Evaluation of four Affibody based near-infrared fluorescent probes for optical imaging of epidermal growth factor receptor positive tumors

1556 Objectives The epidermal growth factor receptor 1 (EGFR) has become an attractive target for cancer molecular imaging and therapy. An Affibody protein with strong binding affinity to EGFR, ZEGFR:1907, has been reported. We are interested in translating Affibody for potential clinical optical imaging of EGFR positive cancers. Thus, in this study four anti-EGFR Affibody based near-infrared (NIR) fluorescent were prepared and their in vivo performance was evaluated in tumor mice. Methods The Affibody analogue, Ac-Cys-ZEGFR:1907, was synthesized using solid phase peptide synthesis method. The purified protein was site-specifically conjugated with NIR fluorescent dye, Cy5.5-mono-maleimide, Alex-Fluor-680-maleimide, IRDye-800CW-maleimide or SRfluor680-maleimide (SR-1004), respectively. Using fluorescence microscopy, the binding specificity of the four NIR fluorescent Affibody probes was studied by high-EGFR-expressing A431 cells and low-EGFR-expressing MCF7 cells. The binding affinities of the four probes (KD) to EGFR were determined by flow cytometry. In vivo optical imaging of the four probes were performed in the mice bearing subcutaneous A431 tumor. Results All four probes exhibited high EGFR binding affinity in nM range. Particularly, Alex680-ZEGFR:1907 showed higher binding affinity than that of other probes, and the KD of the probe was 28.27±4.87nM. In vivo imaging of the four probes revealed that they all showed fast tumor targeting and good tumor-to-normal tissue contrast as early as 0.5 h post-injection (p.i.), the tumor-to-normal ratio reached a peak at 2 to 4 h p.i. by regional of interest (ROI) analysis of images. Ex vivo studies further demonstrated the four probes had high tumor uptakes. Conclusions This work demonstrates that Affibody can be used for tumor EGFR optical imaging. The probes developed are promising for further tumor imaging applications and clinical translation