In 2009, the FDA approved bevacizumab for the treatment of adult patients diagnosed with recurrent glioblastoma. However, the poor permeability of the macromolecules across the blood–brain barrier, determined by multifactorial anatomical and physiological milieu, restricts the clinical therapeutic effect of bevacizumab. The low-density lipoprotein receptor related protein 1 (LRP1) is highly expressed in the endothelial cells of the brain capillary and the glioma cells. Angiopep-2 (ANG) is a 19-aa oligopeptide that can bind to LRP1 and penetrate the blood–brain barrier by receptor-mediated transport. Therefore, ANG can be used as a dual-targeting drug delivery carrier into the brain and the glioma sites. In this study, ANG gene was fused with the C-terminal domain of single-chain antigen binding fragment (scFab) of the anti-VEGF antibody and recombinant scFab-ANG protein was expressed and purified using Rosatte (DE3) strain. We confirmed that ANG could carry anti-VEGF-scFab, penetrate a three-dimensional model of the brain tumor, and cross the hCMEC/D3 monolayer in the in vitro blood–brain barrier model. The animal experiments demonstrated that 3 h after the tail intravenous protein injection, the fluorescent signals in the brains of the mice in the scFab-ANG group were stronger than that in the scFab group. Furthermore, the study of the in situ rat glioma model shows that scFab-ANG could target glioma while anti-VEGF-scFab could not. These findings indicate that scFab-ANG had stronger transepithelial permeability and glioma targeting capacity. Thus, it can be a potential candidate drug for glioblastoma therapy.

中文翻译：

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重组表达的angiopep-2融合抗VEGF单链Fab（scFab）可以穿越血脑屏障并靶向神经胶质瘤。

2009年，FDA批准贝伐单抗用于诊断患有复发性胶质母细胞瘤的成年患者。然而，由多因素解剖学和生理环境确定的大分子穿过血脑屏障的渗透性差，限制了贝伐单抗的临床治疗效果。低密度脂蛋白受体相关蛋白1（LRP1）在脑毛细血管内皮细胞和神经胶质瘤细胞中高表达。Angiopep-2（ANG）是一种19-aa寡肽，可与LRP1结合并通过受体介导的转运作用穿透血脑屏障。因此，ANG可以用作进入脑和神经胶质瘤部位的双重靶向药物递送载体。在这个研究中，将ANG基因与抗VEGF抗体的单链抗原结合片段（scFab）的C端结构域融合，并使用Rosette（DE3）菌株表达并纯化重组scFab-ANG蛋白。我们确认了ANG可以携带抗VEGF-scFab，穿透脑肿瘤的三维模型，并在体外血脑屏障模型中穿过hCMEC / D3单层。动物实验表明，在尾部静脉注射蛋白质后3小时，scFab-ANG组小鼠大脑中的荧光信号强于scFab组。此外，对原位大鼠神经胶质瘤模型的研究表明，scFab-ANG可以靶向神经胶质瘤，而抗VEGF-scFab则不能。这些发现表明scFab-ANG具有更强的跨上皮通透性和胶质瘤靶向能力。从而，