Interleukin-11 receptor is an alternative α-receptor for interleukin-6 and the chimeric cytokine IC7.

The cytokine interleukin 6 (IL-6) signals via the IL-6 α-receptor (IL-6Rα or IL-6R) in complex with the gp130 ß-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.

Synthetic trimeric interleukin-6 receptor complexes with a STAT3 phosphorylation dominated activation profile.

In Interleukin (IL)-6 signalling, IL-6 site I binds to the IL-6 receptor (IL-6R) first, following by IL-6 site II interaction to domain 2/3 of gp130 to form premature trimeric IL-6:IL-6R:gp130 receptor complexes. Formation of the mature hexameric receptor complex is then facilitated by the inter-trimeric interaction of IL-6 site III with domain 1 of the opposing gp130. The two gp130-associated Janus kinases (JAKs) trans-phosphorylate when their spatiotemporal pairing is correct, which causes the activation of STAT, ERK, and AKT pathways in a balanced manner. Since the intracellular domain (ICD) of IL-6R is not needed for STAT/ERK/AKT phosphorylation, we investigated the conditions under which a chimeric IL-6RECD-gp130TMD/ICD receptor protein confers biological activity. For IL-6RECD-gp130TMD/ICD, the extracellular domain (ECD) of IL-6R was fused to the transmembrane domain (TMD) and ICD of gp130. Co-expression of IL-6RECD-gp130TMD/ICD with signalling-deficient gp130 variants did not induce IL-6 signalling, suggesting that the assembly of hexameric complexes failed to dimerize the IL-6R-associated JAKs correctly. By mimicking the premature trimeric receptor complex, IL-6-mediated dimerization of IL-6RECD-gp130TMD/ICD with the single-cytokine-binding variant gp130ΔD1 induced signalling. Of note, IL-6 signalling via these synthetic gp130ΔD1:IL-6RECD-gp130TMD/ICD complexes resulted predominantly in STAT3 phosphorylation. A STAT3-dominated profile was also observed after IL-6-induced signalling mediated by a JAK-deficient IL-6RECD-gp130TMD/ICDΔJAK variant in complex with the JAK-proficient but STAT/ERK/AKT-deficient gp130JAKΔICD variant. Our data showed that effective ERK/AKT signalling could not be executed after intracellular domain swapping from gp130 to the IL-6R. Taken together, the chimeric IL-6R/gp130 receptor may be helpful in the creation of customized synthetic IL-6 signalling.

Unpaired cysteine insertions favor transmembrane dimerization and induce ligand-independent constitutive cytokine receptor signaling.

Naturally occurring gain-of-function (GOF) mutants have been identified in patients for a variety of cytokine receptors. Although this constitutive activation of cytokine receptors is strongly associated with malignant disorders, ligand-independent receptor activation is also a useful tool in synthetic biology e.g. to improve adoptive cellular therapies with genetically modified T-cells. Balanced Interleukin (IL-)7 signaling via a heterodimer of IL-7 receptor (IL-7Rα) and the common γ-chain (γc) controls T- and B-cell development and expansion, whereas uncontrolled IL-7 signaling can drive acute lymphoid leukemia (ALL) development. The ALL-driver mutation PPCL in the transmembrane domain of IL-7Rα is a mutational insertion of the four amino acids proline-proline-cysteine-leucine and leads to ligand-independent receptor dimerization and constitutive activation. We showed here in the cytokine-dependent pre-B-cell line Ba/F3 that the PPCL-insertion in a synthetic version of the IL-7Rα induced γc-independent STAT5 and ERK phosphorylation and also proliferation of the cells and that booster-stimulation by arteficial ligands additionally generated non-canonical STAT3 phosphorylation via the synthetic IL-7Rα-PPCL-receptors. Transfer of the IL-7Rα transmembrane domain with the PPCL insertion into natural and synthetic cytokine receptor chains of the IL-6, IL-12 and Interferon families also resulted in constitutive receptor signaling. In conclusion, our data suggested that the insertion of the mutated PPCL IL-7Rα transmembrane domain is an universal approach to generate ligand-independent, constitutively active cytokine receptors.

Engineered interleukin-6-derived cytokines recruit artificial receptor complexes and disclose CNTF signaling via the OSMR.

Ciliary neurotrophic factor (CNTF) activates cells via the non-signaling α-receptor CNTF receptor (CNTFR) and the two signaling ß-receptors glycoprotein 130 (gp130) and leukemia inhibitory factor receptor (LIFR). The CNTF derivate, Axokine, was protective against obesity and insulin resistance, but clinical development was halted by the emergence of CNTF antibodies. The chimeric cytokine IC7 used the framework of interleukin (IL-)6 with the LIFR-binding site from CNTF to activate cells via IL-6R:gp130:LIFR complexes. Similar to CNTF/Axokine, IC7 protected mice from obesity and insulin resistance. Here, we developed CNTF-independent chimeras that specifically target the IL-6R:gp130:LIFR complex. In GIL-6 and GIO-6, we transferred the LIFR binding site from LIF or OSM to IL-6, respectively. While GIO-6 signals via gp130:IL-6R:LIFR and gp130:IL-6R:OSMR complexes, GIL-6 selectively activates the IL-6R:gp130:LIFR receptor complex. By re-evaluation of IC7 and CNTF, we discovered the Oncostatin M receptor (OSMR) as an alternative non-canonical high-affinity receptor leading to IL-6R:OSMR:gp130 and CNTFR:OSMR:gp130 receptor complexes, respectively. The discovery of OSMR as an alternative high-affinity receptor for IC7 and CNTF designates GIL-6 as the first truly selective IL-6R:gp130:LIFR cytokine, whereas GIO-6 is a CNTF-free alternative for IC7.

Identification of myeloid-derived growth factor as a mechanically-induced, growth-promoting angiocrine signal for human hepatocytes.

Recently, we have shown that after partial hepatectomy (PHx), an increased hepatic blood flow initiates liver growth in mice by vasodilation and mechanically-triggered release of angiocrine signals. Here, we use mass spectrometry to identify a mechanically-induced angiocrine signal in human hepatic endothelial cells, that is, myeloid-derived growth factor (MYDGF). We show that it induces proliferation and promotes survival of primary human hepatocytes derived from different donors in two-dimensional cell culture, via activation of mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3). MYDGF also enhances proliferation of human hepatocytes in three-dimensional organoids. In vivo, genetic deletion of MYDGF decreases hepatocyte proliferation in the regenerating mouse liver after PHx; conversely, adeno-associated viral delivery of MYDGF increases hepatocyte proliferation and MAPK signaling after PHx. We conclude that MYDGF represents a mechanically-induced angiocrine signal and that it triggers growth of, and provides protection to, primary mouse and human hepatocytes.

Bispecific soluble cytokine receptor-nanobody fusions inhibit Interleukin (IL-)6 trans-signaling and IL-12/23 or tumor necrosis factor (TNF) signaling.

At least 0.5% of people in the Western world develop inflammatory bowel disease (IBD). While antibodies that block tumor necrosis factor (TNF) α and Interleukin (IL-)23 have been approved for the treatment of IBD, IL-6 antibodies failed in the phase II clinical trial due to non-tolerable side effects. However, two clinical phase II studies suggest that inhibiting IL-6/soluble IL-6R (sIL-6R)-induced trans-signaling via the cytokine receptor gp130 benefit IBD patients with fewer adverse events. Here we develop inhibitors targeting a combination of IL-6/sIL-6R and TNF or IL-12/IL-23 signaling, named cs130-TNFVHHFc and cs130-IL-12/23VHHFc. Surface plasmon resonance experiments showed that recombinant cs130-TNFVHHFc and cs130-IL-12/23VHHFc bind with high affinity to IL-6/sIL-6R complexes and human TNFα (hTNFα) or IL-12/IL-23, respectively. Immunoprecipitation experiments have verified the higher ordered complex formation of the inhibitors with IL-6/sIL-6R and IL-12. We demonstrated that cs130-TNFVHHFc and cs130-IL-12/23VHHFc block IL-6/sIL-6R trans-signaling-induced proliferation and STAT3 phosphorylation of Ba/F3-gp130 cells, as well as hTNFα- or IL-23-induced signaling, respectively. In conclusion, cs130-TNFVHHFc and cs130-IL-12/23VHHFc represent a class of dimeric and bispecific chimeric cytokine inhibitors that consist of a soluble cytokine receptor fused to anti-cytokine nanobodies.

Respiratory syncytial virus-approved mAb Palivizumab as ligand for anti-idiotype nanobody-based synthetic cytokine receptors.

Synthetic cytokine receptors can modulate cellular functions based on an artificial ligand to avoid off-target and/or unspecific effects. However, ligands that can modulate receptor activity so far have not been used clinically because of unknown toxicity and immunity against the ligands. Here, we developed a fully synthetic cytokine/cytokine receptor pair based on the antigen-binding domain of the respiratory syncytial virus-approved mAb Palivizumab as a synthetic cytokine and a set of anti-idiotype nanobodies (AIPVHH) as synthetic receptors. Importantly, Palivizumab is neither cross-reactive with human proteins nor immunogenic. For the synthetic receptors, AIPVHH were fused to the activating interleukin-6 cytokine receptor gp130 and the apoptosis-inducing receptor Fas. We found that the synthetic cytokine receptor AIPVHHgp130 was efficiently activated by dimeric Palivizumab single-chain variable fragments. In summary, we created an in vitro nonimmunogenic full-synthetic cytokine/cytokine receptor pair as a proof of concept for future in vivo therapeutic strategies utilizing nonphysiological targets during immunotherapy.

Exploring the landscape of synthetic IL-6-type cytokines.

Interleukin-6 (IL-6)-type cytokines not only have key immunomodulatory functions that affect the pathogenesis of diseases such as autoimmune diseases, chronic inflammatory conditions, and cancer, but also fulfill important homeostatic tasks. Even though the pro-inflammatory arm has hindered the development of therapeutics based on natural-like IL-6-type cytokines to date, current synthetic trends might pave the way to overcome these limitations and eventually lead to immune-inert designer cytokines to aid type 2 diabetes and brain injuries. Those synthetic biology approaches include mutations, fusion proteins, and inter-cytokine swapping, and resulted in IL-6-type cytokines with altered receptor affinities, extended target cell profiles, and targeting of non-natural cytokine receptor complexes. Here, we survey synthetic cytokine developments within the IL-6-type cytokine family and discuss potential clinical applications.

Cytokimera GIL-11 rescued IL-6R deficient mice from partial hepatectomy-induced death by signaling via non-natural gp130:LIFR:IL-11R complexes.

All except one cytokine of the Interleukin (IL-)6 family share glycoprotein (gp) 130 as the common ß receptor chain. Whereas Interleukin (IL-)11 signal via the non-signaling IL-11 receptor (IL-11R) and gp130 homodimers, leukemia inhibitory factor (LIF) recruits gp130:LIF receptor (LIFR) heterodimers. Using IL-11 as a framework, we exchange the gp130-binding site III of IL-11 with the LIFR binding site III of LIF. The resulting synthetic cytokimera GIL-11 efficiently recruits the non-natural receptor signaling complex consisting of gp130, IL-11R and LIFR resulting in signal transduction and proliferation of factor-depending Ba/F3 cells. Besides LIF and IL-11, GIL-11 does not activate receptor complexes consisting of gp130:LIFR or gp130:IL-11R, respectively. Human GIL-11 shows cross-reactivity to mouse and rescued IL-6R-/- mice following partial hepatectomy, demonstrating gp130:IL-11R:LIFR signaling efficiently induced liver regeneration. With the development of the cytokimera GIL-11, we devise the functional assembly of the non-natural cytokine receptor complex of gp130:IL-11R:LIFR.

A Hybrid Soluble gp130/Spike-Nanobody Fusion Protein Simultaneously Blocks Interleukin-6 trans-Signaling and Cellular Infection with SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can induce mild to life-threatening symptoms. Especially individuals over 60 years of age or with underlying comorbidities, including heart or lung disease and diabetes, or immunocompromised patients are at a higher risk. Fatal multiorgan damage in coronavirus disease 2019 (COVID-19) patients can be attributed to an interleukin-6 (IL-6)-dominated cytokine storm. Consequently, IL-6 receptor (IL-6R) monoclonal antibody treatment for severe COVID-19 cases has been approved for therapy. High concentrations of soluble IL-6R (sIL-6R) were found in COVID-19 intensive care unit patients, suggesting the involvement of IL-6 trans-signaling in disease pathology. Here, in analogy to bispecific antibodies (bsAbs), we developed the first bispecific IL-6 trans-signaling inhibitor, c19s130Fc, which blocks viral infection and IL-6 trans-signaling. c19s130Fc is a designer protein of the IL-6 trans-signaling inhibitor cs130 fused to a single-domain nanobody directed against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. c19s130Fc binds with high affinity to IL-6:sIL-6R complexes as well as the spike protein of SARS-CoV-2, as shown by surface plasmon resonance. Using cell-based assays, we demonstrate that c19s130Fc blocks IL-6 trans-signaling-induced proliferation and STAT3 phosphorylation in Ba/F3-gp130 cells as well as SARS-CoV-2 infection and STAT3 phosphorylation in Vero cells. Taken together, c19s130Fc represents a new class of bispecific inhibitors consisting of a soluble cytokine receptor fused to antiviral nanobodies and principally demonstrates the multifunctionalization of trans-signaling inhibitors. IMPORTANCE The availability of effective SARS-CoV-2 vaccines is a large step forward in managing the pandemic situation. In addition, therapeutic options, e.g., monoclonal antibodies to prevent viral cell entry and anti-inflammatory therapies, including glucocorticoid treatment, are currently developed or in clinical use to treat already infected patients. Here, we report a novel dual-specificity inhibitor to simultaneously target SARS-CoV-2 infection and virus-induced hyperinflammation. This was achieved by fusing an inhibitor of viral cell entry with a molecule blocking IL-6, a key mediator of SARS-CoV-2-induced hyperinflammation. Through this dual action, this molecule may have the potential to efficiently ameliorate symptoms of COVID-19 in infected individuals.

Exclusive inhibition of IL-6 trans-signaling by soluble gp130FlyRFc.

gp130 is the signal-transducing receptor for the Interleukin (IL)-6 type cytokines IL-6 and IL-11. To induce signaling, IL-6 forms a complex with IL-6 receptor (IL-6R) and IL-11 with IL-11 receptor (IL-11R). Membrane-bound IL-6R and IL-11R in complex with gp130 and the cytokine mediate classic-signaling, whereas trans-signaling needs soluble IL-6R and IL-11R variants. Interleukin (IL)-6 trans-signaling is of particular importance because it drives the development of autoimmune diseases, including rheumatoid arthritis and chronic inflammatory bowel diseases, whereas a role for IL-11 trans-signaling remains elusive. Soluble gp130 selectively inhibits trans-signaling of IL-6 whereas both, classic- and trans-signaling are abrogated by IL-6- and IL-6R-antibodies. Recently, we described an optimized sgp130 variant, which carries three amino acid substitutions T102Y/Q113F/N114L (sgp130FlyFc) resulting in reduced inhibition of IL-11 trans-signaling by increasing the affinity of sgp130 for the site I of IL-6. Moreover, we described that the patient mutation R281Q in gp130 results in reduced IL-11 signaling. Here, we show that the combination of T102Y/Q113F/N114L and R281Q in the new variant sgp130FlyRFc results in complete preservation of IL-11 mediated trans-signaling, whereas inhibition of IL-6 trans-signaling is maintained. Since sgp130Fc (olamkicept) has successfully completed a phase IIa trial in Crohn's disease (CD) and ulcerative colitis, sgp130FlyRFc might serve as second-generation therapeutic to diminish IL-11 trans-signaling cross-reactivity.