Debie Pieterjan, Van Quathem Jannah, Hansen Inge, Bala Gezim, Massa Sam, Devoogdt Nick, Xavier Catarina, Hernot Sophie
In Vivo Cellular and Molecular Imaging Laboratory, Vrije Universiteit Brussel , Laarbeeklaan 103, 1090 Brussels, Belgium.
Laboratory for Cellular and Molecular Imunology, Vrije Universiteit Brussel , Pleinlaan 2, 1050 Brussels, Belgium.
Mol Pharm. 2017 Apr 3;14(4):1145-1153. doi: 10.1021/acs.molpharmaceut.6b01053. Epub 2017 Mar 9.
Advances in optical imaging technologies have stimulated the development of near-infrared (NIR) fluorescently labeled targeted probes for use in image-guided surgery. As nanobodies have already proven to be excellent candidates for molecular imaging, we aimed in this project to design NIR-conjugated nanobodies targeting the tumor biomarker HER2 for future applications in this field and to evaluate the effect of dye and dye conjugation chemistry on their pharmacokinetics during development. IRDye800CW or IRdye680RD were conjugated either randomly (via lysines) or site-specifically (via C-terminal cysteine) to the anti-HER2 nanobody 2Rs15d. After verification of purity and functionality, the biodistribution and tumor targeting of the NIR-nanobodies were assessed in HER2-positive and -negative xenografted mice. Site-specifically IRDye800CW- and IRdye680RD-labeled 2Rs15d as well as randomly labeled 2Rs15d-IRDye680RD showed rapid tumor accumulation and low nonspecific uptake, resulting in high tumor-to-muscle ratios at early time points (respectively 6.6 ± 1.0, 3.4 ± 1.6, and 3.5 ± 0.9 for HER2-postive tumors at 3 h p.i., while <1.0 for HER2-negative tumors at 3 h p.i., p < 0.05). Contrarily, using the randomly labeled 2Rs15d-IRDye800CW, HER2-positive and -negative tumors could only be distinguished after 24 h due to high nonspecific signals. Moreover, both randomly labeled 2Rs15d nanobodies were not only cleared via the kidneys but also partially via the hepatobiliary route. In conclusion, near-infrared fluorescent labeling of nanobodies allows rapid, specific, and high contrast in vivo tumor imaging. Nevertheless, the fluorescent dye as well as the chosen conjugation strategy can affect the nanobodies' properties and consequently have a major impact on their pharmacokinetics.
光学成像技术的进步推动了用于图像引导手术的近红外（NIR）荧光标记靶向探针的发展。由于纳米抗体已被证明是分子成像的优秀候选物，我们在本项目中旨在设计靶向肿瘤生物标志物HER2的近红外共轭纳米抗体，以便在该领域未来应用，并在开发过程中评估染料和染料共轭化学对其药代动力学的影响。IRDye800CW或IRDye680RD通过赖氨酸随机或通过C末端半胱氨酸位点特异性地与抗HER2纳米抗体2Rs15d共轭。在验证纯度和功能后，在HER2阳性和阴性异种移植小鼠中评估了近红外纳米抗体的生物分布和肿瘤靶向性。位点特异性IRDye800CW和IRDye680RD标记的2Rs15d以及随机标记的2Rs15d-IRDye680RD显示出快速的肿瘤积累和低非特异性摄取，在早期时间点导致高肿瘤与肌肉比率（在接种后3小时，HER2阳性肿瘤分别为6.6±1.0、3.4±1.6和3.5±0.9，而接种后3小时HER2阴性肿瘤<1.0，p<0.05）。相反，使用随机标记的2Rs15d-IRDye800CW，由于高非特异性信号，HER2阳性和阴性肿瘤仅在24小时后才能区分。此外，两种随机标记的2Rs15d纳米抗体不仅通过肾脏清除，还部分通过肝胆途径清除。总之，纳米抗体的近红外荧光标记允许在体内进行快速、特异性和高对比度的肿瘤成像。然而，荧光染料以及所选的共轭策略会影响纳米抗体的性质，从而对其药代动力学产生重大影响。
