Duranti Claudia, Carraresi Laura, Sette Angelica, Stefanini Matteo, Lottini Tiziano, Crescioli Silvia, Crociani Olivia, Iamele Luisa, De Jonge Hugo, Gherardi Ermanno, Arcangeli Annarosa
Department of Experimental and Clinical Medicine, Section of Internal Medicine, University of Florence, Florence, Italy.
DIVAL Toscana Srl, Sesto Fiorentino, Florence, Italy.
Oncotarget. 2018 Oct 9;9(79):34972-34989. doi: 10.18632/oncotarget.26200.
Modern molecular imaging techniques have greatly improved tumor detection and post-treatment follow-up of cancer patients. In this context, antibody-based imaging is rapidly becoming the gold standard, since it combines the unique specificity of antibodies with the sensitivity of the different imaging technologies. The aim of this study was to generate and characterize antibodies in single chain Fragment variable (scFv) format directed to an emerging cancer biomarker, the (hERG1) potassium channel, and to obtain a proof of concept for their potential use for molecular imaging. The anti-hERG1scFv was generated from a full length monoclonal antibody and then mutagenized, substituting a Phenylalanine residue in the third framework of the V domain with a Cysteine residue. The resulting scFv-hERG1-Cys showed much higher stability and protein yield, increased affinity and more advantageous binding kinetics, compared to the "native" anti-hERG1scFv. The scFv-hERG1-Cys was hence chosen and characterized: it showed a good binding to the native hERG1 antigen expressed on cells, was stable in serum and displayed a fast pharmacokinetic profile once injected intravenously in nude mice. The calculated half-life was 3.1 hours and no general toxicity or cardiac toxic effects were detected. Finally, the distribution of an Alexa Fluor 750 conjugated scFv-hERG1-Cys was evaluated both in healthy and tumor-bearing nude mice, showing a good tumor-to-organ ratio, ideal for visualizing hERG1-expressing tumor masses . In conclusion, the scFv-hERG1-Cys possesses features which make it a suitable tool for application in cancer molecular imaging.
现代分子成像技术极大地改善了癌症患者的肿瘤检测及治疗后随访。在此背景下，基于抗体的成像正迅速成为金标准，因为它将抗体独特的特异性与不同成像技术的敏感性结合在一起。本研究的目的是生成并表征针对一种新兴癌症生物标志物——（人ether-à-go-go相关基因1）hERG1钾通道的单链可变片段（scFv）形式的抗体，并为其在分子成像中的潜在应用获得概念验证。抗hERG1 scFv由全长单克隆抗体制备而成，然后进行诱变，将V结构域第三个骨架中的苯丙氨酸残基替换为半胱氨酸残基。与“天然”抗hERG1 scFv相比，所得的scFv-hERG1-Cys表现出更高的稳定性和蛋白产量、增加的亲和力以及更有利的结合动力学。因此选择并表征了scFv-hERG1-Cys：它与细胞上表达的天然hERG1抗原具有良好的结合，在血清中稳定，并且在裸鼠静脉注射后显示出快速的药代动力学特征。计算得出的半衰期为3.1小时，未检测到一般毒性或心脏毒性作用。最后，在健康和荷瘤裸鼠中评估了与Alexa Fluor 750偶联的scFv-hERG1-Cys的分布，显示出良好的肿瘤与器官比值，非常适合可视化表达hERG1的肿瘤块。总之，scFv-hERG1-Cys具有使其成为癌症分子成像应用合适工具的特性。
