Dual-labeled Cetuximab-based imaging agent for NIR Fluorescence and PET

Cetuximab (C225) as a chimeric monoclonal antibody specifically targets the epidermal growth factor receptor (EGFR) that often is overexpressed in human malignancies. This phenotype is associated with tumor aggressiveness, treatment resistance, and biological heterogeneity with potential to bypass the blockade of the EGFR signaling pathways. The aim of this work was to prepare and characterize the C225 for near infrared fluorescence (NIRF) and PET imaging using a X-SIGHT Large Stokes Shift Dye (X-SIGHT 670 LSS Dye) and [64Cu]Cu-NOTA as a prerequisite for combination of both imaging methods. C225 was conjugated with the bifunctional chelator 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bz-NOTA) and the X-SIGHT 670 Large Stokes Shift Dye, TFP Ester (XS670). The EGFR-affinity of the immunoconjugate resulted in 94% versus 100% of the unmodified C225 measured with specific enzyme-linked immunosorbent assay (ELISA) on EGFR-positive tumor cell line A431. C225-SCNBz-NOTA was labeled with 64Cu within 30 min with high radiolabeling yield and radiochemical purity. The [64Cu]Cu-NOTA-C225-XS670 showed high accumulation in xenotransplanted squamous cell carcinoma tumors in mice after 24 hours imaged with small animal PET and NIRF. The comparison of both methods in living animals showed the high signal intensity and accumulation of the probe in tumors; however only PET allowed the quantitative characterization of the probe distribution in vivo. The subsequent whole body cryosectioning of the animals into 40 µm sections permitted the direct comparison of the autoradiograms and NIRF images of the tissue cuts. The quantitative comparison of the autoradiograms and the NIRF images after coregistration of the corresponding images yielded a good correlation of the pixel intensities, but the different geometric resolution did not allow a pixel vise comparison. The NIRF images showed a higher differentiation than the autoradiograms. The dual-labeled C225 demonstrated the higher resolution and comparable concentration values of the antibody distribution in whole body cryosections of tumor bearing mice using the combination of 64Cu-radioluminography and NIRF imaging. The dual labeling of antibodies is a promising tool for quantitative evaluation of the long time distribution in animals using NIRF of cryosections beyond the decay of the radionuclide used.

Acknowledgement: This project was partially supported by FP7 project “GIPIO”, Project Reference: 223057