title: A monoclonal antibody ameliorates local inflammation and osteoporosis by targeting TNF-α and RANKL.
abstract: This study aimed to generate a monoclonal antibody (mAb) targeting both tumor necrosis factor-α (TNF-α) and receptor activator of NF-κB ligand (RANKL) and to evaluate the therapeutic effects of this antibody on acute inflammation and osteoporosis. We used hybridoma techniques to generate potential mAbs and enzyme-linked immunosorbent assay (ELISA) to determine their specificity. Crystal violet staining was performed to measure the effective dose of the candidate mAbs. The neutralizing effect of the mAbs was evaluated by TNF-α-mediated cytotoxicity and RANKL-induced osteoclastogenesis assays. We further assessed the therapeutic effect of the mAbs in BALB/c mice with carrageenan-induced acute inflammation and ovariectomy-induced osteoporosis. We successfully generated an IgG1 isotype mAb that recognizes human TNF-α and RANKL, which we named 8G12. The 50% effective dose of 8G12 was approximately 1μg/mL. L929 cells treated with 8G12 exhibited decreased levels of apoptosis (20.04% compared to 63.28% in the positive controls). In addition, treatment with 8G12 inhibited osteoclastogenesis in a dose-dependent manner in vitro. Carrageenan-induced paw edema was significantly reduced in the 8G12-treated mice compared to the positive controls. Treatment with 8G12 also reduced the number of infiltrating leukocytes by more than 50%. The 8G12 treatment not only prevented bone loss but also increased the number, thickness and volume of trabeculae and reduced trabecular separation in ovariectomized mice. Our data suggest that the 8G12 effectively neutralizes the bioactivity of TNF-α and RANKL, ameliorating osteoporosis and inflammation. We therefore propose that 8G12 could be a candidate for generating therapeutic antibodies for treating inflammatory bone diseases.
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 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: 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; 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 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 Immunology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, PR China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Sun
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: zhao-wenming@163.com.
authors.forename: Wenming
authors.initials: W
authors.lastname: Zhao
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage* / Q000302:isolation & purification; D001862:Bone Resorption / Q000209:etiology / Q000517:prevention & control*; D002351:Carrageenan / Q000378:metabolism; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D004195:Disease Models, Animal; D004487:Edema / Q000139:chemically induced / Q000150:complications / Q000188:drug therapy*; D006801:Humans; D006825:Hybridomas; D007249:Inflammation / Q000188:drug therapy*; D051379:Mice; D008807:Mice, Inbred BALB C; D010022:Osteopetrosis / Q000188:drug therapy* / Q000276:immunology; D053245:RANK Ligand / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D053245:RANK Ligand; D014409:Tumor Necrosis Factor-alpha; D002351:Carrageenan
keywords: Acute inflammation; Monoclonal antibody; Osteoporosis; Receptor activator of nuclear factor-κB ligand (RANKL); Tumor necrosis factor-α (TNF-α)