and therapeutics. We describe the use of a llama single-domain antibody fragment, termed Nanobody (R), for the in vivo radioimmunodetection of EGFR overexpressing tumors using single photon emission computed tomography (SPECT) in mice. Methods: Fluorescence-activated cell sorting (FACS) analysis was performed to evaluate the specificity and selectivity of 8B6 Nanobody to bind EGFR on EGFR overexpressing cells. The Nanobody was then labeled with Tc-99m via its C-terminal histidine tail. Uptake in normal organs and tissues was assessed by ex vivo analysis. In vivo tumor targeting of Tc-99m-8B6 Nanobody was evaluated via pinhole SPECT in mice bearing xenografts of tumor cells with either high (A431) or moderate (DU145) overexpression of EGFR. Results: FACS analysis indicated that the 8B6 Nanobody only recognizes cells overexpressing EGFR. In vivo blood clearance of Tc-99m-8B6 Nanobody is relatively fast (half-life, 1.5 h) and mainly via the kidneys. At 3 h postinjection, total kidney accumulation is high (46.6 +/- 0.9%IA) compared to total liver uptake (18.9 +/- 0.6%IA). Pinhole SPECT imaging of mice bearing A431 xenografts showed higher average tumor uptake (5.2+0.5%IA/cm(3)) of Tc-99m-8B6 Nanobody compared to DU145 xenografts (1.8+0.3%IA/cm(3), p<0.001). Conclusion: The EGFR-binding Nanobody investigated in this study shows high specificity and selectivity towards EGFR overexpressing cells. Pinhole SPECT analysis with Tc-99m-8B6 Nanobody enabled in vivo discrimination between tumors with high and moderate EGFR overexpression. The favorable biodistribution further corroborates the suitability of Nanobodies for in vivo tumor imaging.