Effect of Dye and Conjugation Chemistry on the Biodistribution Profile of Near-Infrared-Labeled Nanobodies as Tracers for Image-Guided Surgery

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 preservation, 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 non-specific 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 hours p.i., while <1.0 for HER2-negative tumors at 3 hours p.i., p<0.05). Contrarily, using the randomly-labeled 2Rs15d-IRDye800CW, HER2-positive and -negative tumors could only be distinguished after 24h due to high non-specific 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 have a major impact on the pharmacokinetics of nanobodies.