靶向CD73/PD-L1双特异性抗体的制备及其体外抗肿瘤功能评价

Preparation of CD73/PD-L1 bispecific antibody and evaluation of its antitumor function in vitro

目的制备同时靶向胞外5'-核苷酸酶(cluster of differentiation 73,CD73)与程序性细胞死亡配体1(programmed cell death-ligand 1,PD-L1)的双特异性抗体,并对其体外结合能力和杀伤能力进行评价。方法采用基因工程方法,将PD-L1单链抗体(single-chain fragment variable,scFv)插入至CD73单抗铰链区中,构建抗CD73/PD-L1双特异性抗体(BS-21),通过CHO GS表达系统进行筛选,获得高表达量细胞株,经Protein A及分子筛纯化后,采用分子排阻高效液相色谱法(size exclusion chromatography-high performance liquid chromatography,SEC-HPLC)检测抗体纯度;流式细胞术检测抗体体外结合能力;通过外周血单个核细胞(peripheral blood mononuclear cell,PBMC)体外杀伤人肺癌细胞Calu 1检测抗体体外杀伤能力。结果经加压筛选获得了高产的细胞株;经纯化获得纯度为99.6%的双特异性抗体BS-21,该抗体能同时结合CD73和PD-L1分子;与anti CD73和anti PD-L1组相比,BS-21组显著提高免疫细胞对Calu 1肿瘤细胞的杀伤率(F均为30.36,P均<0.001)。结论双特异性抗体BS-21能同时降低CD73和PD-L1对免疫细胞的免疫抑制作用,具有较好的抗肿瘤功能。

Objective To prepare bispecific antibody targeting cluster of differentiation 73(CD73)and programmed cell death-ligand 1(PD-LI),and evaluate its binding ability and killing ability in vitro.Methods Using genetic engineering method,PD-L1 single-chain fragment variable(scFv)was inserted into the hinge region of CD73 monoclonal antibody to construct anti-CD73/PD-L1 bispecific antibody(BS-21),which was screened by CHO CS expression system to obtain highly expressed cell line.After purified by Protein A and molecular sieve,the purity of antibody was detected by size exclusion chromatography-high performance liquid chromatography(SEC-HPLC),the binding ability of antibody in vitro was detected by flow cytometry,and the killing ability in vitro was detected by using peripheral blood mononuclear cell(PBMC)to kill Calu 1 lung cancer cells in vitro.Results High-yield cell lines were obtained by pressure screening.A bispecific antibody BS-21 with a purity of 99.6%was obtained by purification,which bound to CD73 and PD-L1 molecules simultaneously.Compared with anti CD73 and anti PD-L1 groups,BS-21 group significantly increased the killing rate of immune cells to Calu 1 tumor cells(F=30.36,each P<0.001).Conclusion Bispecific antibody BS-21 reduced the immunosuppressive effect of CD73 and PD-L1 on immune cells simultaneously,and showed good anti-tumor function.