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cDNA cloning of Fab fragment against hepatitis C virus to produce humanized monoclonal antibody

抗丙型肝炎病毒Fab片段的cDNA克隆以产生人源化单克隆抗体

基本信息

批准号：
05454185
负责人：
ESUMI Mariko
金额：
$ 4.1万
依托单位：
Nihon University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454185/
关键词：
Hepatitis C virus Monoclonal antibody cDNA cloning Neutralizing antibody C型肝炎ウィルス

项目摘要

Hepatitis C virus (HCV) is a major causative agent of hepatocellular carcinoma in Japan. About 60% of HCV infection becomes persistent, resulting in development of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Neutralizing antibody against HCV is not clearly identified, because experimental systems of infection and proliferation of HCV are not yet established. Immune response analyzes in patients with acute and chronic hepatitis C suggest that neutralizing antibody is hardly induced in natural infection or a mutant of HCV escaping from neutralizing antibody generates by high mutation rate of HCV.In this work, neutralizing antibody is found out from animals such as mice and chimpanzee actively immunized with recombinant antigens, and is humanized by cDNA cloning of complimentarity-determining (CDR) of the neutralizing antibody.We produced antibody against recombinant HCV envelope glycoproteins, E1 and E2, and synthesized hypervariable region (HVR) 1 peptides, as candidates for neutralizing antibody. Higher antibody response was induced in immunized mice and a chimpanzee, and immunoreactive peptides unique to animals were found in comparison with those of patients. Murine anti-E2 and anti-HVR1 antibodies but not anti-E1 antibody captured HCV.Chimpanzee antiserum against whole immunogens also captured HCV in vitro, and neutralized HCV infectivity in a chimpanzee. The immunized chimpanzee was also protected from HCV infection. Epitope analysis of capturing antibody showed that a reactive peptide exists in HVR1. Althogh HVR1 of HCV is isolate-specific, another HCV isolate was captured with these immunized sera. Now in order to clone cDNA of CDR of antibody, combinatorial antibody library is constructed from spleens of immunized mice and peripheral blood leukocytes of immunized chimpanzee.

丙型肝炎病毒（HCV）是日本肝细胞癌的主要病原体。约60%的HCV感染成为持续性的，导致慢性肝炎、肝硬化和肝细胞癌的发展。由于尚未建立HCV感染和增殖的实验系统，因此尚未明确鉴定抗HCV的中和抗体。对急、慢性丙型肝炎患者的免疫应答分析表明，在自然感染中很难产生中和抗体，或者由于HCV的高突变率而产生了逃避中和抗体的HCV突变体。本工作从用重组抗原主动免疫的小鼠和黑猩猩等动物中发现了中和抗体，我们制备了抗重组HCV包膜糖蛋白E1和E2的抗体，并合成了高变区（HVR）1肽，作为中和抗体的候选肽。在免疫小鼠和黑猩猩中诱导了更高的抗体应答，与患者相比，发现了动物特有的免疫反应性肽。鼠抗-E2和抗-HVR 1抗体能捕获HCV，而抗-E1抗体不能捕获。免疫的黑猩猩也被保护免受HCV感染。捕获抗体的表位分析表明HVR 1中存在反应性肽。尽管HCV的HVR 1是分离株特异性的，但用这些免疫血清捕获了另一株HCV分离株。目前，为了克隆抗体CDR的cDNA，我们从免疫小鼠的脾脏和免疫黑猩猩的外周血白细胞中构建了组合抗体文库。