Molecular imaging of radio- and NIR-labeled EGFR-antibody in tumor bearing mice

Ziel/Aim:

The epidermal growth factor receptor (EGFR) is often 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. Cetuximab (C225) as a chimeric monoclonal antibody specifically targets the EGFR was modified for radio- and near infrared fluorescence (NIRF) imaging as well as for potential applications in radiotherapy using DTPA-, DOTA-, NOTA-modification, radio-labeling with Cu-64, Y-86, Y-90, Lu-177, and for modification with X-SIGHT Large Stokes Shift Dye (X-SIGHT 670 LSS Dye).

Methodik/Methods:

C225 was conjugated with bifunctional chelators based on SCN-Bz and -DTPA, -DOTA, -NOTA, and the C225-NOTA derivative was additionally modified with the X-SIGHT 670 Large Stokes Shift Dye, TFP Ester (XS670). The receptor binding and cell (A431 cells expressing high amounts of EGFR) uptake of Cu-64-NOTA-XS670-C225 was studied in vitro. The EGFR-affinity of the immune-conjugates was measured by a competitive radio-ligand binding assay. The conjugates were labeled with Cu-64, Y-86, Y-90, Lu-177 within 30 min with high radiolabeling yield and radiochemical purity. The biodistribution and –kinetics in vivo were studied by small animal PET or SPECT and ex vivo by autoradiography. Whole body cryo-sectioning of the animals into 40 micrometer sections permitted the direct comparison of the autoradiograms and NIRF images of the tissue sections.

Ergebnisse/Results:

C225 was conjugated with bifunctional chelators based on SCN-Bz and -DTPA, -DOTA, -NOTA, and the C225-NOTA derivative was additionally modified with the X-SIGHT 670 Large Stokes Shift Dye, TFP Ester (XS670). The receptor binding and cell (A431 cells expressing high amounts of EGFR) uptake of Cu-64-NOTA-XS670-C225 was studied in vitro. The EGFR-affinity of the immune-conjugates was measured by a competitive radio-ligand binding assay. The conjugates were labeled with Cu-64, Y-86, Y-90, Lu-177 within 30 min with high radiolabeling yield and radiochemical purity. The biodistribution and –kinetics in vivo were studied by small animal PET or SPECT and ex vivo by autoradiography. Whole body cryo-sectioning of the animals into 40 micrometer sections permitted the direct comparison of the autoradiograms and NIRF images of the tissue sections.

Schlussfolgerungen/Conclusions:

PET allows the quantitative kinetic characterization of biodistribution in small animals, which will be a prerequisite to estimate the dosimetry in animals. SPECT and autoradiography allowed the fine distribution imaging. The dual-labeling of antibodies is a promising tool for quantitative evaluation of the long time distribution in animals using NIRF of cryo-sections beyond the decay of the radionuclide used. Dual-labeled immune-conjugates represent a potential probe for translational application in tumor detection.