Cu-Labeled Trastuzumab Fab-PEG-EGF Radioimmunoconjugates Bispecific for HER2 and EGFR: Pharmacokinetics, Biodistribution, and Tumor Imaging by PET in Comparison to Monospecific Agents.

Heterodimerization of EGFR with HER2 coexpressed in breast cancer (BC) promotes tumor growth, and increased EGFR expression is associated with trastuzumab resistance. Our aim was to construct Cu-labeled bispecific radioimmunoconjugates (bsRIC) composed of trastuzumab Fab, which binds HER2 linked through a polyethylene glycol (PEG) spacer to EGF, and to compare their pharmacokinetic, biodistribution, and tumor imaging characteristics by positron-emission tomography (PET). bsRICs were generated by linking maleimide modified trastuzumab Fab with thiolated EGF through a thioether bond. HER2 and EGFR binding were assessed in vitro in MDA-MB-231 (EGFR/HER2), MDA-MB-468 (EGFR/HER2), MDA-MB-231-H2N (EGFR/HER2), and SKOV3 (EGFR/HER2) cells by competition and saturation cell binding assays to estimate the dissociation constant (K). The elimination of the Cu-NOTA-trastuzumab Fab-PEG-EGF bsRICs from the blood of Balb/c mice was compared to monospecific Cu-NOTA-trastuzumab Fab and Cu-NOTA-EGF. MicroPET/CT imaging was performed in NOD/SCID mice bearing subcutaneous MDA-MB-468, MDA-MB-231/H2N, or SKOV3 human BC xenografts at 24 and 48 h postinjection (p.i.) of bsRICs. Tumor and normal tissue uptake were quantified by biodistribution studies and compared to monospecific agents. The binding of bsRICs to MDA-MB-231 cells was decreased to 24.5 ± 5.2% by excess EGF, while the binding of bsRICs to SKOV3 cells was decreased to 38.6 ± 5.4% by excess trastuzumab Fab, demonstrating specific binding to both EGFR and HER2. Cu-labeled bsRICs incorporating the PEG spacer were eliminated more slowly from the blood than Cu-bsRICs without the PEG spacer and were cleared much more slowly than Cu-NOTA-Fab or Cu-NOTA-EGF. All three tumor xenografts were visualized by microPET/CT at 24 and 48 h p.i. of bsRICs. Biodistribution studies at 48 h p.i. in NOD/SCID mice with MDA-MB-231/H2N tumors demonstrated significantly greater tumor uptake of Cu-NOTA-Fab-PEG-EGF (4.9 ± 0.4%ID/g) than Cu-NOTA-Fab (1.9 ± 0.3%ID/g; P < 0.0001) and Cu-NOTA-EGF (0.7 ± 0.2%ID/g; P < 0.0001). Furthermore, preadministration of an excess of trastuzumab Fab or trastuzumab Fab-PEG-EGF significantly decreased the tumor uptake of Cu-NOTA-Fab-PEG-EGF in SK-OV-3 and MDA-MB-468 xenografts by 4.4-fold (P = 0.0012) and 1.8-fold (P = 0.0031), respectively. Cu-labeled bsRICs bound HER2 or EGFR and were taken up specifically in vivo in tumor xenografts expressing one or both receptors. The PEG linker prolonged the blood residence time contributing to the higher tumor uptake of the bsRICs than monospecific agents.