Preparation of engineered extracellular vesicles with full-length functional PD-1 membrane proteins by baculovirus expression system
Preparation of engineered extracellular vesicles with full-length functional PD-1 membrane proteins by baculovirus expression system
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DOI：
10.1016/j.bbrc.2020.03.187
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发表时间：
2020-06-11
影响因子：
3.1
通讯作者：
Akiyoshi, Kazunari
中科院分区：
文献类型：
--
作者：
Ishikawa, Raga;Yoshida, Shosuke;Akiyoshi, Kazunari
细胞外囊泡（EV）通过转运功能分子促进细胞间的通讯。由于其生物相容性和分子转运能力，将EV修饰为临床使用的药物递送系统是相当感兴趣的。程序性细胞死亡配体1（PD-L1）是用于药物递送至癌症组织的有效靶分子，并结合单跨膜蛋白，程序性细胞死亡蛋白1（PD-1），其是防止自身免疫的免疫检查点。在这项研究中，EV在一个新的表面工程策略进行了修改，纳入重组全长功能PD-1使用杆状病毒系统和新设计的PD-1突变体具有更高的PDL 1亲和力。用重组杆状病毒感染昆虫细胞系Spodoptera frugiperda 9，并将PD-1突变体基因整合到杆状病毒中以表达靶膜蛋白。为了确保有效插入膜中，PD-1的天然信号肽也被杆状病毒包膜糖蛋白的信号肽取代。然后分离并表征表达高亲和力PD-1突变体的工程化EV（PD-1 EV）。免疫染色和共聚焦激光扫描显微镜结果证实了全长功能性PD-1突变体的存在下表达的病毒感染的感染草地贪夜蛾9细胞膜表面和释放的EV膜。此外，信号肽取代显著增加了PD-1 EV与PD-L1之间的结合。PD-1 EV有效结合PD-L1和表达PD-L1的癌细胞，显示出作为靶向PD-L1 EV的新治疗方法的候选者的潜力。(C)2020爱思唯尔公司All rights reserved.
Extracellular vesicles (EVs) facilitate intercellular communication by transporting functional molecules. The modification of EVs for clinical use as drug delivery systems is of considerable interest because of their biocompatibility and molecular transport ability. Programmed cell death ligand 1 (PD-L1) is an effective target molecule for drug delivery to cancer tissues and binds the single-transmembrane protein, Programmed cell death protein 1 (PD-1), an immune checkpoint that guards against autoimmunity. In this study, EVs were modified in a new surface engineering strategy to incorporate recombinant full-length functional PD-1 using a baculovirus system and newly designed PD-1 mutant with higher PDL1 affinity. The insect cell line Spodoptera frugiperda 9 was infected with recombinant baculoviruses incorporating the PD-1 mutant gene to express the target membrane proteins. To ensure an effective insertion into the membrane, the native signal peptide of PD-1 was also replaced with that of the baculovirus envelope glycoprotein. Engineered EVs expressing the high-affinity PD-1 mutants (PD-1 EVs) were then isolated and characterized. Immunostaining and confocal laser scanning microscopy results confirmed the presence of full-length functional PD-1 mutants expressed by viral infection on both infected Spodoptera frugiperda 9 cell membrane surfaces and released EV membranes. Furthermore, the signal peptide substitution drastically increased the binding between PD-1 EVs and PD-L1. PD-1 EVs effectively bound PD-L1 and PD-L1-expressing cancer cells, showing potential as a candidate in new therapy approaches targeting PD-L1 EVs. (C) 2020 Elsevier Inc. All rights reserved.