工程化haPD-1外泌体的制备及功能初步鉴定  

Preparation and preliminary functional identification of engineered haPD-1 exosomes

作　　者：齐田羽 陈磊 孙振[1] 崔恒宓[1,2] QI Tianyu;CHEN Lei;SUN Zhen;CUI Hengmi(Institute of Epigenetics&Epigenomics/College of Animal Science and Technology,Yangzhou University,Yangzhou 225009,China;Jiangsu Innovation Institute for Biomedicine,Nanjing 21l100,China)

机构地区：[1]扬州大学动物科学与技术学院/表观遗传学及表观基因组学研究所,江苏扬州225009 [2]江苏易诺维生物医学研究院,南京211100

出　　处：《扬州大学学报(农业与生命科学版)》2024年第6期65-73,共9页Journal of Yangzhou University:Agricultural and Life Science Edition

基　　金：国家自然科学基金资助项目(81773013)。

摘　　要：为阻断免疫细胞上PD-1与肿瘤细胞上PD-L1结合,阻止免疫逃逸,以利免疫细胞识别并杀伤肿瘤细胞,目前已开发多种单抗药物,但均不可避免地出现副作用或治疗效率不高等问题。为解决这些问题,用慢病毒载体将高亲和PD-1片段(haPD-1)与跨膜蛋白LAMP2B信号肽序列融合,转入HEK293T细胞,使其在膜上分泌表达haPD-1的外泌体,这些外泌体可竞争性地结合肿瘤细胞表面PD-L1,同时拥有自身免疫原性低、可长时间稳定存在的优点。结果表明:haPD-1基因被成功转染至HEK293T细胞;通过细胞上清分离的外泌体经检测符合外泌体特征,并与PD-L1具有良好的亲和力,在体外可被PD-L1阳性HepG2细胞摄取,并可提高NK细胞对PD-L1阳性HepG2肝癌细胞的杀伤力。综上,表达haPD-1的工程化外泌体可能具有类似于PD-L1单抗的功能,可与肿瘤细胞表面的PD-L1结合,阻止肿瘤细胞的免疫逃逸,可应用于肿瘤的免疫治疗。In order to block the binding of PD-1 on immune cells to PD-L1 on tumor cells to achieve the effect of blocking immune escape and enabling immune cells to recognize and kill tumor cells,a variety of monoclonal antibody drugs have been developed,but all of them inevitably produce side-effects or problems such as low therapeutic efficiency.To solve these problems,this experiment used lentiviral vectors to transfer the high-affinity PD-1 fragment(haPD-1)combined with the signal peptide sequence of the transmembrane protein LAMP2B,into HEK293T cells,which secreted exosomes expressing haPD-1 on the membrane.And the haPD-1 on the surface of these exosomes could competitively bind to PD-L1,while the low immunogenicity and long time stability of the exosomes can also avoid the side effects as using monoclonal antibody drugs.The results showed that the lentivirus-transfected HEK293T cells were successfully transfected with the target gene and the exosomes isolated from the supernatant of transfected cells were conformed to be of the characteristics of exosomes,which had a good affinity for PD-L1,and could be uptaken by the PD-L1-positive HepG2 cells in vitro.It can improve the killing power of NK cells against the PD-L1-positive HepG2 cancer cells.Our preliminary research results indicate that engineered exosomes expressing haPD-1 may have functions similar to PD-L1 monoclonal antibody,which can bind to PD-L1 on the surface of tumor cells,prevent immune escape of tumor cells,and can be applied in tumor immunotherapy.

关 键 词：外泌体 PD-1 PD-L1 haPD-1 免疫逃逸 抗肿瘤 

分 类 号：S852.4[农业科学—基础兽医学] Q78[农业科学—兽医学]