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因。大约60％的HCV感染变得持续存在，导致慢性肝炎，肝硬化和肝细胞癌的发展。尚未明确鉴定对HCV中和抗体的中和抗体，因为尚未确定感染和HCV增殖的实验系统。急性和慢性丙型肝炎患者的免疫反应分析表明，在自然感染中中和抗体几乎不会诱导中和HCV突变体从中和抗体中逃脱的抗体，这是通过高的HCV速率产生的，在这项工作的高突变速率中，通过像小鼠和chimpanzee的codna and insigation and Croment and codna and and and and和Chimpanze的抗体中和抗体相结合，并产生了codimnzee and and and codna的抗体。中和抗体的互补性确定性（CDR）。我们产生了针对重组HCV包膜糖蛋白E1和E2的抗体，以及合成的高变量区域（HVR）1肽，作为中和抗体的候选抗体。在免疫小鼠和黑猩猩中诱导了较高的抗体反应，并且与患者相比，发现动物独有的免疫反应性肽。鼠抗E2和抗HVR1抗体，但没有抗E1抗体捕获了HCV.Chimpanzee抗血清针对整个免疫原子的抗体也捕获了整个免疫原子的体外捕获HCV，并且在黑猩猩中捕获了中和HCV感染性。免疫的黑猩猩也受到了HCV感染的保护。捕获抗体的表位分析表明，HVR1中存在反应性肽。 HCV的AlthogH HVR1是分离物特异性的，另一种HCV分离株被这些免疫血清捕获。现在，为了克隆抗体CDR的cDNA，组合抗体库是由免疫小鼠的脾脏和免疫性黑猩猩的外周血白细胞构建的。