Background/purpose. Multi-modality imaging is a major diagnostic component for cancer management, enabling detection, staging of disease, targeting therapy, and monitoring the therapeutic response. The development of a single agent for real-time non-invasive immunoPET imaging and fluorescence guided surgery (FGS) will provide the next generation tool in the surgical management of cancer. Methods. The humanized anti-CEA M5A-IR800 “sidewinder” (M5A-IR800-SW) antibody-dye conjugate has a NIR 800nm dye incorporated into a PEGylated linker and conjugated with the metal chelate p-SCN-Bn-deferoxamine (DFO) for zirconium-89 (89Zr, half-life 78.4 h) PET imaging. The dual-labeled 89Zr-DFO-M5A-SW-IR800 was evaluated for NIR fluorescence imaging, PET/MRI imaging, terminal tissue biodistribution and blood clearance in a human colorectal cancer LS174T xenograft mouse model. Results. The 89Zr-DFO-M5A-SW-IR800 NIR fluorescence imaging showed high tumor and liver localization that was confirmed by PET imaging. Serial PET/MRI imaging was performed at 24 h, 48 h and 72 h and showed tumor localization visible at 24 h that persisted throughout the experiment. However, the PET scans showed higher activity for the liver than the tumor, compared to the NIR fluorescence imaging. This difference is an important finding as it quantifies the expected difference due to the sensitivity and depth of penetration between the 2 modalities.
Real-time intraoperative image-guided cancer surgery promises to improve oncologic outcomes. Tumor-specific antibodies conjugated with near-infrared (NIR) fluorophores have demonstrated the potential to enhance visualization of solid tumor margins and metastatic disease; however, multiple challenges remain, including improvement in probe development for clinical utility. We have developed an NIR-IR800 dye on a PEGylated linker (sidewinder) conjugated to the humanized anti-carcinoembryonic antigen (CEA) antibody (M5A) with extended in vivo serum and tumor persistence. The anti-CEA M5A-sidewinder has a high dye-to-antibody ratio (average of 7 per antibody) that allows, in an orthotopic implanted human pancreatic cancer mouse model increased tumor fluorescence, higher tumor-to-background ratio and extends the surgical scheduling window compared to current antibody dye conjugates. These preclinical results demonstrate the potential of this probe for fluorescence-guided surgery of CEA-positive gastrointestinal cancers.
In proof-of-concept studies, the anti-CEA M5A-IR800 conjugate demonstrated rapid and effective near infrared (NIR) imaging of human colon cancer and pancreatic cancer primary and metastatic lesions in mouse models. A limitation observed from these studies is the antibody-dye conjugate’s rapid clearance from the blood due to the increased hydrophobicity of the IR800 dye. This is a bottleneck for clinical applications, requiring high doses to be administered and a short surgical time window for intraoperative imaging. As a result, we developed a new prototype anti-CEA-swPEG-IR800 conjugate, that incorporates a PEGylated sidearm linker to shield or mask the IR800 dye’s hydrophobicity, a novel approach to extend the blood circulation half-life and in doing so increase tumor sensitivity as well as lower normal hepatic uptake. Results of the anti-CEA-swPEG-IR800 in an orthotopic human pancreatic cancer mouse model demonstrated exceptional optical imaging at lower doses, a much longer in vivo half-life enabling increased tumor fluorescence and higher tumor to background ratios. We propose that our novel anti-CEA-swPEG-IR800 is capable of enhanced optical imaging than currently available agents and will become the next generation optical imaging agent for safe and effective intraoperative image-guided surgery in CEA expressing GI cancers.
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