Novel COX-2 Targeted Nanobodies for Molecular Endoscopic Imaging of Colorectal Adenomas

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality in men and women. Timely detection and diagnosis are key to management of CRC, which is under-diagnosed because colorectal aberrant crypt foci, hyperplastic polyps, and microadenomas are often missed with conventional colonoscopy. The enzyme cyclooxygenase-2 (COX-2) is overexpressed in early stages of colorectal carcinogenesis and plays an important regulatory role in the process, suggesting that it could be a valuable target for enhanced imaging of nascent disease. Thus, we have generated an alpaca-derived library of 73 COX-2-specific nanobody clones. Here, we describe one such nanobody, F9-K45Q-K77Q-ROX, in which two native lysine residues have been mutated followed by conjugation to a fluorophore at the N-terminus with retention of COX-2-selective binding. The site of fluorophore conjugation and COX-2 binding affinity of F9-K45Q-K77Q-ROX were determined by proteomic and microscale thermophoretic analyses, respectively. In cell culture studies using 1483 human head and neck squamous cell carcinoma cells, F9-K45Q-K77Q-ROX accumulated inside cells and bound to intracellular COX-2, as visualized by fluorescence microscopy. In vivo pharmacokinetic, and toxicological analyses revealed that F9-K45Q-K77Q-ROX is detectable in circulation with a plasma half-life of 17.9 min and there is no short-term toxicity associated with single injections of 10 mg/kg, 20 mg/kg, or 40 mg/kg doses at 24 h post-administration. Noninvasive in vivo fluorescence endoscopic imaging validated tumor-specific accumulation of F9-K45Q-K77Q-ROX in azoxymethane/dextran sodium sulfate-induced colorectal adenomas in mice. This work demonstrates the first COX-2-targeted nanobodies including a fluorescent derivative that offers significant promise for targeted endoscopic imaging of COX-2-expressing neoplasms. Significance StatementCurrent colorectal cancer screening procedures, such as white-light colonoscopy, chromoendoscopy, and narrow-band imaging aim to detect solid colon tumors and precursor lesions. However, these methods tend to detect only raised solid tumors and mature cancers, whereas precursor lesions, such as aberrant crypt foci, hyperplastic polyps, and small adenomas are frequently missed. To address the need for better visualization of early lesions, we developed a library of alpaca-derived nanobodies targeted to cyclooxygenase-2 (COX-2), an enzyme that is overexpressed in colorectal adenomas. COX-2-targeted nanobodies bearing a fluorescent tag accumulate and are retained in colonic adenomas, facilitating their endoscopic visualization. This novel COX-2-targeted nanobody platform may also be valuable for early detection of other neoplastic diseases in which COX-2 overexpression occurs. (Word counts 119, limit 120)