抗人类免疫缺陷病毒1型感染的双特异性抗体的构建及其功能研究  

Construction and functional study of a bispecific antibody against human immunodeficiency virus type 1 infection

作　　者：郑钦文 马艳龙 王应丹 吴凡[2] 黄竞荷 ZHENG Qinwen;MA Yanlong;WANG Yingdan;Wu Fan;HUANG Jinghe(Key Laboratory of Medical Molecular Virology(MOE/NHC/CAMS),and Department of Medical Microbiology and parasitology,School of Basic Medical Science,Shanghai Medical College,Fudan University,Shanghai 200032,China;Shanghai Public Health Clinical Center,Fudan University,Shanghai 201508,China)

机构地区：[1]复旦大学上海医学院基础医学院病原生物学系,教育部、卫健委、医科院医学分子病毒学重点实验室,上海200032 [2]上海市(复旦大学附属)公共卫生临床中心,上海201508

出　　处：《微生物与感染》2020年第3期149-157,共9页Journal of Microbes and Infections

基　　金：国家自然科学基金(31771008);“十三五”国家科技重大专项(2017ZX10202102、2018ZX10301403);上海市卫生计生系统“百人计划”(2018BR08)。

摘　　要：人类免疫缺陷病毒(human immunodeficiency virus,HIV)流行毒株的靶向广谱中和抗体(broadly neutralizing antibody,bNAb)的单一疗法最终会导致机体出现病毒逃逸突变,而基于bNAb开发的双特异性或多特异性抗体则表现出较好的中和效力和广谱性。根据已公布的单链可变区基因抗体序列,通过密码子优化后合成一种由单基因编码的双特异性抗体iMab-PGT151,经双酶切和测序对重组质粒进行了验证。酶联免疫吸附试验检测双特异性抗体的结合特异性,检测U87细胞裂解液中的荧光素酶活性以定量分析双特异性抗体对HIV-1假病毒的中和作用;间接免疫荧光染色法检测双特异性抗体iMab-PGT151对人喉癌上皮细胞的反应性;酶联免疫吸附试验检测该抗体对心磷脂的结合能力,验证其自体反应性。结果显示,构建的双特异抗体iMab-PGT151能够成功表达,可分别结合亲本抗体的各个配体,具有双特异性,能100%中和20株假病毒,IC 50值为0.084μg/mL。与亲本抗体相比,该抗体具有更强的中和效力和广谱度,无自体反应性,具有临床适用性。所构建的双特异性抗体iMab-PGT151将可能成为预防和治疗HIV-1感染的有效候选药物之一。The broadly neutralizing antibody(bNAb)against human immunodeficiency virus(HIV)monotherapy eventually results in viral escape mutations in human.Bi-or multi-specific antibodies based on bNAbs show better neutralization potency.According to the published scFv gene antibody sequence,a bispecific antibody iMab-PGT151 encoded by a single gene was synthesized after codon optimization.The recombinant plasmid was verified by sequencing.Enzyme-linked immunosorbent assay(ELISA)was used to detect the binding specificity of iMab-PGT151 and verify its autoreactivity.The luciferase assay in the lysate of U87 cells was used to quantitatively analyze the neutralizing effect of iMab-PGT151 on HIV-1 pseudovirus,and the indirect immunofluorescence test was used to detect the reactivity of iMab-PGT151 to human laryngeal carcinoma epithelial cells.The results showed that iMab-PGT151 could be successfully expressed;it could bind each ligand of the parent antibody separately and had bispecificity;it could neutralize 20 pseudoviruses with IC 50 value of 0.084μg/mL compared with the parent antibody.The antibody had stronger neutralization potency and breadth without autoreactivity.iMab-PGT151 may become one of the promising candidates for the prevention and treatment of HIV-1 infection in the future.

关 键 词：人类免疫缺陷病毒 获得性免疫缺陷综合征 广谱中和抗体 双特异性抗体 

分 类 号：R373[医药卫生—病原生物学]