Dammicco Sylvestre, Goux Marine, Lemaire Christian, Becker Guillaume, Bahri Mohamed Ali, Plenevaux Alain, Cinier Mathieu, Luxen André
GIGA Cyclotron Research Centre in vivo imaging, University of Liège, Liège, Belgium; Chemistry Department, University of Liège, Liège, Belgium.
GIGA Cyclotron Research Centre in vivo imaging, University of Liège, Liège, Belgium.
Nucl Med Biol. 2017 Aug;51:33-39. doi: 10.1016/j.nucmedbio.2017.04.006. Epub 2017 Apr 27.
Nanofitins are low molecular weight, single chain and cysteine-free protein scaffolds able to selectively bind a defined biological target. They derive from Sac7d bacterial protein family and are highly stable over a wide range of pH (0-13) and temperature (Tm ~80°C). Their extreme stability, low cost of production and high tolerability for chemical coupling make Nanofitins a very interesting alternative to antibodies and their fragments. Here, a hexahistidine tagged model Nanofitin (H4) directed against hen egg white lysozyme was radiolabelled and injected in mice to provide a baseline biodistribution and pharmacokinetic profiles to support future Nanofitin development programs.
A single cysteine residue has been genetically inserted in a model Nanofitin and its regioselective radiolabelling has been performed with 4-[F]fluorobenzamido-N-ethylamino-maleimide ([F]FBEM). The synthesis of [F]FBEM has been completely implemented on a radiosynthesis unit (FastLab) including HPLC purification and formulation. Coupling with the [F]FBEM has been achieved on a solid support (Ni magnetic beads) allowing rapid purification at room temperature without organic solvent. PET-MRI studies on C57BL/6 mice were conducted after injection of [F]FBEM-Cys-H4 in order to access the biodistribution of this Nanofitin model.
Radiochemical yield (decay corrected) of 54±7% (n=4) was obtained after optimization for coupling the [F]FBEM to Nanofitin. Pharmacokinetics results of [F]FBEM-Cys-H4 revealed a fast clearance through the liver and the kidneys.
An efficient new method on Ni magnetic beads was developed to radiolabelled his-tagged biomolecules with [F]FBEM. This procedure was applied on a Nanofitin model Cys-H4 and biodistribution kinetic studies were achieved to evaluate the potential use of Nanofitin for diagnostic imaging. Fast clearance indicates that Nanofitins represent very interesting tools for diagnostic imaging.
纳米适配体是低分子量、单链且无半胱氨酸的蛋白质支架，能够选择性地结合特定的生物靶点。它们源自Sac7d细菌蛋白家族，在广泛的pH范围（0 - 13）和温度（熔点约80°C）内具有高度稳定性。其极高的稳定性、低成本的生产以及对化学偶联的高耐受性，使得纳米适配体成为抗体及其片段的极具吸引力的替代品。在此，一种针对鸡蛋清溶菌酶的带有六组氨酸标签的纳米适配体模型（H4）被放射性标记，并注射到小鼠体内，以提供基线生物分布和药代动力学概况，以支持未来的纳米适配体开发项目。
在一个纳米适配体模型中通过基因工程插入了一个半胱氨酸残基，并使用4 - [F]氟苯甲酰胺 - N - 乙氨基 - 马来酰亚胺（[F]FBEM）对其进行区域选择性放射性标记。[F]FBEM的合成已在包括高效液相色谱纯化和制剂的放射性合成单元（FastLab）上完全实现。与[F]FBEM的偶联在固相载体（镍磁珠）上完成，可在室温下快速纯化且无需有机溶剂。在注射[F]FBEM - Cys - H4后，对C57BL / 6小鼠进行了正电子发射断层扫描 - 磁共振成像（PET - MRI）研究，以了解这种纳米适配体模型的生物分布。
在优化[F]FBEM与纳米适配体的偶联后，获得了54±7%（n = 4）的放射性化学产率（衰变校正）。[F]FBEM - Cys - H4的药代动力学结果显示其通过肝脏和肾脏快速清除。
开发了一种在镍磁珠上进行的高效新方法，用于用[F]FBEM对带有组氨酸标签的生物分子进行放射性标记。该方法应用于纳米适配体模型Cys - H4，并进行了生物分布动力学研究，以评估纳米适配体在诊断成像中的潜在用途。快速清除表明纳米适配体是诊断成像中非常有吸引力的工具。
