Plasmids have been deposited at the BCCM/GeneCorner plasmid collection (www.genecorner.ugent.be) hosted by our department. p4x-STAT6-Luc2P (LMBP09396), which contains a STAT6-driven luciferase reporter gene, was purchased from Addgene. pNFconluc, which contains an NF-κB–driven luciferase reporter gene, was a gift from Dr. A. Israel (Institut Pasteur, Paris, France), and pACTbgal (LMBP4341) was from Dr. J. Inoue (Institute of Medical Sciences, Tokyo, Japan). Construction of human and mouse IL-33traps, as well as production of mouse IL-33trap in HEK 293 FreeStyle cells, were described previously (15). Full length human IL-33 was PCR amplified from a human cDNA library and ligated into pCR-Blunt II-TOPO. Splice variants were made by inverse PCR reaction. Subsequently, IL-33 full length and splice variants with a C-terminal 6xHis-tag were PCR amplified and cloned into pJExD by homologous recombination (CloneEZ). The basic bacterial expression vector pJExD, which allows crystal violet-induced expression, was made by modifying the commercial vector pET-Duet1 as follows: lacI and the first T7 promoter and lacO binding site (Eco47III—BamHI) were replaced with a synthetic sequence containing an eilR expression cassette and the crystal violet inducible JExD promoter with eilR binding sites (17). Expression of IL-33 splice variants in Escherichia coli BL21 was induced by addition of 100 nmol/L crystal violet (Sigma-Aldrich, Belgium) for 3.5 h at 37°C. Cells were collected by centrifugation, lysed and soluble IL-33 variants were purified using immobilized metal affinity chromatography using Ni-NTA sepharose (IBA Lifesciences, Germany). Production of truncated mouse IL-33 (residues 109–266) in Escherichia coli BL21 has been described previously (15). Briefly, protein expression was induced with 1 mmol/L isopropyl β-D-1-thiogalactopyranoside, followed by overnight incubation at 28°C. The bacterial pellet was harvested by means of centrifugation, resolubilized, and lysed by means of sonication. The lysate was centrifuged, and soluble IL-33 was purified from the supernatant by using immobilized metal affinity chromatography, followed by size exclusion chromatography (SEC). Protein concentration was determined with a bicinchoninic acid (BCA) protein assay kit.

The ectodomains of the human IL-13Rα1 and IL-4Rα were PCR amplified from a human cDNA library and cloned into pEF6-MycHisA to generate pEF-ShIL13Ra1 and pEF-ShIL4R, respectively. To generate the IL-4/13trap expression plasmid, a human IL-4Rα PCR fragment amplified from pEF-ShIL4R and a linker sequence of 20 times repeating Gly-Gly-Ser (GGS) triplets amplified from pCLG-Duba (LMBP6610) were cloned into the pEF-ShIL13Ra1 vector via 3-way ligation. All constructs were confirmed using DNA sequencing analysis. Human IL-4/13trap and human IL-33trap were produced in HEK293T cells and purified from the medium fraction by immobilized metal affinity chromatography using Ni-NTA sepharose (IBA Lifesciences, Germnay). To reduce the glycosylation complexity and heterogeneity, murine IL-33trap was also produced in suspension growth serum-free adapted HEK293 GlycoDelete cells (18).

HEK293T cells (gift from Dr. Hall, Department of Biochemistry, University of Birmingham, United Kingdom) were seeded at 4 × 10⁴ cells/well in 24-well plates and cultured in Dulbecco's modified Eagle medium supplemented with 10% FCS and 2 mmol/L L-glutamine. The next day, cells were transiently transfected by calcium phosphate precipitation with specific IL-33, IL-4, or IL-13 cytokine receptor expression plasmids. For the IL-33 bioassay, cells were co-transfected with the NF-κB reporter plasmid pNFconluc and the constitutively expressing β-galactosidase plasmid pACTbgal. For the IL-4 and IL-13 bioassay, cells were co-transfected with STAT6 and the STAT6 reporter plasmid p4x-STAT6-Luc2P, as well as the constitutively expressing β-galactosidase plasmid pACTbgal. 24 h later, cells were stimulated with recombinant IL-33, IL-4, or IL-13 for 5 h. For cytokine neutralization experiments, cytokines were incubated for 30 min at room temperature with specific cytokine trap inhibitors before addition to the cells. Cell lysates were analyzed for luciferase activity and normalized based on β-galactosidase levels to correct for potential differences in transfection efficiency.

Potential protein aggregation was measured via size-exclusion chromatography (SEC) coupled with multi-angle laser light scattering (MALLS). Protein samples were injected onto a Superdex 200 Increase10/300 GL column (GE Healthcare), with PBS pH 7.4 as running buffer at 0.5 ml/min, coupled to an inline ultraviolet-detector (Shimadzu), a multi-angle light scattering miniDAWN TREOS instrument (Wyatt) and an Optilab T-rEXrefractometer (Wyatt) at 25°C. A refractive index increment (dn/dc) value of 0.185 ml/g was used for protein concentration and molecular mass determination. Data were analyzed using the ASTRA6 software (Wyatt). Correction for band broadening was applied using parameters derived from BSA injected under identical running conditions. For the analysis of IL-33traps, conjugate analysis was performed using theoretical protein extinction coefficients and a dn/dc value of 0.160 ml/g for the glycan modifier.

Thermostability was measured by ThermoFluor® assay as described (19). Protein samples were diluted in a final volume of 16 μl of PBS buffer and 1 μl of 300X SYPRO Orange (Invitrogen™, Thermo Fisher Scientific) was added. Each experiment was run as a technical triplicate, with a triplicate blank measurement without test protein. Fluorescence in function of the temperature was recorded in a 348-well LightCycler® 480 (Roche Life Science) from 25 to 95°C at 0.02°C/s. Melting temperatures (T_m) were calculated as the V50 value of a Boltzmann sigmoidal curve fitted to the averaged data points of the three replicates in each experiment. Onset temperatures (T_o) were calculated as previously described (20). For graphing, the raw data sets were averaged, blank corrected and then normalized (minimal value at 0%, maximal value at 100%), using Prism 7 Software (GraphPad).

Recombinant cytokine decoy receptor-based biologics such as Etanercept, Rilonacept, and Aflibercept all contain the Fc portion of human IgG1. Fusion to an Fc domain allows dimerisation of the two receptor subunits and provides manufacturing, biological and pharmacological advantages in vivo, including established large-scale affinity purification, half-life extension due to pH-dependent binding to the neonatal Fc receptor (FcRn) (21). For the construction of IL-33trap we used an alternative approach that avoids the need for an Fc to induce receptor dimerisation. More specifically, we cloned a flexible GGS linker sequence between sST2 and sIL-1RAcP to ensure the intramolecular formation of an active sST2/sIL-1RAcP heterodimeric receptor complex (15). Although at that time we did not compare the bioactivity of our linker containing IL-33trap with an IL-33trap variant that does not contain a linker, it is likely that the linker is needed for optimal domain orientation and heterodimerisation between both receptor subunits and consequently the bioactivity of IL-33trap. To further compare the effect of a linker sequence with the effect of fusion to an Fc-moiety on IL-33trap bioactivity, we generated recombinant human IL-33trap variants in which both receptor subunits (sST2 and sIL-1RAcP) were either not separated by a linker or contained a 7x or 12x GGS linker (shown empirically to enable the formation of biologically active IL-33trap), and compared these with the corresponding IL-33trap constructs that also contain a human IgG1 Fc domain at the C-terminus (Figure 1A).

The antagonistic activity of different human IL-33trap constructs was analyzed by measuring their ability to inhibit IL-33-induced activation of an NF-κB-dependent luciferase reporter gene in HEK293T cells that were made IL-33 responsive by transient transfection with human ST2. Prior to cell stimulation, recombinant human IL-33 was incubated for 30 min with equimolar concentrations of each human IL-33trap variant over a range of inhibitors/target ratios. An IL-33trap without Fc and GGS linker had an almost 10-fold reduced activity compared to a similar GGS-less construct fused to an Fc (IC₅₀ of 748 pM and 90 pM, respectively; Figure 1B), suggesting that the Fc-moiety allows the optimal IL-33trap conformation for ligand binding, most likely by mediating dimerisation of two IL-33trap molecules. Importantly, inclusion of a 7x GGS or 12xGGS linker sequence instead of an Fc also enhanced the antagonistic effect of the IL-33trap (IC₅₀ of 194 pM and 42 pM, respectively; Figure 1C), with sIL-1RAcP-12xGGS-sST2 being slightly more potent than sIL-1RAcP-sST2-Fc (IC₅₀ of 42 vs. 90 pM; Figure 1C). Combined use of a 12xGGS linker and an Fc fusion in a single IL-33trap construct (IC₅₀ of 57 pM) did not much further change the potency of a 12xGGS-only or Fc-only construct (IC₅₀ of 42 or 90 pM, respectively; Figures 1C,D). However, additional Fc fusion leads to a more potent molecule in the case of a shorter 7xGGS linker (IC50 67 vs. 194 pM; Figures 1C,D). Collectively, these data illustrate that fusion of either a GGS linker sequence or an Fc-moiety increases the IL-33 antagonistic activity of an sIL-1RAcP-sST2 fusion protein by enabling, respectively, intramolecular and intermolecular interactions between sST2 and sIL-1RAcP that are necessary for the formation of a high affinity IL-33 binding complex. The lower potency of a 7xGGS containing construct (IC₅₀ of 194 pM) compared to a 12xGGS (42 pM) construct suggests that a longer linker increases molecular flexibility, favoring a more optimal intramolecular interaction between sST2 and sIL-1RAcP. Importantly, our data illustrate that insertion of a flexible 12xGGS linker between both IL-33 receptor subunits allows the formation of a single-chain fusion protein with high IL-33 antagonistic activity, circumventing the need for Fc fusion and Fc-mediated dimerisation.

The biophysical behavior of therapeutic proteins is very important for the correct development and optimisation of biologics, since it may impact many aspects of drug function, stability and activity. Excessive aggregates and fragmentation, as well as denaturation or oxidation are indicative of an unstable product, unsuitable for in vivo use. For this reason, we sought to characterize the molecular and thermal stability of the murine 20xGGS-linker containing but Fc-less IL-33trap for which we previously showed an inhibitory effect upon local delivery in a mouse asthma model (15). First, we examined whether IL-33trap is prone to form aggregates during storage. IL-33trap was stored frozen in PBS at −80°C or liquid-stored at 4°C for a time period of 10 days. To determine the presence of potential aggregates, we used size-exclusion chromatography (SEC) coupled with ultraviolet (UV), multi-angle light scattering laser (MALLS) and refractive index (RI) detectors. Protein elution fractions were identified as eluting species with a peak in both UV absorbance and differential RI (dRI) intensity. IL-33trap, either frozen-stored at −80°C or liquid-stored at 4°C for 10 days, was found to be highly homogeneous, adopting monodisperse assemblies, with a protein molecular mass comparable to the theoretical (Figure 2A).

We next assessed the thermostability of IL-33trap using ThermoFluor® assay (19), and melting temperature (T_m) was calculated as the V50 value of a Boltzmann sigmoidal curve fitted to the melting curve (19, 22). The analysis showed a T_m and a T_o of 55 and 46.5°C, respectively which is well above the physiological temperature of 37°C (Figure 2B). Together, our data indicate that IL-33trap is a stable molecule at physiological temperature and does not form aggregates when liquid-stored at 4°C for up to 10 days.

We previously showed that mouse IL-33trap expressed in HEK293T cells is glycosylated (15). To further estimate the degree of glycosylation, we calculated the total protein and glycan molecular weight on IL-33trap using SEC-MALLS and ASTRA6 software (Wyatt), showing that IL-33trap is heavily glycosylated (25–35% of total mass) (Figure 2A). Using NetNglyc 1.0 Server and NetOglyc 4.0 Server prediction (23), 15 potential N-glycosylation and 1–4 potential O-glycosylation sites were found. Although not all of these sites are likely to be occupied, the high number of glycosylation sites leads to significant product heterogeneity, which is further complicated by the inherent difference in glycan chain length and complexity in eukaryotic expression systems. This is a major challenge for efficient purification at high yield, to set sound specifications for product release and hence to assure batch reproducibility. It is also likely to attribute significant heterogeneity in pharmacokinetic behavior of the molecule, due to differential lectin-mediated blood clearance of different glycoforms. Therefore, we decided to reduce the glycosylation complexity and heterogeneity of mouse IL-33trap using HEK293 GlycoDelete technology (18), which reduces glycosylation to short single-branch oligosaccharides that are partially sialylated. Indeed, IL-33trap produced in HEK293 GlycoDelete is notably less glycosylated (6% of the total mass) compared to the original IL-33trap molecule produced in HEK293 FreeStyle cells (25–35% of the total mass) (Figure 2A). We then compared the thermostability and antagonistic activity of the glycosylated and under-glycosylated IL-33trap variants. Reduced glycosylation had only a mild effect on the melting temperature (Figure 2B), and did not affect the ability of IL-33trap to inhibit IL-33-induced NF-κB activation (Figure 2C).

Human IL-33 mRNA can be alternatively spliced in several smaller IL-33 splice variants lacking exon 3, 4, 5 or a combination thereof, which are expressed in different cell types in different proportions (24). In contrast to deletion of exons 3 and 4, absence of exon 5 results in loss of IL-33 activity (25), which is consistent with the fact that exon 5 encodes amino acid residues that are critical for ST2 binding (26). Importantly, binding of inactive IL-33 isoforms to IL-33-neutralizing biologics would serve as a natural sink and decrease drug availability. However, because IL-33trap is a receptor-based biologic, it is expected to exclusively interact with bioactive receptor-binding IL-33 isoforms and to be insensitive to the presence of inactive IL-33 splice variants. To further test this hypothesis, we generated IL-33 splice variants lacking exons 3 and 4 (IL-33Δe3-4) or exons 3, 4 and 5 (IL-33Δe3-5) (Figure 3A), and analyzed their activity in an IL-33 bioassay. As shown previously, IL-33Δe3-4 efficiently induced NF-κB activation, while IL-33Δe3-5 was inactive (Figure 3B). Furthermore, NF-κB activation induced by IL-33Δe3-4 was efficiently inhibited by IL-33trap (Figure 3C). We next performed a competition assay, where we pre-incubated mature IL-33 with IL-33trap in the presence of increasing concentrations of either IL-33Δe3-4 or IL-33Δe3-5 splice variants. Consistent with its receptor-binding capacity, addition of IL-33Δe3-4 reduced the ability of IL-33trap to inhibit IL-33 signaling, while addition of IL-33Δe3-5 had no effect (Figure 3D). These results illustrate that inactive IL-33 splice variants will not act as a sink for IL-33trap, which might offer a significant advantage compared to certain monoclonal antibodies that, depending on the recognized epitope, might not always distinguish between active and inactive IL-33 isoforms.

As for the IL-33trap, a similar approach using single-chain soluble receptor fusion proteins can be used to target other cytokines that signal through a heterodimeric receptor complex. In this regard, we have previously reported the design and validation of TSLP-trap, a fusion protein which consists of the extracellular domains of TSLPR and IL-7Rα fused via a 20xGGS flexible linker (27). Together with IL-33 and IL-25, the epithelium-derived cytokine TSLP is considered a central orchestrator of Th2 responses in atopic disorders, and therefore a promising therapeutic target. Similarly, IL-4 and IL-13 are in the spotlight as interesting dual therapeutic targets in type 2-driven inflammatory disease (28). Importantly, these two cytokines both bind to the type II receptor consisting of IL-4 receptor alpha (IL-4Rα) and IL-13Rα1 (29) (Figure 4A), offering a possibility to simultaneously target two cytokines with one inhibitor. Using a similar design as IL-33trap (15), we generated and validated a new human IL-4/13trap, consisting of the extracellular domain of human IL-13Rα1 fused to the extracellular domain of human IL-4Rα via a flexible 20xGGS linker. The expression construct also contains the human IL-13Rα1 signal sequence at the N-terminus, which allows protein secretion, and a myc/His tag at the C-terminus to facilitate protein purification and detection (Figure 4A). Human IL-4/13trap was produced using HEK293 FreeStyle cells to ensure proper folding, and purified from conditioned media using immobilized metal affinity chromatography and size exclusion chromatography, as described in materials and methods.

To test whether IL-4/13trap displays antagonistic properties, we investigated its ability to inhibit both IL-4 and IL-13 signaling in a cell-based assay. To this end, HEK293T cells were made responsive to either IL-4 or IL-13 by transfection with human IL-4Rα and IL-13Rα1, and downstream STAT6 activation was followed via STAT6-dependent luciferase reporter gene expression. IL-4 as well as IL-13 treatment resulted in robust STAT6 activation (Figure 4B). Importantly, pre-incubation of either IL-4 or IL-13 with equimolar concentration of IL-4/13trap strongly reduced IL-4- and IL-13-induced STAT6-dependent luciferase activity (IC₅₀ of 465 pM and 434 pM, respectively) (Figure 4B). These data convincingly demonstrate that IL-4/13trap behaves as a strong IL-4 and IL-13 antagonist and illustrate the feasibility to develop recombinant single-chain soluble receptor fusion proteins as novel biologics for the inhibition of a wide range of cytokines or other protein ligands that signal via a heterodimeric receptor complex.