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

Highlights • A variety of sgp130Fc muteins was generated.• Introduction of a gp130 SNP (R281Q) into sgp130Fc increases IL-6 specificity.• The sgp130Fc variant sgp130FlyR exclusively affects IL-6 trans-signaling.


Introduction
Interleukin (IL-)6 is the name-giving cytokine of the IL-6 type cytokine family, which consists of nine cytokines. Only IL-6 and IL-11 signal via homodimeric complexes of gp130. Specificity is conveyed by the specific alpha receptors, IL-6 receptor (IL-6R), and IL-11R. Thereby, cells expressing membrane-bound IL-6R can signal via IL-6 in a process called IL-6 classic signaling and cells expressing membrane-bound IL-11R signal via IL-11 classic-signaling. Since the expression pattern of IL-6R and IL-11R are different, the physiological consequences of IL-6 and IL-11 are different. Whereas IL-6 controls inflammatory processes including acute phase response, infection, and autoimmunity but is also involved in tumor development and metabolism [1], IL-11 is mainly involved in regenerative processes [2]. Alpha receptor binding by IL-6 or IL-11 is followed by recruitment of the ubiquitously expressed signaltransducing co-receptor gp130 and initiation of downstream signaling cascades leading to activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT), protein kinase B (PKB or AKT) and mitogen-activated protein kinase (MAPK) pathways. Alternative activation pathways include trans-signaling and trans-activation. In trans-signaling, IL-6/IL-11 binds to soluble IL-6R/IL-11R with the subsequent complex formation and signal initiation with homodimeric gp130 receptors. These complexes can activate downstream signaling cascades in all cells expressing gp130 [3]. Increased or dysregulated IL-6 trans-signaling contributes to the development of many chronic inflammatory diseases including rheumatoid arthritis [4,5], multiple sclerosis [6], inflammatory bowel disease [7][8][9], and cancer [10][11][12]. IL-11 trans-signaling was described in humans but a functional role was not defined yet [13]. In IL-6 trans-presentation, IL-6 binds to membranebound IL-6 receptor on a transmitter cell, which also engages a homodimer of gp130 which is expressed on a juxtacrine receiver cell. Some dendritic cells lack gp130 but express IL-6R and trans-presentation is needed for some CD8 T cell responses [14,15]. For IL-11, trans-presentation was described in cell culture but not yet verified to occur in living organisms [13]. For therapy, targeting IL-6 but not IL-11 is of high interest for many diseases. To date, inhibition of IL-6 signaling antibodies directed against IL-6 and the IL-6R have been approved for e.g. rheumatoid arthritis. Blockade via IL-6 antibodies is, however, associated with many side effects including an increased risk of bacterial and viral infections [16,17]. Mouse studies indicated that blockade of classicsignaling by IL-6/IL-6R antibodies is unfavorable [18] and should be avoided by second-generation therapeutics which selectively target trans-signaling. To date, only sgp130Fc (Olamkicept) has been described to selectively targeting IL-6 trans-signaling (and trans-activation). The sgp130Fc variant olamkicept successfully completed a phase IIa trial in Crohn's disease (CD) and ulcerative colitis (UC) (EudraCT: 2016-000205-36) [19] with promising outcomes including complete remission in 22% of the UC and 14% of the CD patients and clinical response in 44% of the UC and 55% of the CD patients, respectively. Currently, an additional phase II trial in ulcerative colitis (NCT03235752) is ongoing. However, sgp130Fc inhibits IL-11 trans-signaling with similar potency as IL-6 trans-signaling [13]. Of note, the mode of inhibition is completely different for antibodies targeting IL-6 or IL-6R and sgp130Fc targeting the complex of IL-6 and sIL-6R or IL-11 and sIL-11R. IL-6 and IL-11 have three binding sites for their receptors. The site I recognize IL-6R and IL-11R, respectively, while sites II and III establish the contact to gp130. IL-6 or IL-6R antibodies target the site I interface formed between IL-6 and the IL-6R and affect classic, trans-signaling, and transactivation. Like the transmembrane receptor gp130, the fusion protein sgp130Fc, which consists of all six extracellular domains of gp130 fused to an Fc-part of an IgG antibody, targets sites II and III and does not affect IL-6 classic-signaling, unless extremely high concentrations of sIL-6R are used that allow quantitative complexing of IL-6 with sIL-6R [20]. Whereas domain 1 interacts with site III of IL-6, the cytokine-binding module (CBM, domains 2 and 3) interact with IL-6 via site II [21]. Domains 4 to 6 are fibronectin type III (FNIII)-like domains and not directly involved in cytokine binding. However, they are needed for activation of signal transduction and for unknown reasons support binding of IL-6: sIL-6R complexes since shorter sgp130 variants lacking the FNIII domains exhibit reduced inhibitory potency [22]. The Fly mutations (T102Y/Q113F/N114L) are located in the Ig-like D1 domain and increase the affinity towards binding site III of IL-6 of gp130 to increase the inhibitory capacity of sgp130Fc towards IL-6:sIL-6R complexes [23]. Just recently, we found a decreased affinity for Fly towards site III of IL-11 and thereby reduced inhibitory capacity of sgp130Fc towards IL-11: sIL-11R complexes [24]. Only IL-27 of the IL-6 family also interacts with the Ig-like D1 domain of gp130. IL-27 interacts with D1 of gp130 because the IL-27 receptor WSX1 has no Ig-like D1 domain. Therefore, it is possible, that the Fly mutations also affect the binding of gp130 to IL-27. However, we have previously shown that sgp130Fc cannot inhibit IL-27 signaling [25], and hence it is likely that also sgp130 Fly Fc cannot inhibitIL-27 signaling. Albeit at were high concentrations leukemia inhibitory factor (LIF) and oncostatin M (OSM) signaling are slightly affected [26,27], these and all other IL-6 type cytokines including CNTF, OSM, and LIF interact with gp130 via CBM which binding site is not affected by the Fly mutations [21]. Recently SNP R281Q was described for gp130 in a patient presenting with abnormal head shape and craniosynostosis and retained deciduous teeth [28]. We have shown that the R281Q substitution in domain 3 of the gp130 trans-membrane receptor confers reduced IL-11-signaling, while signaling of other IL-6 family members remained intact [28]. Here we incorporated the R281Q SNP into sgp130Fc and demonstrated that is displays an increased selectivity for the inhibition of IL-6 trans-signaling compared to IL-11 trans-signaling. Since sgp130 Fly Fc and the novel variant sgp130 R281Q Fc have reduced inhibitory capacity for IL-11 transsignaling but maintained inhibition of IL-6 trans-signaling, we combined Fly and R281Q mutations in the new variant sgp130 FlyR Fc. This resulted in a highly IL-6 specific trans-signaling inhibitor completely lacking effects on IL-11 mediated trans-signaling.

Transfection, transduction, and selection of cells
CHO-K1 cells were cultured in DMEM medium. For expression of recombinant proteins, 5x10 5 CHO-K1 cells were transfected with Turbofect (Thermo Fisher Scientific, Waltham, MA, USA) and 5 µg plasmid DNA encoding sgp130Fc variants. At 5 h after transfection, the medium was exchanged to DMEM medium without a transfection reagent. For the generation of stable CHO-K1 cell lines, G418 was added to the medium 48 h after transfection. Single clones were selected via limiting dilution. Positive clones expressing Fc fusion proteins were identified by Western blotting using anti-human Fc antibodies. HEK293T cells were cultured in DMEM medium. For expression of recombinant proteins, 2x10 6 cells were transfected with Turbofect and 5 µg plasmid DNA encoding sgp130Fc variants. Mammalian expression plasmids encoding sgp130 R281Q Fc, sgp130 FlyR Fc, HIL-6-TS, IL-11-TS and HIL-11-TS were transfected into Expi-293F™ cells using ExpiFectamine™. Reaching 4.5-5.5 × 10 6 cells/ml, the cells were diluted to a final density of 3 × 10 6 cells/ml in 30 ml Expi293™ expression medium for transfection. 30 μg of the plasmid expression vectors were used for transfection.
Henceforth, the culture was harvested by centrifugation at 450g at 4 • C for 5 min, followed by centrifugation of the resulting supernatant at 4000g at 4 • C for 20 min.

Proliferation assays
Ba/F3-gp130 cells were washed and 5,000 cells of each cell line were cultured for three days in a final volume of 100 µl in the presence of cytokines and inhibitors. The CellTiter-Blue® Reagent was used to determine cellular viability by recording the fluorescence (excitation 560 nm, emission 590 nm) using an Infinite M200 PRO plate reader (Tecan, Crailsheim, Germany) immediately after adding 20 µl of reagent per well (time point 0) and up to 120 min thereafter. Fluorescence values were normalized by subtraction of time point 0 values. All experiments were performed at least three times, and one representative experiment was selected.

Western blotting
Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) or nitrocellulose membranes. Membranes were blocked and probed with the indicated primary antibodies. After washing, membranes were incubated with secondary peroxidaseconjugated antibodies (1:2.500 dilution) or fluorescence-labeled secondary antibodies (1:10.000 dilution). The Immobilon TM Western Reagents (Millipore Corporation, Billerica, MA, USA) and the ChemoCam Imager (INTAS Science Imaging Instruments GmbH, Göttingen, Germany) or the Odyssey Fc Imaging System (LI-CORE Biosciences, Bad Homburg, Germany) were used for signal detection. Control STAT3 and ERK blots were produced on a separate membrane using the same samples.

Expression and purification of recombinant proteins
sgp130Fc was produced in stably transfected CHO-K1 cells (see above) using a roller bottle system, sgp130 R281Q Fc and sgp130 FLYR Fc were produced in Expi293 cells. CHO-K1 culture supernatants were harvested and centrifuged at 1,000g and 4 • C for 30 min, followed by centrifugation of the resulting supernatant at 10,000g at 4 • C for 30 min. The supernatant of the second centrifugation step was filtered (bottle top filter, 0.45-μm pore diameter; Nalgene; Rochester, NY) and purified by affinity chromatography. Before chromatography, the pH values of the filtered cell culture supernatants were adjusted to 7.4. The supernatant was loaded on a protein-A column (HiTrap protein A HP; GE Healthcare) at a flow rate of 1 ml/min. The column was then washed with 30 column volumes of PBS. Proteins were eluted at pH 3.2-3.5 using a 50 mM citric acid buffer. Fractions of 1 ml were collected. Fractions containing the protein peak were pooled, and the pH was adjusted to pH 7 with 1 M Tris. HIL-11 and HIL-6 constructs containing a C-terminal Twin-Strep-Tag were purified using Strep-Tactin resin (IBA cat. #2-5025-001) according to the manufacturer's instructions. Proteins were buffer exchanged to PBS using illustra NAP 25 (GE Healthcare Life Sciences, Munich, Germany) columns. Protein concentration was determined by measuring absorbance at 280 nm, and samples were flash-frozen in liquid nitrogen. Protein quality was assessed by SDS-PAGE and Coomassie staining.

Surface plasmon resonance
For surface plasmon resonance experiments, a Biacore X100 instrument (GE Healthcare Life Sciences) was used. Analysis was performed in multi-cycle mode. Experiments were carried out at 25 • C in PBS pH 7.

Molecular design, expression and purification of sgp130 R281Q Fc and sgp130 FlyR Fc
cDNAs coding for sgp130Fc [27] and sgp130 Fly Fc [24] were used as templates for site-directed mutagenesis to introduce the amino acid exchange R281Q (Fig. 1A). The Fly mutations are located in the Ig-like D1 domain and increase the affinity towards binding site III of IL-6. This may be due to the hydrophobic environment residues T102, Q113 and, N114 are located at site III (Fig. 1B), surrounded by F134, F168 (IL-6R), and L57 (IL-6). Mutation of those residues into Y102, F113, and L114, allows for more hydrophobic contacts e.g. between F113 of gp130 and F168 and F134 of IL-6R, thereby strengthening the binding interface. Superpositioning of IL-11R and IL-11 on the IL-6:IL-6R:gp130 structure (1p9m) indicates that IL-11 site IIIb is more electrostatic in nature. In this case e.g. Q113 of gp130 contacts Q168 of the IL-11R. As a consequence, the Fly mutations weaken the interaction with the IL-11R. Moreover, we have shown that the R281Q substitution in the membrane bound form of gp130 reduced IL-11-signaling, while signaling of other IL-6 family members remained intact [28]. The structural analysis demonstrated that R281 is located at the heart of the site IIb interface formed between gp130 and the IL-6R (Fig. 1B) [28]. There R281 of gp130 interacts via H-bonds and salt bridges with Y257 and D281 of the IL-6R or Y260, D282, and T281 of the IL-11R, respectively. Mutation of R281Q leads to a loss of the electrostatic interactions observed between gp130 and the IL-11R. Hence, R281Q mutation selectively weakens the interaction of gp130 and the IL-11R.
cDNAs coding for sgp130 R281Q Fc and sgp130 Fly,R281Q Fc (sgp130 FlyR Fc) were transiently transfected in Expi293 cells. Secretion of sgp130Fc, sgp130 Fly Fc, sgp130 R281Q Fc, and sgp130 FlyR Fc of transiently transfected Expi293 cells was detected by Western blotting using Fc-antibodies ( Fig. 2A). Following expression, proteins were purified via ProteinA sepharose chromatography ( Fig. 2B and C). Proteins were pure based on SDS-PAGE analysis. In addition to full length proteins some degree of protein degradation was observed based on 55 and 45 kDa bands visible in Fig. 2C via Western blotting. Yields of 3.8 mg/L were obtained for sgp130Fc purified from 800 ml supernatants from stable CHO-K1 cells, while we obtained yields of approximately 13 mg/L for sgp130 Fly Fc and sgp130 FlyR Fc which were purified from 30 ml of transiently transfected Expi293 cells.

sgp130 R281Q Fc and sgp130 FlyR Fc are potent inhibitors of IL-6 transsignaling
sgp130 is a potent inhibitor of IL-6 trans-signaling [27]. Introduction of the three amino acid exchanges T102Y/Q113F/N114L in sgp130 (sgp130 Fly Fc) in binding site I of IL-6 increased affinity of sgp130 towards IL-6:sIL-6R complexes [23]. Using surface plasmon resonance, we compared the binding affinities of the established sgp130Fc, sgp130 Fly Fc, and the novel variants sgp130 R281Q Fc and sgp130 FlyR Fc (Table 1). Binding affinities of sgp130Fc and sgp130 Fly Fc to Hyper IL-6 (HIL-6) were determined previously [23], and are in good agreement with our control determination of sgp130Fc to HIL-6 of 148 pM. HIL-6 is a fusion protein of IL-6 and the soluble IL-6R connected by a flexible peptide linker. HIL-6 mimics trans-signaling and is a potent inducer of cells expressing gp130. Introduction of R281Q in sgp130 R281Q Fc and sgp130 FlyR Fc only minimally affects the binding affinity to HIL-6 compared to sgp130Fc (95 pM, 42 pM vs 148 pM, respectively) (Fig. 3A). This is supported by the capacity of sgp130 R281Q and sgp130 FlyR Fc to inhibit HIL-6 induced STAT3 phosphorylation in Ba/F3-gp130 cells. The inhibitory kinetics of sgp130 R281Q Fc and sgp130 FlyR Fc are indistinguishable from sgp130Fc, about 1 nM sgp130Fc is sufficient for full inhibition of HIL-6 (0.2 nM) induced STAT3 phosphorylation, whereas 0.1 nM already resulted in slightly reduced STAT3 phosphorylation (Fig. 3B). Further support for the good inhibitory capacity of sgp130 R281Q Fc and sgp130 FlyR Fc were obtained from experiments using the inhibition of IL-6:sIL-6R-induced proliferation of Ba/F3-gp130 cells. Ba/F3-gp130 cells are commonly used in IL-6 cytokine research because proliferation depends on IL-6 trans-signaling. As shown previously, sgp130 Fly Fc is a slightly more efficient inhibitor of trans-signaling induced cellular proliferation compared to sgp130Fc with IC50s of 0.27 nM and 0.45 nM, respectively. Importantly, sgp130 R281Q Fc and sgp130 FlyR Fc are also very potent inhibitors of trans-signaling induced cellular proliferation, with IC50s of 0.78 nM and 1.66 nM, respectively (Fig. 3C, Table 1). In conclusion, the established sgp130Fc and sgp130 Fly Fc and the novel sgp130 R281Q Fc and sgp130 FlyR Fc variants are highly effective trans-signaling inhibitors with only minimal differences with regard to affinity and required inhibitory concentrations in cellbased assays.

sgp130 FlyR Fc completely fails to inhibit IL-11 trans-signaling, whereas sgp130 Fly Fc and sgp130 R281Q Fc have reduced IL-11 trans-signaling inhibitory capacity
Like IL-6 in complex with sIL-6R, IL-11 in complex with sIL-11R also induces a homodimeric gp130 receptor signaling complex. Apparently, sgp130Fc is also a potent inhibitor of IL-11 trans-signaling [13]. Our previous results have shown that sgp130 Fly Fc has reduced inhibitory capacity for IL-11 trans-signaling, which is likely a side product resulting from the introduction of affinity increasing amino acids for IL-6 binding [23,24]. Here, we determined the affinities of sgp130Fc, sgp130 R281Q Fc, sgp130 Fly Fc, and sgp130 FlyR Fc to HIL-11 by surface plasmon resonance (Table 1). Whereas the affinity of sgp130Fc to HIL-11 is highly comparable to HIL-6 (69 pM vs. 148 pM), sgp130 R281Q Fc has an about 8fold reduced affinity towards HIL-11 (610 pM). Of note, we have observed a 6.4fold reduction in binding affinity of sgp130 R281Q Fc for HIL-11 in comparison to HIL-6 (610 pM vs. 95 pM). A combination of these mutations in sgp130 FlyR Fc, however, resulted in completely abrogated binding to HIL-11 as shown by surface plasmon resonance (Fig. 4A). Since the D1 domain of gp130 also interacts with IL-27, we further tested sgp130Fc and and sgp130 Fly Fc for IL-27 binding via SPR (Fig. 4B). We found that both proteins bind to IL-27 with comparable affinities of 69 and 58 nM, respectively. Compared to their interaction with HIL-6 this is a reduction of affinity of approximately 1000 × fold. This is well in line with previously published results that sgp130Fc does not inhibit IL-27 signaling (25). In the latter study concentrations of 10 ng/ ml sgp130Fc efficiently blocked HIL-6 induced IL-6 trans-signaling, while 1000 ng/ml sgp130Fc cannot inhibit IL-27 induced signal transduction. Based on our SPR data, concentrations of at least 10 μg/ml sgp130Fc or sgp130 Fly Fc would be required to block IL-27 signaling. Whereas HIL-11 induced STAT3 phosphorylation in Ba/F3-gp130 cells was efficiently inhibited by sgp130Fc, the novel mutant sgp130 R281Q Fc  Table 1 Affinities and inhibitory profiles of sgp130Fc variants for IL-6 and IL-11 transsignaling complexes. Surface plasmon resonance analysis of hyper-IL-11-Fc binding to purified sgp130Fc and cs-130Fc. Hyper-IL-11-Fc was immobilized on a CM-5 chip and increasing concentrations of inhibitors were injected at a flow rate of 30 µl/min. Measurements were carried out on a a Biacore X100 instrument. Ba/F3-gp130 cells were stimulated with the indicated concentrations of cytokines and their respective soluble α-receptors in the presence of decreasing inhibitor concentrations. IC 50 values were determined from three independent experiments. H-IL-11 (10) No inhibition variant inhibited HIL-11 induced STAT3 phosphorylation with lower potency. 1 nM sgp130Fc was sufficient to completely block HIL-6 induced STAT phosphorylation (Fig. 3B), 10 nM of sgp130 R281Q Fc were needed to obtain a reduction in H-IL-11 induced STAT3 phosphorylation (Fig. 4C). As expected from surface plasmon resonance experiments, even the highest inhibitor concentration of up to 100 nM sgp130 FlyR Fc completely failed to inhibit HIL-11 induced STAT3 phosphorylation (Fig. 4C). Finally, the capacity to inhibit IL-11:sIL-11R and HIL-11 induced proliferation of Ba/F3-gp130 cells by sgp130Fc and variants thereof was determined. sgp130Fc inhibits IL-11:sIL-11R and HIL-11 induced cellular proliferation with IC50s of 0.29 nM and 0.12 nM, respectively (Fig. 4D). Inhibition of HIL-11 by sgp130Fc seemed to be more efficient, but only 0.1 nM (10 ng/ml) HIL-11 was used compared to 0.5 nM (10 ng/ml) IL-11 and 2.5 nM (100 ng/ml) sIL-11R. 5 fold fewer HIL-11 molecules compared to IL-11:sIL-11R molecules might explain, why less sgp130Fc was needed to inhibit HIL-11 compared to IL-11:sIL-11R induced proliferation. In addition, the nature of the hypercytokine may contribute to the better inhibition of HIL-11 compared to IL-11:sIL-11R complex. sgp130Fc only binds to the complex of IL-11:sIL-11R but not the individual components IL-11 or sIL-11R. During IL-11: sIL-11R treatment, only a fraction of cytokine and sIL-11R are complexed while probably the majority of the molecules is free. In contrast, due to the covalent linkage of IL-11 and sIL-11R in HIL-11, no diffusion of IL-11 and sIL-11R occurs. Hence, the equilibrium between free IL-11 and sIL-11R and the [IL-11:sIL-11R] complex is strongly shifted towards the complexed state. As a consequence, in comparison, more HIL-11 can be trapped by sgp130 compared to similar concentrations of IL-11 and sIL-11R. Since uncomplexed IL-11 and sIL-11R are not recognized by sgp130 variants, this may allow for free IL-11 and sIL-11R to form a complex in close proximity to membrane bound gp130. This may then lead to immediate recruitment of the complex by membrane bound gp130 resulting in signaling. Thus, the residual complex formation of IL-11 and sIL-11R close to membrane-bound gp130 may explain the reduced effects of sgp130 on IL-11:sIL-11R complexes. Therefore, only the highest concentration of sgp130 Fly Fc (100 nM) was sufficient to at least slightly inhibit proliferation of IL-11:sIL-11R induced proliferation, whereas the highest concentration of sgp130 R281Q Fc and sgp130 FlyR Fc was not able to inhibit IL-11:sIL-11R induced proliferation. Hence, we used the more sensitive HIL-11 induced proliferation of Ba/F3-gp130 cells. Here we were able to reach sufficient concentrations of sgp130 Fly Fc and sgp130 R281Q Fc to demonstrate some inhibition HIL-11 induced cellular proliferation, with sgp130 R281Q Fc being slightly less potent compared to sgp130 Fly Fc (Fig. 3C). Noteworthy, the combination of these mutations in sgp130 FlyR Fc failed to inhibit HIL-11 induced proliferation of Ba/F3-gp130 cells (Fig. 3C, Table 1). In conclusion, HIL-11 induced proliferation supports our findings from surface plasmon resonance and STAT3 phosphorylation demonstrating that the combination of R281Q with Fly in sgp130 FlyR Fc resulted in the first complete IL-6 selective trans-signaling inhibitor based on the original sgp130Fc architecture.

Discussion
In the present study, we describe the strictly IL-6 trans-signaling selective inhibitor sgp130 FlyR Fc, which did not interfere with IL-11 trans-signaling. sgp130 FlyR Fc was generated by introduction of the four amino acid exchanges T102Y/Q113F/N114L/R281Q into sgp130Fc. Several different endogenous soluble isoforms of gp130, implicated in the regulation of IL-6-type cytokine signal transduction, have been detected in human serum [22,[32][33][34]. Due to its implications in disease development, IL-6 is an important drug target and many therapeutic strategies were developed to inhibit excessive IL-6 signaling but only soluble gp130 variants were able to block trans-signaling specifically. We and others demonstrated that sgp130Fc is also a potent inhibitor of IL-11 trans-and cluster-signaling [13,15,35]. Therfore, the dissection of IL-6 and IL-11 trans-signaling effects by sgp130Fc is mandatory, because it cannot be excluded that phenotypes observed in sgp130Fc (mouse) models stem from combined effects on IL-6 and IL-11 signaling. IL-11 is a multifunctional cytokine with effects on hepatocytes, B-cells, macrophages, osteoclasts, cardiac myocytes and fibroblast hematopoietic cells including functions in cardiac regeneration and fibrosis, colon regeneration, bone metabolism and cancer [2,36]. The first generation sgp130Fc (olamkicept) which blocks both IL-6 and IL-11 trans-signaling is currently developed as future therapeutic for chronic inflammatory bowel diseases. The sgp130Fc variant successfully completed a phase IIa trial in Crohn's disease (CD) and ulcerative colitis (UC) [19]. Sgp130Fc induces minor adverse effects including upper respiratory infections, recurrence of herpes labialis and skin and subcutaneous disorders, suggesting that selective inhibition of transsignaling by sgp130Fc is superior over combined inhibition of classic and trans-signaling by antagonistic IL-6 and IL-6R antibodies. Schreiber et al also recognized profound differences in the transcriptional signature in response to olamkicept and an antagonistic IL-6R antibody treatment, which were attributed to differences in the downstream effects of IL-6 classic and trans-signaling [19]. Effects on IL-11 transsignaling might also be observed in this study, therefore, the described IL-6 trans-signaling selectivity of sgp130 FlyR Fc may be the next important step towards second generation sgp130Fc variants. Due to its potential in blocking IL-6 trans-signaling, the first generation sgp130Fc variant was optimized in the past to improve stability and binding affinities for IL-6 [23]. Mutations at IL-6 binding sites II and III were described to improve binding affinities towards IL-6 resulting in improved biological activity [23]. Recently, we have shown that incorporation of the triple mutation FLY (T102Y/Q113F/N114L) at site III of sgp130Fc reduces inhibition of IL-11 trans-signaling, while the inhibitory activity towards IL-6 trans-signaling was maintained [24]. Moreover, we described that the patient mutation R281Q in gp130 also result in reduced IL-11 signaling [28]. Combination of Fly (T102Y/ Q113F/N114L) and R281Q in sgp130 FlyR Fc then resulted in the complete abrogation of IL-11 trans-signaling inhibition whereas inhibitory capacity towards IL-6 trans-signaling is maintained. In summary, sgp130 FlyR Fc may be beneficial for the physiological and pathophysiological dissection of IL-6 family cytokine signaling modes. Furthermore, there are possible therapeutic applications of sgp130 FlyR Fc as second generation IL-6 trans-signaling inhibitor.

Declaration of Competing Interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Juergen Scheller reports financial support was provided by German Research Foundation. Juergen Scheller, Jens M. Moll and Julia Ettich declare that they have a patent #EP-21189174-2 pending to HHU Düsseldorf.