抗PD-L1单抗的研制及其对乙型肝炎病毒的抑制效果初步研究  

Development of monoclonal antibodies against PD-L1 and preliminary investigation on potential application in treatment of chronic hepatitis B virus infection

作　　者：吴勇[1] 张天英[1,2] 宋浏伟 夏宁邵[1] 袁权[1] 

机构地区：[1]厦门大学分子疫苗学与分子诊断学国家重点实验室,国家传染病诊断试剂与疫苗工程技术研究中心,公共卫生学院,厦门361102 [2]厦门大学生命科学学院,厦门361102 [3]厦门万泰凯瑞生物技术有限公司,厦门361000

出　　处：《中国免疫学杂志》2016年第7期1004-1008,1012,共6页Chinese Journal of Immunology

基　　金：国家级科技重大专项(No.2013ZX10002002-001)

摘　　要：目的:获得具有阻断PD-1/PD-L1结合活性的Anti-PD-L1单克隆抗体,并在体内和体外模型中初步探索其应用于慢性HBV感染治疗的潜力。方法:利用大肠杆菌原核表达系统和离子交换柱层析纯化手段,表达纯化获得具有体外结合活性的人源和鼠源的PD-1/PD-L1蛋白,采用纯化后的人PD-L1蛋白作为免疫原免疫BALB/c小鼠,制备小鼠抗人PD-L1的单克隆抗体杂交瘤细胞株,基于间接化学发光免疫法评估了这些抗体与人源和鼠源重组蛋白的结合活性,并定量评估它们对PD-L1体外相互作用的阻断活性。利用慢乙肝病人PBMC体外刺激方法评估其对T细胞功能的促进作用,在HBV转基因小鼠中评估其抑制HBV的效果。结果:本研究获得了8株稳定分泌Anti-PD-L1的单克隆抗体杂交瘤细胞株,其中Anti-PD-L1Ab5和Ab6两株单抗与人和小鼠的PD-L1具有较强的交叉反应性,且均可阻断人和小鼠的PD-1/PD-L1结合活性。在慢乙肝病人PBMC刺激培养实验中,Ab5和Ab6可促进γ干扰素水平升高;在HBV转基因小鼠中单剂注射Ab6,48 h后血清HBV DNA水平降低20倍,血清HBs Ag水平降低至基线水平的30%。结论:获得了2株具有阻断人和小鼠PD-1/PD-L1结合活性的单克隆抗体,其在PBMC体外刺激培养系统中可促进慢乙肝病人的T细胞功能,在HBV转基因小鼠中具有显著的抗病毒效果,具备一定的治疗应用潜力。Objective： To get specific monoclonal antibodies（ m Abs） against PD-L1 which can block PD-1 /PD-L1 binding,and explore the feasibility of its application on the treatment of chronic HBV infection preliminarily by in vitro and in vivo model. Methods： E. coli expression and series chromatography purification system were employed to get human and mouse PD-1 / PD-L1 that had binding activity in vitro. By immunizing BALB / c mouse with purified recombination proteins of PD-L1,m Ab hybridoma cell lines against PD-L1 were obtained. The reactivity with human / mice PD-L1 of individual antibody and the interaction blocking activity of the m Abs to PD-1 / PD-L1 in vitro were examined by indirect chemiluminescence immune assay. Results： 8 cell lines against PD-L1 were obtained and 2 Anti-PDL1 m Abs（ Ab5 Ab6） performed strong immune activity to human / mice PD-L1 and blocking activity to PD-1 / PD-L1. In the PBMC stimulation experiment of chronic HBV patient,Ab5 and Ab6 could promote the γ-IFN levels. With HBV infecting mice model,intravenous injections of these m Abs induced dramatically decrease of HBV DNA copies about 20 times,HBs Ag levels in serum reduced to 30% of the baseline level. Conclusion： We obtained 2 PD-L1 m Abs with the reactivity to human / mice PD-L1 and blocking activity to PD-1 / PD-L1. The 2 m Abs can promote T cell function in PBMC stimulation culture of chronic HBV patient,have significant antiviral effect in HBV transgenic mice and can be candidates for immunotherapy applications.

关 键 词：程序性细胞死亡蛋白配体1 治疗性单克隆抗体 乙型肝炎病毒 免疫治疗 

分 类 号：R392[医药卫生—免疫学]