64Cu-Labeled Trastuzumab Fab-PEG24-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 64Cu-labeled bispecific radioimmunoconjugates (bsRIC) composed of trastuzumab Fab, which binds HER2 linked through a polyethylene glycol (PEG24) 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 (EGFRmod/HER2low), MDA-MB-468 (EGFRhigh/HER2neg), MDA-MB-231-H2N (EGFRmod/HER2mod), and SKOV3 (EGFRlow/HER2high) cells by competition and saturation cell binding assays to estimate the dissociation constant (Kd). The elimination of the 64Cu-NOTA-trastuzumab Fab-PEG24-EGF bsRICs from the blood of Balb/c mice was compared to monospecific 64Cu-NOTA-trastuzumab Fab and 64Cu-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. 64Cu-labeled bsRICs incorporating the PEG24 spacer were eliminated more slowly from the blood than 64Cu-bsRICs without the PEG spacer and were cleared much more slowly than 64Cu-NOTA-Fab or 64Cu-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 64Cu-NOTA-Fab-PEG24-EGF (4.9 ± 0.4%ID/g) than 64Cu-NOTA-Fab (1.9 ± 0.3%ID/g; P < 0.0001) and 64Cu-NOTA-EGF (0.7 ± 0.2%ID/g; P < 0.0001). Furthermore, preadministration of an excess of trastuzumab Fab or trastuzumab Fab-PEG24-EGF significantly decreased the tumor uptake of 64Cu-NOTA-Fab-PEG24-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. 64Cu-labeled bsRICs bound HER2 or EGFR and were taken up specifically in vivo in tumor xenografts expressing one or both receptors. The PEG24 linker prolonged the blood residence time contributing to the higher tumor uptake of the bsRICs than monospecific agents.

Bispecific radioimmunoconjugates exploit receptor heterogeneity for positron emission tomography of tumors expressing HER2 and/or EGFR.

HER2 heterogeneity is a challenge for molecular imaging or treating HER2-positive breast cancer (BC). EGFR is coexpressed in some tumors exhibiting HER2 heterogeneity. Bispecific radioimmunoconjugates (bsRICs) that bind HER2 and EGFR were constructed by linking trastuzumab Fab through polyethyleneglycol (PEG24) to EGF. We established s.c. tumors in NOD-SCID mice that homogeneously or heterogeneously expressed HER2 and/or EGFR by the inoculation of HER2-positive/EGFR-negative SK-OV-3 cells, EGFR-positive/HER2-negative MDA-MB-468 cells or mixtures of these cells. [64Cu]Cu-NOTA-trastuzumab Fab-PEG24-EGF were compared to [64Cu]Cu-NOTA-trastuzumab Fab or [64Cu]Cu-NOTA-EGF for the PET imaging of HER2 and/or EGFR-positive tumors. [64Cu]Cu-NOTA-trastuzumab Fab-PEG24-EGF bsRICs imaged tumors expressing HER2 or EGFR or heterogeneously expressing these receptors, while monospecific agents only imaged HER2-or EGFR-positive tumors. Our results indicate that bsRICs labeled with 64Cu are able to exploit receptor heterogeneity for tumor imaging. PET may select patients for radioimmunotherapy with bsRICs complexed to the ß-particle emitter, 177Lu or Auger electron-emitter, 111In in a theranostic approach.