64Cu-Labeled Trastuzumab Fab-PEG24-EGF Radioimmunoconjugates Bispecific for HER2 and EGFR: Pharmacokinetics, Biodistribution, and Tumor Imaging by PET in Comparison to Monospecific Agents

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• 作者：Luke Yongkyu Kwon ; Deborah A. Scollard ; Raymond M. Reilly
• 刊名：Molecular Pharmaceutics
• 出版年：2017
• 出版时间：February 6, 2017
• 年：2017
• 卷：14
• 期：2
• 页码：492-501
• 全文大小：522K
• ISSN：1543-8392

文摘

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 (EGFR^mod/HER2^low), MDA-MB-468 (EGFR^high/HER2^neg), MDA-MB-231-H2N (EGFR^mod/HER2^mod), and SKOV3 (EGFR^low/HER2^high) cells by competition and saturation cell binding assays to estimate the dissociation constant (K_d). 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.