Stanford University, Stanford Cancer Center, Molecular Imaging Program at Stanford, Department of Radiology and Bio-X Program, 1201 Welch Road, Lucas Expansion, P095, Stanford, California 94305-5344, USA.
J Biomed Opt. 2010 May-Jun;15(3):036007. doi: 10.1117/1.3432738.
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.
亲和体蛋白是一种具有三螺旋束结构的工程化蛋白支架。亲和体分子体积小（7kD），在体内靶向过表达的癌症生物标志物方面具有巨大潜力。为了开发基于亲和体的分子探针用于表皮生长因子受体（EGFR）阳性肿瘤的体内光学成像，一种抗-EGFR 亲和体分子 Ac-Cys-Z（EGFR：1907）（7kD）被特异性地与近红外荧光染料 Cy5.5-单马来酰亚胺偶联。使用荧光显微镜，通过高 EGFR 表达的 A431 细胞和低 EGFR 表达的 MCF7 细胞检查探针 Cy5.5-Z（EGFR：1907）的结合特异性。通过流式细胞术确定 Cy5.5-Z（EGFR：1907）（K（D））与 EGFR 的结合亲和力为 43.6+/-8.4 nM。在体内成像研究中，探针在 A431 肿瘤中早在注射后 0.5 小时（p.i.）即可快速靶向肿瘤并具有良好的肿瘤对比度，而 MCF7 肿瘤几乎不可见。离体成像研究还表明，Cy5.5-Z（EGFR：1907）在注射后 24 小时具有高肿瘤、肝和肾摄取率。总之，Cy5.5-Z（EGFR：1907）对 EGFR 具有良好的亲和力和高特异性。Cy5.5-Z（EGFR：1907）能够迅速达到良好的肿瘤与正常组织对比度，从而证明了其在癌症的 EGFR 靶向分子成像中的潜力。
