Stotz Sophie, Bowden Gregory D, Cotton Jonathan M, Pichler Bernd J, Maurer Andreas
Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Eberhard Karls University, 72076 Tuebingen, Germany.
Cluster of Excellence iFIT (EXC 2180) "Image Guided and Functionally Instructed Tumor Therapies", Eberhard Karls University, 72076 Tuebingen, Germany.
Pharmaceuticals (Basel). 2021 Sep 3;14(9):897. doi: 10.3390/ph14090897.
There is a need for versatile in vivo nuclear imaging reporter systems to foster preclinical and clinical research. We explore the applicability of the SNAPTag and novel radiolabeled small-molecule ligands as a versatile reporter gene system for in vivo nuclear imaging. SNAPTag is a high-affinity protein tag used in a variety of biochemical research areas and based on the suicide DNA repair enzyme O-methylguanine methyl transferase (MGMT). Its ligands are well suited for reporter gene imaging as the benzyl guanine core scaffold can be derivatized with fluorescent or radiolabeled moieties for various applications. Three guanine-based SNAPTag ligands ([F]FBBG, [F]FBG and [F]FBG) were synthesized in high yields and were (radio)chemically characterized. HEK293 cells were engineered to express the SNAPTag on the cell surface and served as cell model to assess target affinity by radiotracer uptake assays, Western blotting and SDS-PAGE autoradiography. A subcutaneous HEK293-SNAPTag xenograft model in immunodeficient mice was used for in vivo evaluation of [F]FBBG and [F]FBG while the biodistribution of [F]FBG was characterized in naïve animals. The results were validated by ex vivo biodistribution studies and immunofluorescence staining of the xenografts. All three radiotracers were produced in high radiochemical purity, molar activity and good yields. Western blot analysis revealed successful SNAPTag expression by the transfected HEK293 cells. In vitro testing revealed high target affinity of all three tracers with an up to 191-fold higher signal in the HEK293-SNAPTag cells compared to untransfected cells. This was further supported by a prominent radioactive protein band at the expected size in the SDS-PAGE autoradiograph of cells incubated with [F]FBBG or [F]FBG. The in vivo studies demonstrated high uptake in HEK293-SNAP xenografts compared to HEK293 xenografts with excellent tumor-to-muscle ratios (7.5 ± 4.2 for [F]FBBG and 10.6 ± 6.2 for [F]FBG). In contrast to [F]FBG and its chemical analogue [F]FBG, [F]FBBG showed no signs of unspecific bone uptake and defluorination in vivo. Radiolabeled SNAPTag ligands bear great potential for clinical applications such as in vivo tracking of cell populations, antibody fragments and targeted radiotherapy. With excellent target affinity, good stability, and low non-specific binding, [F]FBBG is a highly promising candidate for further preclinical evaluation.
需要通用的体内核成像报告系统来促进临床前和临床研究。我们探索了SNAP标签和新型放射性标记的小分子配体作为体内核成像通用报告基因系统的适用性。SNAP标签是一种高亲和力蛋白质标签，用于多种生化研究领域，基于自杀性DNA修复酶O-甲基鸟嘌呤甲基转移酶（MGMT）。其配体非常适合报告基因成像，因为苄基鸟嘌呤核心支架可以用荧光或放射性标记部分进行衍生化，以用于各种应用。三种基于鸟嘌呤的SNAP标签配体（[F]FBBG、[F]FBG和[F]FBG）以高产率合成并进行了（放射性）化学表征。对HEK293细胞进行工程改造，使其在细胞表面表达SNAP标签，并用作细胞模型，通过放射性示踪剂摄取试验、蛋白质免疫印迹和SDS-PAGE放射自显影来评估靶标亲和力。在免疫缺陷小鼠中建立皮下HEK293-SNAP标签异种移植模型，用于对[F]FBBG和[F]FBG进行体内评估，同时在未处理的动物中表征[F]FBG的生物分布。通过离体生物分布研究和异种移植的免疫荧光染色对结果进行了验证。所有三种放射性示踪剂均以高放射化学纯度、摩尔活性和良好产率制备。蛋白质免疫印迹分析显示转染的HEK293细胞成功表达了SNAP标签。体外测试显示所有三种示踪剂都具有高靶标亲和力，与未转染的细胞相比，HEK293-SNAP标签细胞中的信号高191倍。在用[F]FBBG或[F]FBG孵育的细胞的SDS-PAGE放射自显影图中，预期大小处出现明显的放射性蛋白条带，进一步支持了这一点。体内研究表明，与HEK293异种移植相比，HEK293-SNAP异种移植中的摄取量很高，肿瘤与肌肉的比率极佳（[F]FBBG为7.5±4.2，[F]FBG为10.6±6.2）。与[F]FBG及其化学类似物[F]FBG相比，[F]FBBG在体内没有显示出非特异性骨摄取和脱氟的迹象。放射性标记的SNAP标签配体在体内追踪细胞群体、抗体片段和靶向放疗等临床应用中具有巨大潜力。由于具有出色的靶标亲和力、良好的稳定性和低非特异性结合，[F]FBBG是进一步临床前评估的极具前景的候选物。
