Jürgen Scheller

The cytokine interleukin-6 orchestrates inflammation through two distinct pathways of receptor activation: anti-inflammatory classic signalling via membrane-bound interleukin-6 receptor and pro-inflammatory trans-signalling via soluble interleukin-6 receptor. Current interleukin-6-targeted therapies block both pathways, thereby disrupting essential homeostatic functions initiated by classic signalling. We here show a high-affinity, human-specific antibody that selectively inhibits interleukin-6 trans-signalling. This antibody maintains classic signalling-induced expression of hepatic acute phase genes while completely blocking trans-signalling-induced activation of endothelial cells. By targeting the domain D2 of membrane-bound and soluble human interleukin-6 receptor, the antibody selectively prevents formation of the trans-signalling receptor complex without interfering with ligand binding. This selective blockade demonstrates that human interleukin-6 classic and trans-signalling proceed through distinct receptor assemblies. Our findings describe mechanistic insight into interleukin-6 receptor assembly and provide a strategy to suppress inflammatory interleukin-6 activity while preserving regenerative and anti-inflammatory signalling, thus offering both conceptual and potential therapeutic advances in the targeted modulation of cytokine signalling networks.

Synthetic engineering has shown that cytokine receptors from different families can be functionally combined to a large extent. We and others have developed fully synthetic cytokine ligand–receptor systems. Here, our synthetic cytokine receptor (SyCyR) system has been used to generate highly effective non-natural cytokine receptor complexes for the interleukin (IL)-6-type signaling receptors gp130, LIFR, and the erythropoietin receptor (EpoR). Epo promotes the formation of red blood cells in the bone marrow, while IL-6 plays a role in the immune response and regulation of inflammation. Their synergistic effects in combination could contribute to effectiveness of anemia treatment in future in vivo studies. Activation of these SyCyRs is based on homo- and heterodimeric GFP-mCherry fusion proteins as synthetic cytokine ligands and VHH nanobodies (variable domain of single-domain antibodies) against GFP and mCherry fused to the transmembrane and intracellular domains of engineered cytokine receptors. Although synthetic cytokine signaling was achieved for most designs, gp130:LIFR and gp130:EpoR designs were more efficient compared to LIFR:EpoR designs. The gp130:EpoR signaling combines typical signaling signature of both pathways. Taken together, our data show that synthetic cytokine receptors for members of the IL-6 family and EpoR are generally feasible.

The sirtuin SIRT4 has been implicated in the control of autophagy and mitochondrial quality control via mitophagy. However, the role of SIRT4 in regulating autophagy/mitophagy induced by different stressors is unclear. Here, we show that cells expressing SIRT4(H161Y), a catalytically inactive, dominant‐negative mutant of SIRT4, fail to upregulate LC3B‐II. These cells also exhibit a reduced autophagic flux upon treatment with different inducers of mitophagy/autophagy, that is, CoCl ₂ ‐triggered pseudohypoxia, CCCP (carbonyl cyanide 3‐chlorophenylhydrazone)/oligomycin‐mediated respiratory chain inhibition, or rapamycin treatment. Interestingly, SIRT4(H161Y) expression upregulated protein levels of HDAC6, which is involved in mitochondrial trafficking and autophagosome‐lysosome fusion, and inhibited the conversion of OPA1‐L to OPA1‐S, which is associated with increased mitochondrial fusion and decreased mitophagy. Both HDAC6 and OPA1 are SIRT4 interactors. However, the pharmacological inhibition of HDAC6 using Tubacin or of OPA1 using MYLS22 did not restore the stress‐induced upregulation of LC3B‐II levels upon autophagy/mitophagy treatment in SIRT4(H161Y)‐expressing cells. Remarkably, inhibition of autophagosome–lysosome fusion and thus disruption of late autophagic flux by BafA1 treatment also failed to restore LC3B‐II levels upon autophagy/mitophagy treatment, suggesting an inhibitory effect of SIRT4(H161Y) on the initiation/early phase of autophagy. Consistent with this, we demonstrate that SIRT4(H161Y) promotes the phosphorylation of ULK1 at S638 and S758 (mTORC1 targets), both of which mediate an important inhibitory regulation of autophagy initiation. Thus, our data suggest a positive regulatory function of SIRT4 in the ULK1‐dependent early regulation/initiation of stress‐induced autophagic flux, presumably via modulation of AMPK/mTORC1 signaling.

Nanobodies are small, single-domain antibody fragments derived from heavy chain - only antibodies. They combine high binding affinity with advantages such as compact size, stability, solubility, and flexible epitope recognition, making them attractive tools in molecular biology and therapeutic applications. In this study, we engineered and optimized nanobodies for controlled activation of synthetic cytokine receptors, aiming to expand options for receptor customization. Specifically, we used nanobodies as extracellular domains of the gp130 receptor to induce dimerization upon antigen binding. To enable receptor activity, we introduced framework mutations that promote the formation of an i-shaped nanobody (iBody) dimer, adapted from i-shaped antibodies. These mutations enhanced dimerization and enabled low-level ligand-independent receptor activation. AlphaFold modeling identified the key amino acids responsible for forming the iBody interface. Additional modifications reduced intermolecular affinity, thereby minimizing background activation while preserving the structural features necessary for ligand-induced stimulation. This approach effectively broadened the receptor's activation range. Importantly, these framework mutations were not limited to the gp130-specific nanobody GP11 but were also functional in AIP3, an anti-idiotypic nanobody targeting palivizumab. Here, the modified nanobody fusion receptor could be activated by palivizumab, overcoming prior steric hindrance.

Introduction: Although Interleukin (IL)-6-type cytokine signaling is critical for maintaining the body's homeostasis, aberrant signaling has been observed in numerous diseases including autoimmunity and cancer. Currently, all approved biologics that inhibit IL-6-type cytokines specifically target the key pro-inflammatory mediator IL-6 or its receptor (IL6R). Historically, direct inhibition of glycoprotein 130 (gp130)—the shared transmembrane receptor for IL-6-type cytokines—was avoided due to concerns that broad suppression might cause more harm than benefit. However, this view is being reconsidered in light of the clinical success of Janus kinase (JAK) inhibitors, which broadly disrupt cytokine signaling, including pathways mediated by gp130.Methods: Here we developed four single domain antibodies (sdAb), consisting out of a camelid-derived nanobody and a human Fc-fragment, and characterized them by direct protein interaction analysis, epitope binding, epitope binning, as well as inhibition of cytokine-induced stimulation and proliferation of appropriate Ba/F3 cell lines and trans-migration in HT-29 cells.Results: The four sdAb-Fc constructs GP01-, GP11- GP13- and GP20-Fc bind directly to gp130 in the cytokine binding module (CBM) and largely inhibit IL-6-type cytokine signaling by interfering with the high-affinity binding site of IL-6, IL-11, CLCF1, CT1, CNTF, OSM and LIF. Furthermore, we functionally demonstrate the inhibitory effect of the selected nanobodies in cell-based transmigration assays of the human colorectal cancer cell line HT-29.Discussion: In summary, our study has identified and characterized four novel inhibitory high-affinity gp130 nanobodies with potential for use in cytokine-dependent autoimmunity or cancer therapy.

The cytokine interleukin 6 (IL‐6) signals via the IL‐6 α‐receptor (IL‐6Rα or IL‐6R) in complex with the gp130 β‐receptor. Cell type restricted expression of the IL‐6R limits the action of IL‐6 mainly to hepatocytes and some immune cells. Here, we show that IL‐6 also binds to the IL‐11 α receptor (IL‐11Rα or IL‐11R) and induces signaling via IL‐11R:gp130 complexes, albeit with a lower affinity compared to IL‐11. Antagonistic antibodies directed against IL‐11R, but not IL‐6R, inhibit IL‐6 signaling via IL‐11R:gp130 receptor complexes. Notably, IL‐11 did not cross‐react with IL‐6R. IL‐11R has also been identified as an alternative α receptor for the CNTF/IL‐6‐derived chimeric cytokine IC7, which has recently been shown to induce weight loss in mice. Accordingly, the effects of therapeutic monoclonal antibodies against IL‐6 or IL‐6R, which both block IL‐6 signaling, may be slightly different. These findings provide new insights into IL‐6 signaling and therefore offer new potential therapeutic intervention options in the future.

Inflammation with expression of interleukin 6 (IL-6) in the central nervous system (CNS) occurs in several neurodegenerative/neuroinflammatory conditions and may cause neurochemical changes to endogenous neuroprotective systems. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are two neuropeptides with well-established protective and anti-inflammatory properties. Yet, whether PACAP and VIP levels are altered in mice with CNS-restricted, astrocyte-targeted production of IL-6 (GFAP-IL6) remains unknown. In this study, PACAP/VIP levels were assessed in the brain of GFAP-IL6 mice. In addition, we utilised bi-genic GFAP-IL6 mice carrying the human sgp130-Fc transgene (termed GFAP-IL6/sgp130Fc mice) to determine whether trans-signalling inhibition rescued PACAP/VIP changes in the CNS. Transcripts and protein levels of PACAP and VIP, as well as their receptors PAC1, VPAC1 and VPAC2, were significantly increased in the cerebrum and cerebellum of GFAP-IL6 mice vs. wild type (WT) littermates. These results were paralleled by a robust activation of the JAK/STAT3, NF-κB and ERK1/2MAPK pathways in GFAP-IL6 mice. In contrast, co-expression of sgp130Fc in GFAP-IL6/sgp130Fc mice reduced VIP expression and activation of STAT3 and NF-κB pathways, but it failed to rescue PACAP, PACAP/VIP receptors and Erk1/2MAPK phosphorylation. We conclude that forced expression of IL-6 in astrocytes induces the activation of the PACAP/VIP neuropeptide system in the brain, which is only partly modulated upon IL-6 trans-signalling inhibition. Increased expression of PACAP/VIP neuropeptides and receptors may represent a homeostatic response of the CNS to an uncontrolled IL-6 synthesis and its neuroinflammatory consequences.

Interleukin-6 (IL-6) is a highly pro-inflammatory cytokine involved in the etiopathology of several inflammatory diseases and cancer. As so, the inhibition of IL-6 signaling pathways has emerged as an attractive therapeutic avenue for the treatment of several chronic diseases. Since IL-6 trans-signaling was described as the pathological branch of IL-6, selective inhibitors were developed. Next-generation variants with increased trans-signaling specificity and potency emerged as great candidates for the treatment of several diseases, with reduced off-target effects. The highly time-consuming and costly processes involving recombinant protein production, however, have hampered the progress of anti-cytokine pharmaceuticals in clinic so far. Herein, we developed gene therapeutic modalities of IL-6-trans-signaling inhibitors as alternatives for sustained recombinant protein secretion. By using an IL-6-dependent lymphoma cell line and xenograft tumor model, we demonstrated the superior inhibitory potential of second-generation anti-IL-6 trans-signaling therapeutic. We compared the efficiency of distinct gene delivery modalities using a bioluminescent biomarker probe and observed consistent protein production via cell-based delivery. When delivered intratumorally, genetically engineered sgp130^FlyRFc-secreting cells significantly reduced tumor burden and increased animal survival, representing a promising therapeutic avenue to be explored in clinically relevant gene delivery applications.