Imaging of EGFR expression in murine xenografts using site-specifically labelled anti-EGFR ¹¹¹In-DOTA-Z_EGFR:2377 Affibody molecule: Aspect of the injected tracer amount

Introduction: Overexpression of epidermal growth factor receptor (EGFR) is a prognostic and predictive biomarker in a number of malignant tumours. Radionuclide molecular imaging of EGFR expression in cancer could influence patient management. However, EGFR expression in normal tissues might complicate in vivo imaging. The aim of this study was to evaluate if optimization of the injected protein dose might improve imaging of EGFR expression in tumours using a novel EGFR-targeting protein, the DOTA-Z_EGFR:2377 Affibody molecule. Methods: An anti-EGFR Affibody molecule, Z_EGFR:2377, was labelled with ¹¹¹In via the DOTA chelator site-specifically conjugated to a C-terminal cysteine. The affinity of DOTA-Z_EGFR:2377 for murine and human EGFR was measured by surface plasmon resonance. The cellular processing of ¹¹¹In-DOTA-Z_EGFR:2377 was evaluated in vitro. The biodistribution of radiolabelled Affibody molecules injected in a broad range of injected Affibody protein doses was evaluated in mice bearing EGFR-expressing A431 xenografts. Results: Site-specific coupling of DOTA provided a uniform conjugate possessing equal affinity for human and murine EGFR. The internalization of ¹¹¹In-DOTA-Z_EGFR:2377 by A431 cells was slow. In vivo, the conjugate accumulated specifically in xenografts and in EGFR-expressing tissues. The curve representing the dependence of tumour uptake on the injected Affibody protein dose was bell-shaped. The highest specific radioactivity (lowest injected protein dose) provided a suboptimal tumour-to-blood ratio. The results of the biodistribution study were confirmed by γ-camera imaging. Conclusion: The ¹¹¹In-DOTA-Z _EGFR:2377 Affibody molecule is a promising tracer for radionuclide molecular imaging of EGFR expression in malignant tumours. Careful optimization of protein dose is required for high-contrast imaging of EGFR expression in vivo.