The GEM-handle as convenient labeling strategy for bimodal single-domain antibody-based tracers carrying 99mTc and a near-infrared fluorescent dye for intra-operative decision-making.

Intra-operative fluorescence imaging has demonstrated its ability to improve tumor lesion identification. However, the limited tissue penetration of the fluorescent signals hinders the detection of deep-lying or occult lesions. Integrating fluorescence imaging with SPECT and/or intra-operative gamma-probing synergistically combines the deep tissue penetration of gamma rays for tumor localization with the precision of fluorescence imaging for precise tumor resection. In this study, we detail the use of a genetically encoded multifunctional handle, henceforth referred to as a GEM-handle, for the development of fluorescent/radioactive bimodal single-domain antibody (sdAb)-based tracers. A sdAb that targets the urokinase plasminogen activator receptor (uPAR) was engineered to carry a GEM-handle containing a carboxy-terminal hexahistidine-tag and cysteine-tag. A two-step labeling strategy was optimized and applied to site-specifically label IRDye800CW and 99mTc to the sdAb. Bimodal labeling of the sdAbs proved straightforward and successful. 99mTc activity was however restricted to 18.5 MBq per nmol fluorescently-labeled sdAb to prevent radiobleaching of IRDye800CW without impeding SPECT/CT imaging. Subsequently, the in vivo biodistribution and tumor-targeting capacity of the bimodal tracer were evaluated in uPAR-positive tumor-bearing mice using SPECT/CT and fluorescence imaging. The bimodal sdAb showed expected renal background signals due to tracer clearance, along with slightly elevated non-specific liver signals. Four hours post-injection, both SPECT/CT and fluorescent images achieved satisfactory tumor uptake and contrast, with significantly higher values observed for the anti-uPAR bimodal sdAb compared to a control non-targeting sdAb. In conclusion, the GEM-handle is a convenient method for designing and producing bimodal sdAb-based tracers with adequate in vivo characteristics.

The Design and Preclinical Evaluation of a Single-Label Bimodal Nanobody Tracer for Image-Guided Surgery.

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a "multifunctional single attachment point" (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and 111In-labeling of DTPA, the tracer's characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer's ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [111In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications.