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.

Tryptophan (W) at position 37 of murine IL-12/IL-23 p40 is mandatory for binding to IL-12Rß1 and subsequent signal transduction.

Interleukin (IL)-12 and IL-23 are composite cytokines consisting of p35/p40 and p19/p40, respectively, which signal via the common IL-12 receptor ß1 (IL-12Rß1) and the cytokine-specific receptors IL-12Rß2 and IL-23R. Previous data showed that the p40 component interacts with IL-12Rß1, whereas p19 and p35 subunits solely bind to IL-23R and IL-12Rß2, resulting in tetrameric signaling complexes. In the absence of p19 and p35, p40 forms homodimers and may induce signaling via IL-12Rß1 homodimers. The critical amino acids of p19 and p35 required for binding to IL-23R and IL-12Rß2 are known, and two regions of p40 critical for binding to IL-12Rß1 have recently been identified. In order to characterize the involvement of the N-terminal region of p40 in binding to IL-12Rß1, we generated deletion variants of the p40-p19 fusion cytokine. We found that an N-terminal deletion variant missing amino acids M23 to P39 failed to induce IL-23-dependent signaling and did not bind to IL-12Rß1, whereas binding to IL-23R was maintained. Amino acid replacements showed that p40W37K largely abolished IL-23-induced signal transduction and binding to IL-12Rß1, but not binding to IL-23R. Combining p40W37K with D36K and T38K mutations eliminated the biological activity of IL-23. Finally, homodimeric p40D36K/W37K/T38K did not interact with IL-12Rß1, indicating binding of homodimeric p40 to IL-12Rß1 is comparable to the interaction of IL-23/IL-12 and IL-12Rß1. In summary, we have defined D36, W37, and T38 as hotspot amino acids for the interaction of IL-12/IL-23 p40 with IL-12Rß1. Structural insights into cytokine-cytokine receptor binding are important to develop novel therapeutic strategies.