Generation and Characterization of Novel Pan-Cancer Anti-uPAR Fluorescent Nanobodies as Tools for Image-Guided Surgery.

Fluorescence molecular imaging plays a vital role in image-guided surgery. In this context, the urokinase plasminogen activator receptor (uPAR) is an interesting biomarker enabling the detection and delineation of various tumor types due to its elevated expression on both tumor cells and the tumor microenvironment. In this study, anti-uPAR Nanobodies (Nbs) are generated through llama immunization with human and murine uPAR protein. Extensive in vitro characterization and in vivo testing with radiolabeled variants are conducted to assess their pharmacokinetics and select lead compounds. Subsequently, the selected Nbs are converted into fluorescent agents, and their application for fluorescence-guided surgery is evaluated in various subcutaneous and orthotopic tumor models. The study yields a panel of high-affinity anti-uPAR Nbs, showing specific binding across multiple types of cancer cells in vitro and in vivo. Lead fluorescently-labeled compounds exhibit high tumor uptake with high contrast at 1 h after intravenous injection across all assessed uPAR-expressing tumor models, outperforming a non-targeting control Nb. Additionally, rapid and accurate tumor localization and demarcation are demonstrated in an orthotopic human glioma model. Utilizing these Nbs can potentially enhance the precision of surgical tumor resection and, consequently, improve survival rates in the clinic.

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.

Design and Validation of Site-Specifically Labeled Single-Domain Antibody-Based Tracers for in Vivo Fluorescence Imaging and Image-Guided Surgery.

Near-infrared fluorescence molecular imaging has become an established preclinical technique to investigate molecular processes in vivo and to study novel therapies. Furthermore, fluorescence molecular imaging is gaining significant interest from clinicians as an intra-operative guidance tool. This technique makes use of targeted fluorescent tracers as contrast agents that recognize specific biomarkers expressed at the site of disease. Single-domain antibodies have shown to possess excellent properties for in vivo imaging in comparison to conventional antibodies. In this chapter, we describe a method for site-specific conjugation of a near-infrared fluorophore to single-domain antibodies by exploiting cysteine-maleimide chemistry. As opposed to random conjugation, site-specific conjugation results in a homogenously labeled fluorescent tracer and avoids inference with antigen binding.

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.

Corticostriatal dysfunction and social interaction deficits in mice lacking the cystine/glutamate antiporter.

The astrocytic cystine/glutamate antiporter system xc- represents an important source of extracellular glutamate in the central nervous system, with potential impact on excitatory neurotransmission. Yet, its function and importance in brain physiology remain incompletely understood. Employing slice electrophysiology and mice with a genetic deletion of the specific subunit of system xc-, xCT (xCT-/- mice), we uncovered decreased neurotransmission at corticostriatal synapses. This effect was partly mitigated by replenishing extracellular glutamate levels, indicating a defect linked with decreased extracellular glutamate availability. We observed no changes in the morphology of striatal medium spiny neurons, the density of dendritic spines, or the density or ultrastructure of corticostriatal synapses, indicating that the observed functional defects are not due to morphological or structural abnormalities. By combining electron microscopy with glutamate immunogold labeling, we identified decreased intracellular glutamate density in presynaptic terminals, presynaptic mitochondria, and in dendritic spines of xCT-/- mice. A proteomic and kinomic screen of the striatum of xCT-/- mice revealed decreased expression of presynaptic proteins and abnormal kinase network signaling, that may contribute to the observed changes in postsynaptic responses. Finally, these corticostriatal deregulations resulted in a behavioral phenotype suggestive of autism spectrum disorder in the xCT-/- mice; in tests sensitive to corticostriatal functioning we recorded increased repetitive digging behavior and decreased sociability. To conclude, our findings show that system xc- plays a previously unrecognized role in regulating corticostriatal neurotransmission and influences social preference and repetitive behavior.