Synthesis and evaluation of (64)Cu-radiolabeled NOTA-cetuximab ((64)Cu-NOTA-C225) for immuno-PET imaging of EGFR expression

OBJECTIVE: Epidermal growth factor receptor (EGFR) is overexpressed in a wide variety of solid tumors, serving as a well-characterized target for cancer imaging or therapy. In this study, we aimed to design and synthesize a radiotracer, (64)Cu-NOTA-C225, targeting EGFR for tumor positron emission tomography (PET) imaging. METHODS: Cetuximab (C225) was conjugated to a bifunctional chelator, p-isothiocyanatobenzyl-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and further radiolabeled with copper-64 for PET imaging. (64)Cu-NOTA-IgG and Cy5.5-C225 were also synthesized as control probes. A431 and A549 mouse models were established for micro-PET and/or near-infrared fluorescence (NIRF) imaging. RESULTS: (64)Cu-NOTA-C225 exhibited stability in vivo and in vitro up to 24 h and 50 h post-injection, respectively. A431 tumors with average standard uptake values (SUVs) of 5.61±0.69, 6.68±1.14, 7.80±1.51 at 6, 18 and 36 h post-injection, respectively, which were significantly higher than that of moderate EGFR expressing tumors (A549), with SUVs of 0.89±0.16, 4.70±0.81, 2.01±0.50 at 6, 18 and 36 h post-injection, respectively. The expression levels of A431 and A549 were confirmed by western blotting. Additionally, the tracer uptake in A431 tumors can be blocked by unlabeled cetuximab, suggesting that tracer uptake by tumors was receptor-mediated. Furthermore, NIRF imaging using Cy5.5-C225 showed that the fluorescence intensity in tumors increased with time, with a maximal intensity of 8.17E+10 (p/s/cm(2)/sr)/(μW/cm(2)) at 48 h post-injection, which is consistent with the paradigm from micro-PET imaging in A431 tumor-bearing mice. CONCLUSIONS: The (64)Cu-NOTA-C225 PET imaging may be able to specifically and sensitively differentiate tumor models with different EGFR expression levels. It offers potentials as a PET radiotracer for imaging of tracer EGFR-positive tumors.