Kwon Luke Yongkyu, Scollard Deborah A, Reilly Raymond M
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, Ontario, Canada.
STTARR Innovation Centre, University Health Network , Toronto, Ontario, Canada.
Mol Pharm. 2017 Feb 6;14(2):492-501. doi: 10.1021/acs.molpharmaceut.6b00963. Epub 2017 Jan 18.
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.
在乳腺癌（BC）中共表达的表皮生长因子受体（EGFR）与人类表皮生长因子受体2（HER2）的异源二聚化促进肿瘤生长，且EGFR表达增加与曲妥珠单抗耐药相关。我们的目的是构建由曲妥珠单抗Fab组成的铜标记双特异性放射免疫缀合物（bsRIC），其通过聚乙二醇（PEG）间隔臂与HER2结合，并通过正电子发射断层扫描（PET）比较它们的药代动力学、生物分布和肿瘤成像特征。通过硫醚键将马来酰亚胺修饰的曲妥珠单抗Fab与硫醇化的表皮生长因子（EGF）连接，生成bsRIC。通过竞争和饱和细胞结合试验在体外评估MDA-MB-231（EGFR/HER2）、MDA-MB-468（EGFR/HER2）、MDA-MB-231-H2N（EGFR/HER2）和SKOV3（EGFR/HER2）细胞中HER2和EGFR的结合，以估计解离常数（K）。将铜-氮杂环三乙酸（Cu-NOTA）-曲妥珠单抗Fab-PEG-EGF bsRIC从Balb/c小鼠血液中的清除情况与单特异性Cu-NOTA-曲妥珠单抗Fab和Cu-NOTA-EGF进行比较。在注射bsRIC后24小时和48小时，对携带皮下MDA-MB-468、MDA-MB-231/H2N或SKOV3人BC异种移植瘤的非肥胖糖尿病/严重联合免疫缺陷（NOD/SCID）小鼠进行微型PET/CT成像。通过生物分布研究对肿瘤和正常组织摄取进行定量，并与单特异性药物进行比较。过量的EGF使bsRIC与MDA-MB-231细胞的结合降低至24.5±5.2%，而过量的曲妥珠单抗Fab使bsRIC与SKOV3细胞的结合降低至38.6±5.4%，表明其对EGFR和HER2均有特异性结合。与不含PEG间隔臂的铜标记bsRIC相比，含有PEG间隔臂的铜标记bsRIC从血液中的清除更慢，且比Cu-NOTA-Fab或Cu-NOTA-EGF清除慢得多。在注射bsRIC后24小时和48小时，微型PET/CT均可观察到所有三种肿瘤异种移植瘤。在注射后48小时对携带MDA-MB-231/H2N肿瘤的NOD/SCID小鼠进行的生物分布研究表明，Cu-NOTA-Fab-PEG-EGF的肿瘤摄取（4.9±0.4%注射剂量/克）显著高于Cu-NOTA-Fab（1.9±0.3%注射剂量/克；P<0.0001）和Cu-NOTA-EGF（0.7±0.2%注射剂量/克；P<0.0001）。此外，预先给予过量的曲妥珠单抗Fab或曲妥珠单抗Fab-PEG-EGF可使SK-OV-3和MDA-MB-468异种移植瘤中Cu-NOTA-Fab-PEG-EGF的肿瘤摄取分别显著降低约4.4倍（P = 0.0012）和1.8倍（P = 0.0031）。铜标记的bsRIC与HER2或EGFR结合，并在体内被表达一种或两种受体的肿瘤异种移植瘤特异性摄取。PEG连接子延长了血液停留时间，导致bsRIC比单特异性药物具有更高的肿瘤摄取。
