Development and characterization of 89Zr-labeled panitumumab for immuno-positron emission tomographic imaging of the epidermal growth factor receptor

Abstract

The epidermal growth factor receptor (EGFR) is overexpressed in the majority of malignancies and has been associated with poor outcomes. Panitumumab, an anti-EGFR monoclonal antibody that binds to the extracellular binding domain of EGFR, is increasingly used with radiotherapy and chemotherapy but has associated toxicities. The purpose of this study was to develop and characterize a novel targeted imaging agent for the EGFR using radiolabeled panitumumab. Flow cytometry studies were performed to evaluate EGFR expression in several cell lines. Desferrioxamine-Bz-NCS (DFO) was conjugated to panitumumab and labeled with (89)Zr. Cell uptake studies were performed in four cell lines. For biodistribution studies and micro-positron emission tomography/computed tomography (PET/CT), mouse xenograft models were generated using the same cell lines. PET was performed, and tumors and select organs were harvested for biodistribution studies. Panitumumab was radiolabeled with (89)Zr with high radiochemical purity and specific activity and was found to be stable in serum. Cell binding studies demonstrated that radiotracer uptake in cells correlated with the degree of EGFR expression. MicroPET/CT imaging studies demonstrated a high intensity of (89)Zr-panitumumab in A431 and HCT 116 tumors in comparison with the EGFR-negative tumors. Biodistribution studies confirmed the results from the imaging studies. (89)Zr-panitumumab imaging of EGFR-positive tumors demonstrated levels of radiotracer uptake associated with EGFR expression.

Figure 1

Flow cytometric analysis of epidermal growth factor receptor (EGFR) expression. The A431 epidermoid carcinoma, HCT116 colorectal cancer, T47D breast cancer, and MDA-MB435 breast cancer cell lines were evaluated for EGFR expression. Panitumumab was used as the primary antibody, and FITC-conjugated goat antihuman IgG was used as the secondary antibody. Data are shown as cell number on the ordinate access and EGFR intensity on the abscissa.

Figure 2

Cell uptake studies with ⁸⁹Zr-panitumumab. Cell uptake curves of cell number versus percentage of administered activity in A431, HCT116, T47D, and MDA-MB435 cells (n = 3).

Figure 3

Biodistribution of ⁸⁹Zr-panitumumab in A431, HCT116, T47D, and MDA-MB435 xenograft models at 24 (A) and 120 (B) hours postinjection. A 1 mg blocking dose of panitumumab was administered 120 minutes prior to administration of ⁸⁹Zr-panitumumab. Data are expressed as percent injected dose per gram ± standard deviation, n = 5 for each time point.

Figure 4

Representative microPET/CT images obtained at 24 (A) and 120 (B) hours. Tumors were located in the right flank of each mouse. Maximum intensity projected reconstructions are demonstrated in the upper panels, and the axial slice at the center of the tumor is shown in the bottom panel. The scale, expressed as standardized uptake value (SUV), is demonstrated at the far right. A 1 mg blocking dose of panitumumab was administered 120 minutes prior to injection of ⁸⁹Zr-panitumumab.

Figure 5

A graphical analysis of ⁸⁹Zr-panitumumab uptake in xenograft tumor models expressed as standardized uptake value (SUV) ± standard deviation for each tumor type.

Figure 6

Immunofluorescent staining to evaluate the relative levels of epidermal growth factor receptor (EGFR) expression for each tumor type. EGFR staining is demonstrated in red, with a counterstain for the nucleus in blue.

Figure 7

Linear correlation between cell line epidermal growth factor receptor (EGFR) expression (as determined by FACS, as shown in Figure 1) and standardized uptake value (SUV) of tumor xenografts with the same cell lines (from Figure 4B).