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

Abstract

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.

Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics

“…Nanobodies can readily be conjugated to radionuclides and fluorochromes, using either sortagging or classical chemical conjugation strategies (Figure 6 B). Such nanobody-radionuclide and nanobody-NIRF conjugates are useful tools for imaging of tumors antigens or tumor-associated stromal cells, such as the mannose receptor of macrophages (MMR, CD206) ( 11 , 16 , 105 – 107 ). Moreover, such conjugates also have therapeutic potential, e.g., by local delivery of ionizing radiation to the tumor or by thermal cytotoxicity via a photosensitive, NIRF.…”

Tracers for Fluorescence-Guided Surgery: How Elongation of the Polymethine Chain in Cyanine Dyes Alters the Pharmacokinetics of a Dual-Modality c[RGDyK] Tracer

“…This concept has been successfully applied to nanoparticles, antibody analogs, and peptides (8)(9)(10)(11)(12)(13), as exemplified by the plurality of hybrid tracers in the literature (14)(15)(16)(17). For example, for the arginylglycylaspartic acid (RGD) vector, it has been documented that the introduction of different pendant moieties on an otherwise stable conjugated system influences the pharmacokinetics (18)(19)(20)(21)(22).…”

Chemically-defined camelid antibody bioconjugate for the magnetic resonance imaging of Alzheimer's disease

“…For example, the obtained unlabeled and over-labeled VHHs can result in a saturation effect, in a modification of the VHH properties for its target, or in a significant impact on their pharmacokinetics, as recently shown with a near-infrared fluorescent VHH. 54 In our case, the functional properties of the conjugate mixtures were indeed affected compared to the starting VHH, with a significant decrease of the Aß recognition when one to two DOTA/Gd are attached. This effect could be due to steric hindrance in the vicinity of the antigen recognition site, which increased when more amino groups are derivatized.…”