Herstellung und Charakterisierung hochaffiner Inhibitoren für die Zytokine IL-6 und LIF basierend auf den Liganden-bindenden Domänen ihrer Rezeptor-Untereinheiten

Abstract

Cytokines are small secreted proteins produced by different cell types in nearly all tissues. They are used as intercellular mediators being involved in the regulation of immunity and inflammation. Dysregulated cytokine production plays an important role in the development and progression of different diseases. Dysregulated IL-6 production can lead to chronic inflammatory diseases like rheumatoid arthritis or to different types of cancer. While the importance of fused cytokine receptors for the treatment of dysregulated cytokine production has already been noticed, their mode of action has not been studied in detail. This should be carried out in the present study for the IL-6 inhibitor IL-6 receptor fusion protein (IL-6-RFP) constructed in our group. The first part of this work presents a detailed biochemical characterization of the IL-6 inhibitor IL-6-RFP. This inhibitor comprises the ligand-binding domains of glycoprotein 130 (gp130) and interleukin-6 receptor alpha (IL-6R alpha), which were fused by a linker. In this work, it could be shown, that IL-6-RFP is a highly specific inhibitor which does not influence the bioactivities of the cognate IL-6-type cytokines OSM and LIF. Furthermore, a two-fold molar excess of IL-6-RFP over IL-6 is already sufficient to trap IL-6 completely in a binary IL-6/IL-6-RFP complex. The complex IL-6/IL-6-RFP is more stable and has a higher binding affinity than the complex of IL-6 and its soluble receptors sgp130 and sIL-6R alpha. Accordingly, IL-6 acts as an inhibitor by binding IL-6 in solution with a higher affinity than its soluble receptors and thereby neutralizing it. Because of its high affinity, IL-6-RFP resolves IL-6 from its receptor complex on the cell surface as could be shown by live cell imaging. Next, the stoichiometry of the complex IL-6/IL-6-RFP was analysed by blue native PAGE and gel filtration. It could be shown that the complex IL-6-/IL-6-RFP is built up in analogy to the hexameric IL-6 receptor complex, the structure of which was revealed by x-ray structure analysis in other studies. It was stated, that the IL-6 receptor complex is composed of two molecules of each IL-6, IL-6R alpha and gp130. The first part of the present study highlights that IL-6-RFP is a promising IL-6 inhibitor for the treatment of diseases caused by dysregulated IL-6 expression. IL-6-RFP is an inhibitor for a cytokine recruiting its receptors with all three receptor binding sites (sites I, II and III). But many cytokines bind to their receptors by using only their sites II and III. LIF, OSM, IL-27, IL-31 and other cytokines belong to this group. The aim of the second part of this work was to reveal how those cytokines can be blocked with the help of fused soluble receptors. As a prototype for such a new site II/III inhibitor an inhibitor for LIF should be constructed comprising the ligand-binding domains of the respective receptors. It was revealed that the first five domains of the murine LIFR [LIFR(D1-D5)] were sufficient for a potent binding of human LIF. For inhibition of human LIF the murine LIFR was used since it is known that the murine LIFR binds human LIF with an extraordinarily high affinity. For the precipitation of murine LIF the fusion protein mLIF-RFP was well suited. It is composed of the first N-terminal domains of the murine LIFR fused to the cytokine-binding module of murine gp130. In cell culture experiments it was demonstrated that a five-fold molar surplus of LIFR(D1-D5) or mLIF-RFP over human or murine LIF efficiently repressed the phosphorylation of STAT3. LIFR(D1-D5) and mLIF-RFP are specific inhibitors and do not inhibit the the bioactivity of IL-6. Furthermore, mLIF-RFP does not repress the bioactivity of the cognate cytokine OSM. For the complete inhibition of gene induction after stimulation of MEF cells with human or murine LIF an addition of LIFR(D1-D5) or mLIF-RFP in a molar ratio of 1:1 was already sufficient. The construction of the site II/III inhibitor mLIF-RFP is a strategy which can be applied for the development of other cytokine inhibitors based on the fusion of ligand binding receptor domains. These site II/III inhibitors could become useful tools for the investigation of the cytokines´ functions or for the treatment of diseases caused by dysregulated cytokine expression