靶向CD96/PD1双特异性抗体的构建与鉴定  

Bispecific antibody targeting CD96 and PD1

作　　者：马佳兵 赵文旭 徐艳敏 沈俊杰 陈雪娇 田茂鲜 钱程 MA Jiabing;ZHAO Wenxu;XU Yanmin;SHEN Junjie;CHEN Xuejiao;TIAN Maoxian;QIAN Cheng(College of Life Sciences and Medicine,Zhejiang Sci-Tech University,Hangzhou 310018,China;Chongqing Institute of Precision Medicine and Biotechnology Co.,Ltd.,Chongqing 400030,China;Chongqing Precision Biotech Co.,Ltd.,Chongqing 400030,China;Center for Precision Medicine of Cancer,Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment,Chongqing University Cancer Hospital,Chongqing 400030,China)

机构地区：[1]浙江理工大学生命科学与医药学院,杭州310018 [2]重庆精准生物产业技术研究院有限公司,400030 [3]重庆精准生物技术有限公司,400030 [4]重庆大学附属肿瘤医院精准医学研究中心,400030

出　　处：《免疫学杂志》2022年第3期269-276,共8页Immunological Journal

基　　金：国家科技部“科技助力经济2020”重点专项(SQ2020YFF0401839)。

摘　　要：目的构建可以同时靶向免疫检查点分子CD96和PD1(programmed cell death protein 1)的双特异性抗体,为肿瘤免疫治疗提供一种新的思路。方法构建表达抗CD96和PD1的ScFv-Fc结构的质粒,使用CHO真核表达系统生产抗体。经过Protein A纯化后,使用SDS-PAGE和分子排阻色谱鉴定抗体。外源构建CHO-PD1和293T-CD962种靶细胞,流式细胞术检测CD96和PD1的ScFv-Fc抗体与上述靶细胞以及T细胞上抗原的结合能力,并采用Fortebio分子互作仪进行抗原抗体结合解离动力学验证。分别采用Knobs-into-holes(KIH)突变策略构建ScFv-KIH双特异性抗体结构,以及类DVD-Ig(Dual variable domain immunoglobulin)分子结构,构建ScFv串联型(ScFv)2-Fc结构的双特异性抗体,并分别对表达的2种双特异性抗体进行了功能验证。结果抗CD96和PD1的ScFv-Fc抗体可特异性识别靶细胞和T细胞上的靶抗原,构建生产的2种结构的双特异性抗体均能正常表达,且能同时靶向CD96和PD12种抗原。结论ScFv-KIH和(ScFv)2-Fc型的双特异性抗体表达成功,并可同时识别CD96和PD1抗原。This study was performed to construct bispecific antibodies(BsAbs)that can simultaneously target CD96 and PD1(programmed cell death protein 1)antigen,and provide a new idea for tumor immunotherapy.Plasmids expressing the ScFv-Fc structure of CD96 and PD1 were constructed,and stably transfect into CHO cells to produce antibodies.The antibodies were purified using affinity chromatography,and verified by SDS-PAGE and SEC.Flow cytometry was used to detect the binding of the purified antibody to target cells(CHO-PD1 and 293 TCD96)and T cells;Fortebio was used to detect the interaction between antibody and antigen.Furthermore,we introduced mutation of the Knobs-into-holes(KIH)heavy chain matching strategy to construct the ScFv-KIHBsAb and the(ScFv)2-FcBsAb based on DVD-Ig(Dual variable domain immunoglobulin).The size of CD96 and PD1 antibodies were correct,and effectively form the dimer,which could specifically recognize the antigens on target cells and T cells;the size of two kinds of BsAbs werecorrect and could target CD96 and PD1 two antigenssimultaneously.In summary,we successful constructScFv-KIH and(ScFv)2-FcBs Abs,which can recognizeCD96 and PD1 antigens at the same time.

关 键 词：CD96 PD1 双特异性抗体 肿瘤免疫治疗 Knobs-into-holes 

分 类 号：R730.3[医药卫生—肿瘤]