Construction, Expression And Characterization Of Anti-CD20 Chimeric Monoclonal Antibody

Non-Hodgkin's lymphoma(NHL) is a common hematological malignancy with high mortality. Traditional chemotherapy is partly effective to it, but the side-effect of chemicals is too drastic. While, monoclonal antibody against CD20 antigen on the membrane of B-cell NHLs and normal B-lymphocyte showed satisfactory curative effect in clinical treatment of NHL. After binding to CD20, the chimeric monoclonal antibody against CD20 can kill B-cell NHLs and normal B-lymphocyte cells by induce apoptosis, ADCC, CDC and other cytotoxicities. The anti-CD20 antibody has slightly side-effect and will not kill other tissues. A chimeric antibody against CD20 on B cell was constructed and expressed in CHO cell and its characters was studied. VH and VL genes were synthesised according to reported sequence; CH and CL genes were amplified from normal B cell RNA by RT-PCR. They were connected by overlap extension PCR and cloned into pIRES bicistronic expression vector. The vector was transfected into CHO cell for expression. High expression level clones were picked by ELISA. Among them, about 24 clones were found to secrete antibody.1B3, 1B4, 1D3, 2A2, 2D4, 3A2, 4F10 were more productive than other clones. The expression product of 1B3 was purified by proteinA affinity chromatography . We reckoned that the expression concentration of 1B3 was about 2.13mg/L. Reduced SDS-PAGE analysis showed that the relative molecular weight of chimeric antibody was coincidence with expectation. Its purity was about 95%.ELISA assay revealed that the chimeric antibody being capable of binding specifically to human lymphoma cell lines expressing CD20 antigen. Its affinity constant was 0.113nM. Cytotoxicity experiment revealed that the chimeric antibody alone induced direct cytotoxicity in CD20-expressing human lymphoma cell line.