RESUMO
Monoclonal antibodies are an important addition to the medicinal treatment paradigm for IBD patients. While effective, these agents show a high degree of primary and secondary non-response, and methods to predict response are highly desired. Information on drug distribution at the target level is often lacking. Fluorescent endoscopic imaging using labelled antibody drugs may provide insight regarding drug distribution, target engagement and drug response, but these assessments require stable and functional fluorescently-conjugated probes. Infliximab, vedolizumab, adalimumab and ustekinumab were conjugated to IRDye 800CW, IRDye 680LT and ZW800-1. The resulting 12 tracer candidates were analysed and characterised on SE-HPLC, SDS-PAGE, iso-electric focussing (IEF) and ELISA in order to evaluate their feasibility as candidate clinical tracers for cGMP development. Major differences in the conjugation results could be seen for each conjugated drug. For Infliximab, 2 conjugates (800CW and 680LT) showed formation of aggregates, while conjugates of all drugs with ZW800-1 showed reduced fluorescent brightness, reduced purification yield and formation of fragments. All 6 of these candidates were considered unfeasible. From the remaining 6, ustekinumab-680LT showed reduced binding to IL23, and was therefore considered unfeasible. Out of 12 potential tracer candidates, 5 were considered feasible for further development: vedolizumab-800CW, vedolizumab-680LT, adalimumab-800CW, adalimumab-680LT and ustekinumab-800CW. Infliximab-680LT and ustekinumab-680LT failed to meet the standards for this panel, but may be rendered feasible if tracer production methods were further optimized.
RESUMO
Tumor-positive resection margins are present in up to 23% of head and neck cancer (HNC) surgeries, as intraoperative techniques for real-time evaluation of the resection margins are lacking. In this study, we investigated the safety and potential clinical value of fluorescence-guided imaging (FGI) for resection margin evaluation in HNC patients. We determined the optimal cetuximab-800CW dose by quantification of intrinsic fluorescence values using multi-diameter single-fiber reflectance, single-fiber fluorescence (MDSFR/SFF) spectroscopy. Methods: Five cohorts of three HNC patients received cetuximab-800CW systemically: three single dose cohorts (10, 25, 50 mg) and two cohorts pre-dosed with 75 mg unlabeled cetuximab (15 or 25 mg). Fluorescence visualization and MDSFR/SFF spectroscopy quantification was performed and were correlated to histopathology. Results: There were no study-related adverse events higher than Common Terminology Criteria for Adverse Events grade-II. Quantification of intrinsic fluorescence values showed a dose-dependent increase in background fluorescence in the single dose cohorts (p<0.001, p<0.001), which remained consistently low in the pre-dosed cohorts (p=0.6808). Resection margin status was evaluated with a sensitivity of 100% (4/4 tumor-positive margins) and specificity of 91% (10/11 tumor-negative margins). Conclusion: A pre-dose of 75 mg unlabeled cetuximab followed by 15 mg cetuximab-800CW was considered the optimal dose based on safety, fluorescence visualization and quantification of intrinsic fluorescence values. We were able to use a lower dose cetuximab-800CW than previously described, while remaining a high sensitivity for tumor detection due to application of equipment optimized for IRDye800CW detection, which was validated by quantification of intrinsic fluorescence values.
