The Construction And Eukaryotic High-level Expression Of Anti-human VEGF165 Mouse/human Chimeric Antibody

Neovascularization is a critical step in growth and metastasis of the tumors. Vascular endothelial growth factor (VEGF), which is the major one of the various angiogenic factors secreted by tumors, plays a important role in the tumors angiogenesis. VEGF165, that is encoded of 165 amino acids and generated by alternative splicing from a single VEGF gene containing eight exons, is the predominant functional isoforms in vivo of VEGF. Neutralizing antibodies to human VEGF had been showed have inhibitory activity on the growth of primary and metastatic solid tumors in experimental tumor model of mice. To investigate the effects of anti-VEGF neutralizing antibodies on growth and metastasis of tumor, the anti-VEGF neutralizing monoclonal antibodies (McAbs) were generated with human VEGF 165 as antigen. Firstly, recombinant human VEGF165 with biological activity was expressed in E. coli and injected into BALB/c mice. 14 strains anti-human VEGF165 McAbs were obtained employing hybridoma technique. They can specifically bind to human VEGF165 in Western blot assay and ELISA. The neutralizing activities of each McAbs were assayed using miles' assay, CAM assay, and endothelial cell growth assay. It was shown that 9 of 14 McAbs have neutralizing activity to VEGF. 4 of these 9 McAbs significantly inhibited the growth of primary...