抗人PD-L1 单克隆抗体的制备及其应用

目的:获得能应用于临床诊断以及阻断PD-L1 与PD-1 结合的抗人PD-L1 单克隆抗体。方法:采用重组表达的人PD-L1 蛋白免疫BALB/ c 小鼠,通过杂交瘤细胞融合技术获得稳定分泌抗人PD-L1 单抗的阳性细胞株,ELISA 方法鉴定抗体的特异性、亲和力、亚型等方面特性;免疫印迹、间接免疫荧光方法对肿瘤细胞进行检测;肿瘤杀伤实验验证抗体阻断活性。结果:共获得2 株抗人PD-L1 单抗,抗体效价分别为1 2.56 106 和1 3 105 ,亲和力分别为1.5 109 L/ mol 和2.5 108 L/ mol,均与PD-L2 蛋白无交叉反应。免疫印迹、间接免疫荧光证实抗体有诊断作用。杀伤实验显示抗体有阻断作用。结论:共获得两株稳定分泌高效价、高特异性的抗人PD-L1 单抗的杂交瘤细胞株,能作为诊断抗体应用于肿瘤表型检测和预后有效性的评估。抗体的阻断功能可应用于联合CIK 细胞免疫治疗。

Preparation and application of anti-human PD-L1 monoclonal antibodies

Objective:To obtain a high specificity and high affinity anti-human PD-L1 monoclonal antibody which can be used for clinical diagnosis and block PD-L1 and PD-1 binding.Methods: BALB/ c mice were immunized with recombinant PD-L1 protein.The positive cell clones stably secreting anti-human PD-L1 monoclonal antibody were obtained by classical hybridoma cell fusion technique.The specificity,affinity,subtype and other characteristics of the antibody were identified by ELISA.Immunofluorescence and indirect immunofluorescence were used to detect the tumor cells.Antibody blocking activity was confirmed by tumor killing test.Results:Two cell strains stably secreting monoclonal antibodies against human PD-L1 were screened out.Ab1 and Ab2 had high titer and affinity.The antibody titers were 1 2.56 106 and 1 3 105 ,and the affinity was 1.5’109 L/ mol and 2.5’108 L/ mol respecttively.There was no cross reaction between these two antibodies and PD-L2.Immunoblotting,indirect immunofluorescence confirmed that the antibody can be used to the diagnosis.Experiment showed that PD-L1 antibodies can increases tumor-killing activity of CIK cells.Conclusion:Two hybridoma cell lines capable of stably secreting highly specific and high affinity anti-human PD-L1 monoclonal antibody are obtained.They can specifically bind to PD-L1 molecules on tumor cells and can be used to the diagnosis of tumor phenotype and prognosis.Antibody blocking function can be applied to combined CIK cell immunotherapy.