Method details

[⁶⁴Cu]Cu-NOTA-trastuzumab Fab-PEG₂₄-EGF specifically binding to both HER2 and EGFR or monospecific [⁶⁴Cu]Cu-NOTA-trastuzumab Fab or [⁶⁴Cu]Cu-NOTA-EGF monospecific for HER2 or EGFR, respectively were synthesized and characterized as reported.¹⁷ Groups of NOD-SCID mice (n = 5) with s.c. tumors on the right flank homogeneous for HER2 or EGFR, or on the left flank heterogeneous for HER2 and EGFR were injected i.v. (tail vein) with 15–20 MBq (10 μg) of these bispecific or monospecific agents. Mice were sedated with isoflurane 2% in oxygen and imaged at 24 h and 48 h post-injection (p.i.) on a PET tomograph (Siemens MicroPET Focus 220). Images (supine position) were acquired for 20–60 min then were reconstructed by filtered back-projection and ordered subset expectation maximization methods, followed by a maximum a posteriori probability reconstruction algorithm with no correction for attenuation or partial-volume effects. The full-width-at-half-maximum resolution of the PET tomograph was 1.37 mm in the transaxial plane at the center of the field of view. Immediately after PET imaging, mice were transferred to an eXplore Locus Ultra Preclinical CT Scanner (GE Healthcare) for a whole-body CT scan using routine acquisition parameters (16 s anatomical scan at 80 kVp, 50 mA, and voxel size of 150 × 150 × 150 μm. MicroPET and CT images were co-registered using Inveon Research Workplace software (Siemens). Immediately after the 48 h imaging time point, mice were sacrificed and tumors and samples of blood and normal tissues were collected, weighed and ⁶⁴Cu measured in a γ-counter. Tumor and normal tissue uptake were expressed as percentage injected dose per gram (%ID/g).