Novel Inhibitors for Murine and Human Leukemia Inhibitory Factor Based on Fused Soluble Receptors

Abstract

Proinflammatory cytokines such as tumor necrosis factor (TNF), 2 interleukin-1␤ (IL-1␤), or interleukin-6 (IL-6) have been identified as promising therapeutic targets in the treatment of chronic inflammation. A dimeric soluble TNF receptor is currently used for the treatment of inflammatory diseases caused by elevated TNF expression (1). Whereas TNF signals through a receptor homotrimer, most cytokines signal through receptor complexes consisting of two or more different receptor subunits. In this case, the respective cytokine can be inhibited by using fusion proteins composed of the different soluble receptors, as we and others showed for the inhibition of IL-6 (2-4).All cytokines signaling through the common receptor subunit gp130 belong to the family of IL-6-type cytokines (5), which includes IL-6, IL-11, IL-27, LIF, OSM, ciliary neurotrophic factor, cardiotrophin-1, cardiotophin-like cytokine, and neuropoietin. IL-6-type cytokines contain distinct receptorbinding sites that were discovered by mutagenesis studies on IL-6, ciliary neurotrophic factor, and LIF (6 -8). The IL-6-type cytokines can be subdivided into those containing three (I, II, and III) or two (II and III) receptor-binding sites. Site I determines the specificity of ␣-receptor binding. The ␣-receptor is not capable of transferring the signal into the cell but is crucial for increasing the binding affinity of the cytokine to its signaling receptors. Site II seems to be the universal gp130-binding site of all IL-6-type cytokines. Depending on the cytokine, site III is used for the recruitment of LIFR, OSMR, or a second gp130 molecule (5). The IL-6 inhibitor IL-6-RFP (2, 3) was designed to block a cytokine containing all three receptor-binding sites.However, there are also IL-6-type cytokines, which do not need to recruit an ␣-receptor analogous to human IL-6R␣, and thus do not seem to have a functional site I. One example for a cytokine belonging to this group is the leukemia inhibitory factor (LIF). In this study we present an approach to construct inhibitory receptor fusion proteins for human and murine LIF as prototypes of inhibitors targeting cytokines whose receptors only bind to the site II and III of the cytokine without occupying site I. We designate these inhibitors "site II/III inhibitors."LIF signals through a heterodimer of LIFR and gp130. Janus tyrosine kinases that are constitutively associated with the cytoplasmic parts of gp130 (9) and LIFR (10) are activated upon ligand binding and phosphorylate the receptors and the recruited transcription factor STAT3. Activated STAT3 dimerizes and translocates into the nucleus, where it induces LIF target genes (11).We wanted to integrate only those receptor domains of gp130 and LIFR into the inhibitory receptor fusion proteins that are necessary for LIF binding. For gp130, which includes six extracellular domains (D1-D6), it has been clearly shown that domains D2 and D3 forming the cytokine-binding module (CBM) are necessary and sufficient for LIF binding (12). In contrast, there are contradi...

Targeting JAK/STAT Signaling Pathway in Inflammatory Diseases

“…By contrast, a similar approach taken to develop a murine LIF fusion protein (i.e. mLIF-RFP) showed that mLIF-RFP or hLIF-RFP blocked STAT3 activation induced by murine or human LIF, respectively, but RFP inhibited neither IL-6 nor OSMinduced STAT3 phosphorylation in the several cell lines that were tested, including COS-7, HEK and mouse embryo fibroblast cells [168]. The results of these studies, when taken together, suggested the distinct possibility that similar fusion protein strategies might be employed for creating potent and specific inhibitory biologics for other pro-inflammatory cytokines such as IL-27/IL-28 [9,169] IL-31 [170], as well OSM [171], that were reported to either activate JAK/STAT or as in the case of IL-32 [1,24,172] induce TNF-, IL-1 and IL-6 gene expression via SAP/MAPK activation [172].…”

“…IL-6RFP) [168]. In structural studies IL-6RFP was shown to be more stable than an IL-6/sIL6R /sgp130 complex.…”

Definition and Characterization of an Inhibitor for Interleukin-31

“…These mediators can be subdivided in two subgroups: those recruiting three receptor components via sites 1, 2 and 3, and a second subgroup (IL-31, OSM, LIF, IL-27), requiring the involvement of only two receptor subunits recognized through interactions with the sites 2 and 3 (14). A recent report described the possibility of neutralizing the LIF response by using a mouse construction inhibiting interactions between the receptor subunits and sites 2 and 3 of the cytokine (40). Our findings extend this approach to the human IL-31 receptor and emphasize the specificity of the observed neutralizing response.…”

Insights into IL-23 biology: From structure to function

“…This strategy was successfully applied to generate inhibitors for the IL-6-type family members IL-6 and leukemia inhibitory factor (LIF) and might be transferred to the IL-23 signaling complex [134][135][136], by fusion of sIL-23R and sIL-12Rβ1.…”