Towards real-time antibody-based vital optical imaging of early oesophageal neoplasia

Background: The incidence of oesophageal cancer is rising worldwide, yet the surveillance of patients at risk is very ineffective when using white-light reflectance endoscopy. Overexpression of proteins such as the intracellular enzyme Cyclooxygenase-2 (COX-2) and the tyrosine kinase receptor c-Met has been reported in oesophageal premalignant lesions and adenocarcinomas. The goal of this study is to develop optical imaging probes that target diagnostic biomarkers for the detection of early oesophageal neoplasia in vivo.
Materials and Methods: Probes based on anti-COX-2 [AS66], anti-c-Met [EP1454Y] and (Met13) monoclonal IgG antibodies were synthesised by addition of Cy5 fluorescent dye. For anti-COX-2 probes, and the cell-penetrating peptide TAT was also added to allow intracellular access. Cy5-labelled GE-137 (Edinburgh Molecular Imaging), a peptide-based probe which binds c-Met, was also evaluated. Binding and competition assays were used to determine Kd values. The binding efficiency of each probe was investigated in two oesophageal adenocarcinoma-derived cell lines with high (OE33) and low (FLO-1) COX-2 and c-Met expression. The specificity of each probe was investigated using siRNA knockdown assays. The ability to detect oesophageal cancer in vivo was tested in mice bearing OE33 or FLO-1 xenografts using an in-house fluorescence imaging device, following intravenous administration of the probes at a well-tolerated dose of 0.5 mg/kg.
Results: Low Kd values in the same order of magnitude as the native antibodies were obtained for the conjugated probes, representing high binding affinity. All probes demonstrated significantly higher binding and retention in OE33 as compared to FLO-1 cells (P < 0.001). However, the fluorescence signal from the Cy5-COX-2-TAT probe in OE33 cells was comparable to its control, Cy5-COX-2 without TAT, suggesting high off-target binding of the anti-COX-2 antibody. Imaging of OE33 cells in which either COX-2 or c-Met was selectively knocked down supported earlier data, demonstrating high specificity of anti-c-Met antibodies and low specificity of anti-COX-2 [AS66]. In vivo studies showed tumour uptake and retention of the Cy5-Met13 in OE33 but not FlO-1 tumour xenografts (P < 0.0001) as well as high c-Met specificity when compared to a Cy5-IgG control (P < 0.001). However, Cy5-COX-2-TAT showed both poor uptake and lack of COX-2 specificity in vivo when compared to controls: non-specific Cy5-IgG-TAT and Cy5-COX-2 without TAT. Finally, high tumour- but low c-Met specificity was observed for GE-137, with high TBR ratios recorded in both OE33 and FLO-1 xenografts.
Conclusions: We have presented a feasible new approach to optical imaging of c-Met and COX-2. Successful conjugate synthesis and TAT-facilitated internalisation in vitro serve as a proof of concept for the future development of an anti-COX-2 fluorescent probe, on the condition of selecting an antibody with better specificity. Furthermore, a non-invasive means of optical imaging of c-Met in vivo, presented in this thesis, can have important diagnostic implications for various types of gastrointestinal cancer.

Advisor: Vallis, K
Vojnovic, B