Affibody Molecule for Positron Emission Tomography Imaging of Human Epidermal Growth Factor Receptor: Validation in Mouse Models and Human Cancer Tissues

Background: Tumor heterogeneity and changes in epidermal growth factor receptor (EGFR) expression status over time post challenges for the design of strategies for effective anti-EGFR monoclonal antibodies in the treatment of non-small-cell lung cancer (NSCLC). Therefore, there is an urgent need to develop techniques for real-time and comprehensive tumor EGFR profiling especially in lung cancer precision medicine trials. Radionuclide imaging of EGFR expression in tumors may screen patients for EGFR-targeting therapies and predict response or resistance to certain treatments.Methods: EGFR-specific Affibody molecule (ZEGFR:1907) was radiolabeled with 68Ga. The radioligands were characterized in vitro and in mice bearing subcutaneous tumors with varying levels of EGFR expression: HCC827 (EGFR overexpression), H1975 (moderate-high), A549 (moderate), H358 (low), and H520 (negative). In vivo tumor targeting activity using PET imaging and biodistribution were conducted in tumor-bearing nude mice. Autoradiography, western blot, immunofluorescence, and immunohistochemistry were performed in human tumor samples. Statistical analyses were performed using GraphPad Prism 7.0. One-way or two-way analysis of variance (ANOVA) followed by the Bonferroni’s multiple comparisons test was used. Statistical significance was set at P < 0.05.Results: 68Ga-NOTA-ZEGFR:1907 showed higher uptake in high EGFR-expressing cells (HCC827, H1975) when compared to cells with moderate to low EGFR (A549, H358) or without EGFR (H520). Radionuclide imaging showed probe accumulation was preferential in EGFR-expressing tumors, particularly in HCC827, H1975 xenografts. A549 and H358 xenografts were mildly and indistinctly visualized. EGFR-negative H520 xenografts were barely visible at any time-point. Biodistribution showed a significantly higher accumulation in HCC827 tumors when compared to H520 tumors (3.20 ± 0.10 %ID/g vs. 0.81 ± 0.08 %ID/g at 2h, P< 0.05). Specific binding to EGFR could be competitively blocked by excess un-radiolabeled affibody molecules in cell uptake, PET imaging and biodistribution assays. Autoradiography showed the regions with high radiotracer uptake partly overlapped with the area of positive EGFR immunofluorescence and immunohistochemistry. Finally, the overall accumulation of autoradiography was positively correlated with immunohistochemistry score.Conclusion: Affibody-based radiotracer 68Ga-NOTA-ZEGFR:1907 is suitable for identification of EGFR expression, showing great potential for further applications and clinical translation.