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

https://doi.org/10.1016/j.intimp.2005.05.010Get rights and content

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.

Introduction

Tocilizumab is a humanized anti-human IL-6 receptor (IL-6R) monoclonal antibody that inhibits IL-6 functions as reported previously [1]. In the clinical trials, tocilizumab shows significant improvement of disease activity of Castleman's disease, rheumatoid arthritis, Crohn's disease and system-onset juvenile idiopathic arthritis [2], [3], [4], [5].

IL-6 is a multifunctional cytokine and plays essential roles in host defense mechanisms through the immune system, hematopoiesis and the central nervous system [6]. IL-6 exerts its biological activities through two membrane molecules, a ligand-binding 80 kD chain (IL-6R) and a non-ligand-binding signal transducer gp130. After binding of IL-6 to membrane bound IL-6R (mIL-6R), IL-6/IL-6R complex associates with gp130 and then the signal is transmitted into the cell. In addition, soluble IL-6R (sIL-6R), which lacks trans-membrane and cytoplasmic domains, can associate with gp130 in the presence of IL-6 and transduce the signal through gp130. Namely, both mIL-6R and sIL-6R play essential roles in IL-6 signaling [7].

IL-6 and related cytokines, such as leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and IL-11 exhibit multiple functions and redundancy in biological activities with each other. All of these cytokine receptors utilize gp130 in their signal transduction systems, and constitute a cytokine receptor super family [8].

In this paper, we indicate that tocilizumab has the binding activity to both sIL-6R and mIL-6R and the neutralizing activity to IL-6, and that it does not have the ability to inhibit other IL-6 family cytokines in which their receptors are highly homologous to IL-6R.

Section snippets

Antibodies

Tocilizumab, MT18 (non-neutralizing anti-human IL-6R monoclonal antibody) [9] and recombinant soluble human IL-6R (sIL-6R) [10] were prepared in our laboratory.

Binding activity to sIL-6R and inhibitory activity of IL-6 binding to sIL-6R

Binding activity to sIL-6R and inhibitory activity of IL-6 binding to sIL-6R were measured by ELISA as previously described [1].

Dissociation constant analysis

Each 10 μL of 125I-tocilizumab (11 nmol/L, prepared in our laboratory) and tocilizumab (11, 33, 77, 165, 341 and 11000 nmol/L) were added to the IL-6R-expressing human myeloma cell line, U266B1 cells (200 μL, 5.5

Binding activity to sIL-6R

The binding activity of tocilizumab to sIL-6R was examined using the sIL-6R-coated plates. The results showed that tocilizumab is able to bind to sIL-6R in a dose-dependent manner at the concentration between 0.001 and 0.1 μg/mL. In contrast, hIgG1 did not show any binding activity to sIL-6R (Fig. 1A).

Inhibitory effect on the binding of IL-6 and sIL-6R

The binding of IL-6 (10 ng/mL) to sIL-6R was suppressed by the addition of tocilizumab in a dose-dependent manner at the concentration between 0.002 and 4 μg/mL. Complete inhibition was achieved

Discussion

There are two ways in IL-6 signal transduction, one is mIL-6R-mediated and the other is sIL-6R-mediated. In the cells expressing IL-6R on their cell surface, IL-6 binds to mIL-6R, this formed complex associates with the second IL-6 receptor called gp130, and then a signal is transmitted into the cells. Similarly, IL-6 can form a complex with sIL-6R in the body fluids and transmit a signal through gp130 [7]. Therefore, it is essential to inhibit both mIL-6R- and sIL-6R-mediated signaling for the

Cited by (274)

View all citing articles on Scopus
View full text