抗磷脂酰肌醇蛋白聚糖3抗体定点偶联药物的制备及其抗肿瘤活性评价  

Preparation of site-specific antibody-drug conjugate targeting glypican 3 and evaluation of its anti-tumor activity

作　　者：孙召朋 沈明月 唐昭娜 袁灿 王欣 彭秀娥 王翠 惠希武 刘伯宁 姚兵 SUN Zhaopeng;SHEN Mingyue;TANG Zhaona;YUAN Can;WANG Xin;PENG Xiue;WANG Cui;HUI Xiwu;LIU Boning;YAO Bing(CSPC Megalith Biopharmaceutical Co.,Ltd.,Shijiazhuang 050000,Hebei Province,China;不详)

机构地区：[1]石药集团巨石生物制药有限公司,河北石家庄050000 [2]石药集团新型药物制剂与辅料国家重点实验室,河北石家庄050000

出　　处：《中国生物制品学杂志》2023年第10期1192-1197,共6页Chinese Journal of Biologicals

基　　金：河北省科技厅重点资助项目(20372407D,20372402D)。

摘　　要：目的通过杂交瘤技术获得抗磷脂酰肌醇蛋白聚糖3(glypican 3,GPC3)单克隆抗体,并制备抗体偶联药物(antibody-drug conjugate,ADC),评价ADC的抗肿瘤活性。方法采用免疫雄性BALB/c小鼠的方式获得高亲和力抗GPC3单克隆抗体,采用ELISA、流式细胞术检测抗体对抗原的亲和力及在肿瘤细胞的内吞率;并通过定点偶联技术获得DAR(drug-to-antibody ratio)为2的ADC药物,细胞杀伤试验检测其对肝癌细胞HepG2和Hep3b的增殖抑制效果。结果抗体16C8-8E6在蛋白水平有很高的亲和力,EC_(50)为2.51 ng/mL,细胞水平(高表达GPC3的稳转株4E1、Hep3b、HepG2)亲和力明显高于原研抗体GC33(t分别为14.9、13.0和12.9,P均<0.05),在高表达GPC3的稳转株4E1的荧光强度为20542±107;内吞率优于原研抗体GC33,48 h内吞率达73.9%。16C8-8E6-ADC对肝癌细胞HepG2和Hep3b有一定的抑制增殖活性。结论成功获得靶向GPC3的单克隆抗体,为靶向GPC3的免疫疗法研究奠定了基础。Objective To obtain monoclonal antibody against glypican 3(GPC3)by hybridoma technique and prepare antibody-drug conjugate(ADC)to investigate the anti-tumor activity of ADC.Methods Monoclonal antibody against GPC3with high affinity was obtained by immunizing male BALB/c mice and detected for its affinity to antigen and endocytosis rate in tumor cells by ELISA and flow cytometry.ADC drugs with DAR(drug-to-antibody ratio)of 2 were obtained by sitespecific enzyme coupling technique,and the inhibitory effects on proliferation of tumor cells HepG2 and Hep3b were detected by cell killing experiments.Results Antibody 16C8-8E6 had a high affinity at protein level with the EC_(50)of 2.51 ng/mL,and the affinity at cell level(stable strains 4E1,Hep3b and HepG2 with high expression of GPC3)was significantly higher than that of the original antibody GC33(t=14.9,13.0 and 12.9,respectively,each P<0.05).The fluorescence intensity of stable strain 4E1 with high expression of GPC3 was 20542±107;The endocytosis rate was better than that of the original antibody GC33,which reached 73.9%within 48 h.16C8-8E6-ADC showed certain inhibitory activity on the proliferation of HepG2 and Hep3b cells.Conclusion The monoclonal antibody targeting GPC3 was successfully obtained,which lays a foundation of the research of immunotherapy targeting GPC3.

关 键 词：磷脂酰肌醇蛋白聚糖3 免疫偶联药物 单克隆抗体 肝癌 肿瘤 

分 类 号：R392.11[医药卫生—免疫学]