In vivo optical imaging of glioma using a novel uPAR-targeting fluorescent single-domain antibody

Introduction
Fluorescence molecular imaging plays a vital role in clinical applications such as image-guided surgery [1]. This technology applies targeted fluorescent agents recognizing biomarkers expressed by diseased or healthy tissue. The urokinase plasminogen activator receptor (uPAR) is a molecular target with increased expression in many human tumors [2]. Targeting moieties such as single-domain antibodies (sdAbs), characterized by high target affinity and fast systemic clearance [3], are excellent candidates for molecular imaging with major advantages over small molecules, peptides, and antibodies.

Methods
Following the llama immunization with uPAR protein, a collection of sdAbs was retrieved and characterized in vitro. The lead compound (sdAb15) with high uPAR affinity, and non-targeting control sdAb, were labeled with a novel NIR fluorophore - s775z [4], and tested in subcutaneous and later orthotopic model of human glioma. 2 nmol of sdAb15-s775z was injected IV in tumor-bearing or sham-operated mice (n=4 per group) which were killed 1h later. Two other groups of tumor-bearing mice (n=4 per group) were given either the control sdAb or sdAb15-s775z plus a 60x molar excess of unlabeled sdAb15. Brains were removed for ex vivo assessment with BLI and fluorescence camera. The signal and target-to-background ratios (TBRs) were quantified using ImageJ.

Results/Discussion
13 sdAbs specific for the human variant of uPAR were retrieved. Their thermal stability ranged between 59.5 and 73.6°C, and uPAR-affinity, as determined by SPR, spanned between 203 and 2.1 nM. Based on the stability, binding characteristics, flow cytometry studies, and production yield, the lead sdAb15 was selected, labeled with s775z, and tested in the glioma tumor models.
NIR fluorescent signals in the orthotopic model could clearly be observed in tumor-bearing mice injected with sdAb15-s775z. Contrarily, no signal was detected in mice injected with the control sdAb, mice injected with an excess of unlabeled sdAb15 or sham-operated mice. The signal of sdAb15-s775z colocalized with the BLI and GFP signals. Ex vivo TBRs, calculated using healthy brain tissue as background, reached 2.57±0.65 for sdAb15-s775z, and 0.99±0.39 or 1.34±0.09 for mice injected with an excess of sdAb15 or control sdAb, respectively. TBR for sham-operated mice was determined at 1.10±0.03.

Conclusions
Intravenous injection of fluorescently labeled anti-uPAR sdAb15 enabled localization of gliomas and clear delineation between tumor and healthy brain tissue. SdAb15 is relevant for future clinical translation, e.g., as a contrast agent to predict tumor aggressiveness non-invasively or to guide surgical procedures in real-time. These findings could enhance the accuracy of surgical tumor debulking and increase the patient survival rate.

Acknowledgement
The study was funded by Stichting Tegen Kanker, Fonds voor Wetenschappelijk Onderzoek, SRP50, IOF, and SofinaBoël Fund.

References
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[2] Smith HW and Marshall CJ. 'Regulation of cell signalling by uPAR'. Nat Rev Mol Cell Biol (2010) 11:23–36.
[3] Muyldermans S. 'Applications of Nanobodies'. Annu Rev Anim Biosci (2021) 9:401–421.
[4] Li DH, Schreiber CL, and Smith BD. 'Sterically shielded heptamethine cyanine dyes for bioconjugation and high-performance near-infrared fluorescence imaging'. Angew Chem Int Ed Engl (2020) 59:12154–12161.