Minerva Imaging, Copenhagen, Denmark.
Dept. of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Dept. of Biomedical Sciences, Rigshospitalet and University of Copenhagen, Denmark.
Theranostics. 2019 Jun 9;9(15):4409-4420. doi: 10.7150/thno.32883. eCollection 2019.
Antibody-based PET tracers are exceptionally well-suited for determination of the biodistribution and quantification of therapeutic antibodies. The continued expansion in antibody-based therapeutics has accordingly driven the development towards more robust conjugation strategies in order to reliably predict the performance of such agents. We therefore aimed to evaluate the effect of site-specific labeling by enzymatic remodeling on the stability, immuno-reactivity and tumor-targeting properties of the monoclonal antibody (mAb) trastuzumab and compare it to conventional, random labeling in a HER2-positive xenograft mouse model. : Trastuzumab was conjugated with the -SCN-Bn-Desferrioxamine (SCN-Bn-DFO) chelator randomly on lysine residues or site-specifically on enzymatically modified glycans using either β-galactosidase or endoglycosidase S2 prior to Zr radiolabeling. Zr-DFO-trastuzumab was injected into SK-OV-3 tumor-bearing NMRI nude mice. The antibody dose was titrated with either 100 µg or 500 µg of unlabeled trastuzumab. Mice underwent small animal PET/CT imaging 24, 70 and 120 hours post-injection for longitudinal assessment. Parallel experiments were conducted with an isotype control matched antibody. imaging was supported by conventional biodistribution and HER2 immuno-histochemistry. Furthermore, site-specifically labeled Zr-DFO-trastuzumab was evaluated in a panel of subcutaneous patient-derived xenograft (PDX) models. Additionally, the affinity, stability and immuno-reactivity were assessed for all tracers. : Site-specific labeling significantly increased PET tumor uptake (One-way ANOVA, ) at all time-points when compared to random labeling. Mean tumor uptakes were 6.7 ± 1.7, 13.9 ± 3.3 and 15.3 ± 3.8 % injected dose per gram tissue (%ID/g) at 70 hours post-injection, for random, β-galactosidase or endoglycosidase S2 labeled probes, respectively. Co-injection with unlabeled trastuzumab increased the circulation time of tracers but did not alter tumor uptake notably. Site-specific probes presented with a superior stability and immuno-reactivity compared to the randomly labeled probe. biodistribution confirmed the data obtained by PET imaging, and site-specific Zr-DFO-trastuzumab successfully detected HER2-positive tumors in PDX mouse models. : Zr-DFO-trastuzumab is well-matched for specific immuno-PET imaging of HER2-positive tumors and site-specific labeling of trastuzumab by the SiteClick technology minimizes the impact of the DFO chelator on immuno-reactivity, stability and biodistribution. These findings support further development of site-specifically radiolabeled mAbs for immuno-PET.
基于抗体的 PET 示踪剂非常适合用于确定治疗性抗体的生物分布和定量。随着抗体治疗的不断扩展，人们相应地开发了更强大的偶联策略，以便可靠地预测这些药物的性能。因此，我们旨在通过酶重塑来评估定点标记对曲妥珠单抗单克隆抗体 (mAb) 的稳定性、免疫反应性和肿瘤靶向特性的影响，并将其与 HER2 阳性异种移植小鼠模型中的常规随机标记进行比较。曲妥珠单抗通过β-半乳糖苷酶或内切糖苷酶 S2 酶促修饰聚糖上的定点或赖氨酸残基上的随机方式与 -SCN-Bn-去铁胺（SCN-Bn-DFO）螯合剂偶联，然后进行 Zr 放射性标记。Zr-DFO-曲妥珠单抗被注射到 SK-OV-3 荷瘤 NMRI 裸鼠中。用 100μg 或 500μg 未标记的曲妥珠单抗滴定抗体剂量。注射后 24、70 和 120 小时进行小动物 PET/CT 成像以进行纵向评估。用匹配的同种型对照抗体进行平行实验。放射性配体成像得到常规生物分布和 HER2 免疫组织化学的支持。此外，还在一系列皮下患者来源的异种移植 (PDX) 模型中评估了定点标记的 Zr-DFO-曲妥珠单抗。此外，还评估了所有示踪剂的亲和力、稳定性和免疫反应性。定点标记与随机标记相比，在所有时间点均显著增加了 PET 肿瘤摄取（单向方差分析，）。在 70 小时时，随机、β-半乳糖苷酶或内切糖苷酶 S2 标记探针的肿瘤摄取分别为 6.7±1.7、13.9±3.3 和 15.3±3.8%注入剂量/克组织（%ID/g）。与未标记的曲妥珠单抗共同注射可延长示踪剂的循环时间，但对肿瘤摄取没有明显影响。与随机标记探针相比，定点探针具有更好的稳定性和免疫反应性。生物分布证实了通过 PET 成像获得的数据，并且定点 Zr-DFO-曲妥珠单抗成功地在 PDX 小鼠模型中检测到了 HER2 阳性肿瘤。Zr-DFO-曲妥珠单抗非常适合用于 HER2 阳性肿瘤的特异性免疫 PET 成像，并且 SiteClick 技术对曲妥珠单抗的定点标记最大限度地减少了 DFO 螯合剂对免疫反应性、稳定性和生物分布的影响。这些发现支持进一步开发用于免疫 PET 的定点放射性标记 mAb。
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