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

丙型肝炎病毒（HCV）是日本肝细胞癌的主要病原体。约60%的丙型肝炎病毒感染持续存在，导致慢性肝炎、肝硬化和肝细胞癌的发展。由于HCV感染和增殖的实验系统尚未建立，因此抗HCV的中和抗体尚未明确确定。急性和慢性丙型肝炎患者的免疫应答分析表明，在自然感染或HCV高突变率产生的HCV逃离中和抗体的突变体时，很难诱导中和抗体。本研究从小鼠和黑猩猩等动物中提取重组抗原主动免疫的中和抗体，并对中和抗体的互补决定（CDR）进行cDNA克隆，使其人源化。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物。在免疫小鼠和黑猩猩中诱导了更高的抗体反应，并且与患者相比，发现了动物特有的免疫反应肽。小鼠抗e2和抗hvr1抗体捕获HCV，但抗e1抗体不捕获HCV。黑猩猩抗全免疫原血清也能在体外捕获丙型肝炎病毒，并中和黑猩猩的丙型肝炎病毒传染性。接种疫苗的黑猩猩也不受丙型肝炎病毒感染。捕获抗体的表位分析表明，HVR1中存在一个活性肽。虽然HCV的HVR1是分离特异性的，但用这些免疫血清捕获了另一种HCV分离物。现在为了克隆抗体CDR的cDNA，利用免疫小鼠的脾脏和免疫黑猩猩的外周血白细胞构建了组合抗体文库。

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.