To neutralize the pathological activities of tumor necrosis factor-α (TNF-α) and receptor activator of NF- κB ligand (RANKL), we engineered a humanized 8G12 (h8G12) antibody to target both TNF-α and RANKL and identified its characterizations. Standard molecular biological and complementarity determining regions (CDRs)-grafting techniques were used to engineer the h8G12 antibody. Enzyme linked immunosorbent assay (ELISA) and western blotting were employed to determinate its binding activation and specificity. TNF-α-mediated cytotoxicity and RANKL-induced osteoclastogenesis assays were used to evaluate its neutralizing effects. The cDNA sequences were established by grafting the murine monoclonal antibody (mAb) 8G12 CDRs into the heavy and light chain (HC and LC) variable regions (VH and VL) frameworks of the human mAbs 3DGG-B and 1I9R-L, respectively. Recombnant plasmids were transfected into Chinese hamster ovary (CHO) cells to produce h8G12 antibody, which could recognize both TNF-α and RANKL simutaneously. In addition, the h8G12 antibody reduced the TNF-α-mediated apoptosis of L929 cells by 25.84%. Furthermore, the h8G12 antibody also significantly inhibited leukocytes infiltration in a murine contact allergic inflammation model. Corresponding with the inhibition of apoptosis, the h8G12 antibody significantly reduced the number of osteoclast-like cells in a dose-dependent manner. These results demonstrated that the h8G12 antibody neutralized activities of both TNF-α and RANKL and might be a potential candidate for inflammatory bone diseases, such as rheumatoid arthritis (RA).
This study was supported by the State Key Development Program for Basic Research of China (Grant No. 2010CB529106) and the National Nature Science Foundation of China (Grant No. 31370936).