title: Engineering and characterization of a humanized antibody targeting TNF-α and RANKL.
abstract: To neutralize the pathological activities of tumor necrosis factor-α (TNF-α) and receptor activator of NF-κB ligand (RANKL), we engineered and characterized a humanized 8G12 (h8G12) antibody that targeted TNF-α and RANKL. Standard molecular biological and complementarity determining region (CDR)-grafting techniques were used to engineer the h8G12 antibody, and enzyme-linked immunosorbent assays (ELISAs) and Western blotting were employed to determine its binding activation and specificity. TNF-α-mediated cytotoxicity and RANKL-induced osteoclastogenesis assays were used to evaluate the neutralizing effects of the antibody. 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) of the human mAbs 3DGG_B and 1I9R_L, respectively. The recombinant plasmids were transfected into Chinese hamster ovary (CHO) cells to produce the h8G12 antibody, which could simultaneously recognize TNF-α and RANKL. In addition, the h8G12 antibody reduced the TNF-α-mediated apoptosis of L929 cells by 25.84%. Furthermore, the h8G12 antibody significantly inhibited leukocyte infiltration in a murine allergic contact inflammation model. Concurrent 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 the activities of TNF-α and RANKL and that it might be a potential candidate for the treatment of inflammatory bone diseases, such as rheumatoid arthritis (RA).
authors.affiliation: Department of Immunology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, PR China; Department of Pathogeny Biology and Immunology, School of Basic Medical Sciences, Inner Mongolia Medical University, Jinshan Development Zone, Hohhot, Inner Mongolia 010110, PR China.
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Immunology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, PR China.
authors.forename: Yuxuan
authors.initials: Y
authors.lastname: Du
authors.affiliation: Department of Rheumatology and Clinical Immunology, The First Affiliated Hospital of Xiamen University, No. 55 Zhenhai Road, Xiamen, Fujian 361003, PR China.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Qian
authors.affiliation: Department of Immunology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, PR China.
authors.forename: Haitao
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Immunology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, PR China.
authors.forename: Shentao
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Immunology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, PR China.
authors.forename: Xulong
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Rheumatology & Immunology, Clinical Immunology Center, Peking University People's Hospital, No. 11 Xizhimen South Street, Beijing 100044, PR China.
authors.forename: Zhanguo
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Immunology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, PR China. Electronic address: huihui_yuan@163.com.
authors.forename: Huihui
authors.initials: H
authors.lastname: Yuan
authors.affiliation: Department of Immunology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, PR China. Electronic address: zhao-wenming@163.com.
authors.forename: Wenming
authors.initials: W
authors.lastname: Zhao
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage* / Q000235:genetics / Q000276:immunology*; D002478:Cells, Cultured; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004306:Dose-Response Relationship, Immunologic; D016503:Drug Delivery Systems / Q000379:methods; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D010010:Osteoclasts / Q000187:drug effects / Q000276:immunology*; D015202:Protein Engineering / Q000379:methods*; D053245:RANK Ligand / Q000235:genetics / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D053245:RANK Ligand; D014409:Tumor Necrosis Factor-alpha
keywords: Dual targets; Humanized antibody; Receptor activator of NF-κB ligand (RANKL); Tumor necrosis factor-α (TNF-α)