Near-Infrared Fluorescence Imaging of Pancreatic Cancer Using a Fluorescently Labelled Anti-CEA Nanobody Probe: A Preclinical Study

Abstract

Molecular fluorescence-guided surgery using near-infrared light has the potential to improve the rate of complete resection of cancer. Typically, monoclonal antibodies are being used as targeting moieties, however smaller fragments, such as single-domain antibodies (i.e., Nanobodies®) improve tumor specificity and enable tracer injection on the same day as surgery. In this study, the feasibility of a carcinoembryonic antigen-targeting Nanobody (NbCEA5) conjugated to two zwitterionic dyes (ZW800-1 Forte [ZW800F] and ZW800-1) for visualization of pancreatic ductal adenocarcinoma (PDAC) was investigated. After site-specific conjugation of NbCEA5 to the zwitterionic dyes, binding specificity was evaluated on human PDAC cell lines with flow cytometry. A dose escalation study was performed for both NbCEA5-ZW800F and NbCEA5-ZW800-1 in mice with subcutaneously implanted pancreatic tumors. Fluorescence imaging was performed up to 24 h after intravenous injection. Furthermore, the optimal dose for NbCEA5-ZW800-1 was injected in mice with orthotopically implanted pancreatic tumors. A dose-escalation study showed superior mean fluorescence intensities for NbCEA5-ZW800-1 compared to NbCEA5-ZW800F. In the orthotopic tumor models, NbCEA5-ZW800-1 accumulated specifically in pancreatic tumors with a mean in vivo tumor-to-background ratio of 2.4 (SD = 0.23). This study demonstrated the feasibility and potential advantages of using a CEA-targeted Nanobody conjugated to ZW800-1 for intraoperative PDAC imaging.

NANOBODY® Molecule, a Giga Medical Tool in Nanodimensions

“…VHHs are also suitable for optical imaging, such as near-infrared (NIR) imaging using NIR fluorescent dyes. NIR imaging has been widely developed for molecularly guided precision surgery based on the imaging of specific markers [84]. While complete resection of cancer significantly improves favorable outcomes, distinguishing tumor tissue from benign surrounding tissue is still challenging.…”

“…Thus, due to their short circulation time, VHHs are more suitable for non-invasive, shortterm in vivo imaging than fluorochrome-labeled conventional mAbs and offer a promising technology for tumor-specific fluorescence delivery [88]. Studies have shown that ZW800-Fluorescent VHH targeting CEA facilitates visualization of cell lines and patient-derived pancreatic cancers, which better mimic a clinically relevant tumor microenvironment [84,89]. Similarly, a VHH conjugated with IRDye800 targeting carbonic anhydrase IX (CAIX), a specific membrane-bound protein that is upregulated under hypoxic conditions, was able to detect precancerous ductal carcinoma in situ (DCIS) [90].…”