基于酶催化抗体药物偶联物中抗体的定点突变及鉴定  

Transglutaminase-based antibody-drug conjugation: antibody site-specific mutation and identification

作　　者：马招兄 徐瑶[1] 赵虹[2] 孙福谋[1] 张鑫荣[1] 王旻[1] 张娟[1] 

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009 [2]浙江省中医院,浙江杭州310006

出　　处：《药学学报》2017年第3期403-408,共6页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金面上项目资助(81473125);江苏省自然科学基金项目资助(BK20161459);华海药业研究生创新基金(1010040003);江苏省研究生科研创新计划(硕士)(KYZZ15_0190);大学生创新实验训练项目基金(SY15090)

摘　　要：谷氨酰胺转移酶催化谷氨酰胺与赖氨酸及其衍生物形成异肽键,可用于抗体与小分子药物的定点偶联。本文在构建的抗人CD24嵌合抗体c G7的基础上,定点突变c G7获得具有4个可催化的谷氨酰胺位点的嵌合抗体c G7Q。本研究采用overlap PCR技术将抗体重链CH段第297位天冬酰胺突变成谷氨酰胺,构建含突变重链的真核表达载体。将含突变重链与轻链的载体共转染CHO-s细胞,筛选稳定高产单克隆细胞株,表达及鉴定目的蛋白c G7Q,表面等离子共振法与流式细胞术分析c G7Q与人CD24分子的结合能力,乳酸脱氢酶释放实验鉴定定点突变对抗体依赖细胞介导的细胞毒作用(ADCC)的影响,结果显示,经改造的c G7Q保持母体单抗的结构和特异性结合能力及部分ADCC效应,为后续制备靶向CD24定点偶联药物奠定基础。Transglutaminase（TG） posttranslational modification of antibody permits more precisely conjugating. Based on the amino acid sequence of an anti-CD24 antibody（cG7）, this article is aimed to generate a deglycosylated cG7 mutant（cG7Q）. Firstly, we introduced additional glutamines at position 297（N297Q） by site-directed mutagenesis, and then transfected the recombinant plasmids into CHO-s cells via electroporation method and screened by Dot blot assay. Subsequently, cG7Q was expressed and purified through Protein A affinity chromatography, further identified by SDS-PAGE electrophoresis and Western blot. Its affinity was detected with surface plasmon resonance and flow cytometry assay, and ADCC effect was determined by lactate dehydrogenase（LDH） release. Eventually, a cG7 mutant, cG7Q was successfully expressed with sequence-specific conjugation sites for further study.

关 键 词：抗体 定点突变 谷氨酰胺转移酶 抗体药物偶联物 

分 类 号：R966[医药卫生—药理学]