Anti-CD19Fab-(CP)_(3)新型二聚化抗体的构建及靶向性观察  

Construction and targeting effect observation of novel Anti-CD19Fab-(CP)_(3)dimeric antibody

作　　者：雷晓敏 范冬梅 袁向飞 卢杨 张砚君 王建祥 熊冬生 LEI Xiaomin;FAN Dongmei;YUAN Xiangfei;LU Yang;ZHANG Yanjun;WANG Jianxiang;XIONG Dongsheng(Institute of Hematology&Blood Diseases Hospital,Chinese Academy of Medical Sciences&Peking Union Medical College,Tianjin 300020,China;不详)

机构地区：[1]中国医学科学院血液病医院(中国医学科学院血液学研究所)、北京协和医学院、天津市血液病细胞治疗研究重点实验室、实验血液学国家重点实验室、国家血液系统疾病临床研究中心、细胞生态海河实验室,天津300020 [2]天津医学健康研究院 [3]天津市急腹症器官损伤与中西医修复重点实验室

出　　处：《山东医药》2023年第28期14-17,31,共5页Shandong Medical Journal

基　　金：国家自然科学基金重点项目(81830005);细胞生态海河实验室创新基金项目(22HHXBSS00032);中国医学科学院医学与健康科技创新工程项目(2021-I2M-1-041)。

摘　　要：目的构建并表达Anti-CD19Fab-(CP)_(3)二聚化抗体,鉴定其蛋白分子量及相对含量并观察其靶向性。方法通过基因克隆技术构建原核表达载体PAYZ-Anti-CD19Fab-(CP)_(3)及阳性对照质粒PAYZ-Anti-CD19Fab-(CPP)_(3),将质粒转化至大肠杆菌16C9中进行表达。表达产物经过透析及Protein G亲和层析柱纯化,采用SDS-PAGE电泳法及液相串联质谱法(LC-MS)鉴定Anti-CD19Fab-(CP)_(3)二聚化抗体分子量及相对含量;采用流式细胞术检测二聚化抗体与CD19^(+)B系淋巴瘤Raji细胞的结合能力以及二聚化抗体对CD19鼠源亲本全抗的竞争能力。结果成功构建并表达Anti-CD19Fab-(CP)_(3),所得产物纯化后经SDS-PAGE及LC-MS法鉴定其主要成分为二聚化抗体,含量为76.2%;Anti-CD19Fab-(CP)_(3)二聚化比例高于对照Anti-CD19Fab-(CPP)3,差异有统计学意义(P<0.05)。同浓度下,与Anti-CD19Fab-(CPP)_(3)及Anti-CD19Fab相比,Anti-CD19Fab-(CP)_(3)对Raji细胞的结合能力更强,亲和力更好,差异有统计学意义(P均<0.05)。结论通过在Fab的CH1尾端引入(CP)_(3)的改构方式可有效形成高比例二聚化抗体,显著提高抗体对靶细胞的靶向性,未来可应用于多价抗体及相关药物研发。Objective To construct and express Anti-CD19Fab-(CP)_(3)dimeric antibody,to identify its protein molecular weight and relative content,and to observe its targeting effect.Methods Gene cloning technology was used to construct prokaryotic expression vectors PAYZ-Anti-CD19Fab-(CP)_(3)and positive plasmid PAYZ-Anti-CD19Fab-(CPP)_(3),and the recombinant plasmids were transformed into Escherichia coli 16C9 for expression,respectively.The expression products were purified by dialysis and Protein G affinity chromatography.In addition,SDS-PAGE electrophoresis and liquid-chromatography-tandem mass spectrometry(LC-MS)were used to detect the molecular weight and relative content.The binding ability of dimeric antibody to CD19^(+)B-cell lymphoma Raji cells and the competitive effect of dimeric antibody on murine parent full-length antibody were conducted by flow cytometry.Results The Anti-CD19Fab-(CP)_(3)was successfully constructed and expressed.The purified Anti-CD19Fab-(CP)_(3)was identified by SDS-PAGE and LC-MS as dimeric antibody mainly,and the content was 76.2%.Anti-CD19Fab-(CP)_(3)had a significant higher proportion of dimers than Anti-CD19Fab-(CPP)_(3),with statistically significant difference(P<0.05).Flow cytometry showed that Anti-CD19Fab-(CP)_(3)had a stronger binding affinity to Raji cells than Anti-CD19Fab-(CPP)_(3) and Anti-CD19Fab at the same concentration,with statistically significant difference(all P<0.05).Conclusions A high proportion dimeric antibody can be effectively formed by introducing(CP)_(3)into the CH1 tail of Fab,significantly improving the targeting effect of the antibody to the target cells,which can be applied in the development of polyvalent antibodies and related drugs in the future.

关 键 词：Anti-CD19Fab-(CP)_(3) Anti-CD19Fab-(CPP)_(3) 二聚化抗体 靶向性 

分 类 号：R965[医药卫生—药理学]