Affibody
protein is an engineered
protein scaffold with a three-helical bundle structure. Affibody molecules of small size (7 kD) have great potential for targeting overexpressed
cancer biomarkers in vivo. To develop an Affibody-based
molecular probe for in vivo optical imaging of
epidermal growth factor receptor (EGFR) positive
tumors, an anti-EGFR Affibody molecule, Ac-Cys-Z(EGFR:1907) (7 kD), is site-specifically conjugated with a near-IR fluorescence
dye, Cy5.5-mono-maleimide. Using fluorescent microscopy, the binding specificity of the probe
Cy5.5-Z(EGFR:1907) is checked by a high-EGFR-expressing A431 cell and low-EGFR-expressing MCF7 cells. The binding affinity of
Cy5.5-Z(EGFR:1907) (K(D)) to EGFR is 43.6+/-8.4 nM, as determined by flow cytometry. For an in vivo imaging study, the probe shows fast
tumor targeting and good
tumor contrast as early as 0.5 h postinjection (p.i.) for A431
tumors, while MCF7
tumors are barely visible. An ex vivo imaging study also demonstrates that
Cy5.5-Z(EGFR:1907) has high
tumor, liver, and kidney uptakes at 24 h p.i.. In conclusion,
Cy5.5-Z(EGFR:1907) shows good affinity and high specificity to the EGFR. There is rapid achievement of good
tumor-to-normal-tissue contrasts of
Cy5.5-Z(EGFR:1907), thus demonstrating its potential for EGFR-targeted molecular imaging of
cancers.