title: Tocilizumab inhibits signal transduction mediated by both mIL-6R and sIL-6R, but not by the receptors of other members of IL-6 cytokine family.
abstract: To characterize the biological activity of tocilizumab, a humanized anti-human interleukin-6 receptor (IL-6R) monoclonal antibody, we examined its binding activity to both soluble IL-6R (sIL-6R) and membrane bound IL-6R (mIL-6R) and its neutralizing activity to other IL-6 family cytokines. ELISA assay demonstrated that tocilizumab bound to sIL-6R and inhibited IL-6 binding to sIL-6R in a dose-dependent manner. The dissociation constant (Kd value) for IL-6R was determined to be 2.54+/-0.12 nmol/L by Scatchard analysis. In addition, tocilizumab had the ability to dissociate IL-6 and sIL-6R from their preformed complex. The immune complex of tocilizumab and sIL-6R did not transmit signaling. Moreover, tocilizumab suppressed the IL-6/sIL-6R complex-induced proliferation of human gp130-transfected cell, BAF-h130. In addition, tocilizumab had the ability to bind to human IL-6R expressing COS-7 cells and to suppress the growth of the IL-6-dependent myeloma cell line, KPMM2. Finally, to analyze the specificity of this antibody, the effects on signal transduction of IL-6 family cytokines such as interleukin-11 (IL-11), oncostatin M (OSM), leukemia inhibitory factor (LIF), and ciliary neurotrophic factor (CNTF) were examined using murine transfectant cell lines (BaF/IL-6R, BaF/IL-11R, BaF/OSMR, BaF/LIFR and BaF/CNTFR) that proliferate depending on IL-6, IL-11, OSM, LIF and human CNTF, respectively. Tocilizumab inhibited the proliferation of BaF/IL-6R induced by IL-6, but did not inhibit the proliferation of BaF/IL-11R, BaF/OSMR, BaF/LIFR and BaF/CNTFR cells induced by their corresponding cytokines. These lines of evidence indicate that tocilizumab is able to bind to both sIL-6R and mIL-6R and to inhibit IL-6 binding to its receptors, leading to the blockade of the IL-6 signaling through both sIL-6R and mIL-6R, but not block the signaling of other IL-6 family cytokines.
authors.affiliation: Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co. Ltd., 1-135, Komakado, Gotemba, Shizuoka, 412-8513, Japan. miharamsh@chugai-pharm.co.jp
authors.forename: Masahiko
authors.initials: M
authors.lastname: Mihara
authors.forename: Keiko
authors.initials: K
authors.lastname: Kasutani
authors.forename: Makoto
authors.initials: M
authors.lastname: Okazaki
authors.forename: Akito
authors.initials: A
authors.lastname: Nakamura
authors.forename: Shigeto
authors.initials: S
authors.lastname: Kawai
authors.forename: Masamichi
authors.initials: M
authors.lastname: Sugimoto
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Matsumoto
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Ohsugi
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized; D019556:COS Cells; D049109:Cell Proliferation / Q000187:drug effects; D002522:Chlorocebus aethiops; D016207:Cytokines / Q000276:immunology* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015850:Interleukin-6 / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D018121:Receptors, Cytokine / Q000276:immunology / Q000378:metabolism; D019947:Receptors, Interleukin-6 / Q000276:immunology* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects* / Q000276:immunology; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D016207:Cytokines; D015850:Interleukin-6; D018121:Receptors, Cytokine; D019947:Receptors, Interleukin-6; C502936:tocilizumab