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. Author manuscript; available in PMC: 2025 Feb 27.
Published in final edited form as: J Immunol. 2025 Jan 28;214(1):180–191. doi: 10.1093/jimmun/vkae022

Development of anti-murine IL-18 binding protein antibodies to stimulate IL-18 bioactivity

Arnaud Huard *,†,‡, Sébastien Fauteux-Daniel *,†,‡, Jérémie Goldstein *,†,‡, Praxedis Martin *,†,‡, Matthias Jarlborg *,†,‡, Julie Andries §,, Assunta Caruso *,†,‡, Alejandro Díaz-Barreiro *,†,‡, Emiliana Rodriguez *,†,‡, Laurie Vaillant *,†,‡, Savvas N Savvides §,, Cem Gabay *,†,‡,||
PMCID: PMC7617445  EMSID: EMS200183  PMID: 40018678

Summary

Interleukin (IL)-18 is an immunoregulatory cytokine that acts as a potent inducer of T helper 1 and cytotoxic responses. IL-18 activity is regulated by its decoy receptor IL-18 binding protein (IL-18BP) which forms a high affinity complex with IL-18 to block binding of the cognate receptors. A disbalance between IL-18 and IL-18BP associated with excessive IL-18 signaling can lead to systemic inflammation. Indeed, the severity of CpG-induced macrophage activation syndrome (MAS) is exacerbated in IL-18BP KO mice. On the contrary, targeting IL-18BP can have promising effects to enhance immune responses against pathogens and cancer. We generated monoclonal rabbit anti-mouse IL-18BP antibodies labeled from 441 to 450. All antibodies, except from antibody 443, captured mIL-18BP when used in a sandwich ELISA. Using an IL-18 bioassay, we showed that antibody 441 did not interfere with the regulatory effect of mIL-18BP, whereas all other antibodies displayed different levels of antagonism. Further experiments were performed using antibody 445 endowed with potent neutralizing activity and antibody 441. Despite binding to IL-18BP with the same affinity, antibody 445, but not antibody 441, was able to release IL-18 from preformed IL-18-IL-18BP complexes. Administration of antibody 445 significantly aggravated the severity of CpG-induced MAS as compared to antibody 441. Additional experiments using naïve WT, IL-18BP KO, and IL-18 KO mice confirmed the specificity of the neutralizing effect of antibody 445 towards IL-18BP. Our studies led to the development of a monoclonal anti-IL-18BP antibody with neutralizing activity that results in the promotion of IL-18 activities.

Keywords: Interleukin (IL)-18, IL-18 binding protein, monoclonal antibodies, macrophage activation syndrome

Introduction

Inflammation is a double-edged process that plays a critical role in the fight against various infectious pathogens and noxious agents but when in excess can lead to the pathogenesis of inflammatory disorders and serious organ damage (1). Inflammatory responses are regulated by different cytokines via the stimulation of cellular responses leading to the production of inflammatory mediators, chemotaxis, leukocyte migration, and lymphocyte activation. Interleukin (IL)-18 is a prototypical inflammatory cytokine that belongs to the family of IL-1 cytokines. IL-18 stimulates the production of interferon (IFN)-γ by T lymphocytes and NK cells and activates cell cytotoxic activities of CD8+ T cells and NK cells (2, 3), thus contributing to host defence mechanisms. IL-18 has also been shown to contribute to the pathogenesis of several autoinflammatory diseases, including adult-onset Still’s disease (4), systemic juvenile idiopathic arthritis (5), macrophage activation syndrome (MAS) (6), NLRC4 gain-of-function mutations associated with MAS and severe enterocolitis (7), and other conditions (8).

IL-18 is synthesized as a pro-protein that must be proteolytically processed in its N-terminal pro-domain to become biologically active. In the context of canonical inflammasome activation, caspase-1 cleaves pro-IL-18 to generate mature IL-18. Caspase-1 processing of pro-IL-18 creates important conformational changes that allow IL-18 to bind to its cognate receptors (9). In addition to canonical inflammasomes, a non-canonical inflammasome pathway involving human caspase-4 and 5 and mouse caspase-11 (functionally homologous to human caspase-4 and 5) has been described a few years ago (10), (11). Intracellular LPS binds to these caspases leading to Gasdermin-D cleavage and cell membrane pore formation. The subsequent efflux of K+ via cell membrane pores activates the nucleotide-binding domain and leucine-rich repeat pyrin domain-containing protein 3 (NLRP3) inflammasome with subsequent pro-IL-1β and pro-IL-18 cleavage and release. In addition, it has recently been shown that human caspase-4 and caspase-5, but not mouse caspase-11, can directly process pro-IL-18 and to a lesser extent pro-IL-1β (12), (13).

Mature IL-18, but not pro-IL-18, binds to IL-18 binding protein (IL-18BP), a naturally secreted soluble decoy receptor that forms a high affinity complex with IL-18, serving as a homeostatic sink to prevent the interaction of IL-18 with its cell surface receptors (14). IL-18BP has a unique immunoglobulin (Ig) domain and is highly glycosylated. The human IL18BP gene encodes four isoforms IL-18BPa, b, c and d. IL-18BPa is the predominant isoform and, like hIL-18BPc, can antagonize IL-18 activity, in opposition to isoforms b and d that lack a complete Ig domain and cannot bind IL-18. In the mouse, two isoforms are produced by alternative splicing but with identical Ig domain: mIL-18BPc and d, and both are active to block mouse IL-18. In addition, mouse IL-18BPd shares a common C-terminal domain with human IL-18BPa and is also able to inhibit human IL-18 (15). The production of IL-18BP is upregulated by IFN-γ, forming a negative feedback loop that controls IL-18 activity (16). IL-18BP shows significant amino-acid sequence differences in its binding domain compared to IL-18Rα, which could explain its higher binding affinity for IL-18 (17). Furthermore, IL-18BP is found in high amounts in the circulation, thus serving as a very effective sequestration mechanism for circulating IL-18 under physiologic and inflammatory conditions.

The administration of recombinant human IL-18BPa (tadekinig alfa) led to significant improvement in patients with some autoinflammatory diseases (reviewed in (8)). On the other hand, strategies aimed at targeting IL-18BP could be of value to enhance immune responses in the case of cancer immunotherapy (18). Indeed, it has recently been shown that the administration of a monoclonal anti-IL-18BP antibody displayed beneficial effects by increasing immune responses against cancer cells in a heterotopic model of cancer in mice (19).

Here, we describe the development of several monoclonal antibodies against mouse IL-18BPd. One such anti-IL-18BP antibody showed no effect, while all others exhibited various degrees of IL-18BP neutralization, abrogating its ability to sequester IL-18, as assessed by an in vitro bioassay. The administration of a neutralizing monoclonal anti-IL-18BP antibody markedly increased the severity of a mouse model of MAS dependent on IL-18, recapitulating the phenotypic manifestations observed in IL-18BP deficient mice with aggravated cachexia, splenomegaly, anaemia, and IFN-γ production (20, 21).

Materials and Methods

Monoclonal anti-mouse IL-18BP antibody production

Rabbits were immunized with mouse IL-18BP protein to generate anti-IL-18BP antibodies. Blood was taken at various time points and enriched for B lymphocytes. The isolated B-cells were sorted by FACS into single cells and cultivated for 7 days. Subsequently, the B-cell supernatants were removed and used for testing. Direct ELISAs were developed in the 384-microtiterplate format to identify antibodies against mouse IL-18BP. These assays were used to screen B-cell supernatants. Positive samples were selected for further processing. Plasmids representing unique antibody sequences were created and used to produce recombinant rabbit antibodies. Ten candidates have been retained and further tested by sandwich IL-18BP ELISA (see below) as well as for their functional activity using stably transfected RAW 264.7 cells expressing mIL-18R (see below). The two antibody clones 441 and 445 were retained for further in vitro and in vivo experiments following their preliminary functional characterization. These antibodies were murinized to obtain IgG2a antibodies with the addition of a LALAPG mutation in the Fc region to prevent binding to Fcγ receptors and complement activation (22) by FairJourney Biologics (Porto, Portugal).

Cell culture

RAW 264.7 cells were grown in cell culture medium (Dulbecco’s modified eagle’s medium (DMEM) (Gibco, Waltham, MA, USA) containing 10% FCS, supplemented with penicillin/streptomycin and maintained in a tissue culture incubator at 37°C and 5% CO2 in a humid environment. Cells were removed by pipetting up and down, and then dispensed at a density of 1 x 106 cells per well in a 6-well plate in 2.5 mL cell medium and grown for 18 h prior to transfection.

Vector preparation

Vector backbone pcDNA3.1(+) Geneticin_A009 coding for mouse IL-18Rα, or vector backbone pcDNA3.1(+) Hygromycin_A069 coding for mouse IL-18Rβ were customized by Thermo Fisher Scientific (Waltham, MA, USA). The commercially available vector pcDNA3.1(+) (Thermo Fisher Scientific) was used as Geneticin-expressing empty vector control. To generate the Hygromycin-expressing empty vector, the insert coding for mouse IL-18Rβ was removed. In brief, 2 μg DNA were digested with NheI (New England Biolabs) and BamHI (New England Biolabs, Ipswich, MA, USA)) in the appropriate buffer for 1 h at 37°C. The vector was purified using Qiaquick Gel extraction kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. DNA was blunted by incubating with Klenow fragment (Thermo Fisher) in the appropriate buffer for 15 min at 37°C according to manufacturer’s instructions, followed by inactivation at 75°C for 10 min and purified with GeneJet PCR Purification kit (Thermo Fisher) according to manufacturer’s instructions. The DNA was ligated using T4 DNA Ligase (New England Biolabs) in the appropriate buffer according to manufacturer’s instructions overnight at 16°C. DH5α bacteria were transformed by heat shock at 42°C for 45 seconds. Transformed bacteria were grown on agar plates containing ampicillin, colonies were picked, and DNA was sequenced.

3 μg of each of pcDNA3.1(+)_Geneticin_A009 coding for mouse IL-18Rα, empty pcDNA3.1(+) empty vector, pcDNA3.1(+)_Hygromycin_A069 coding for mouse IL-18Rβ and pcDNA3.1(+)_Hygromycin empty vector were linearized with PvuI (New England Biolabs) or SSpI (New England Biolabs) in the appropriate buffer for 1 h at 37°C. Restriction enzymes were then inactivated at 65°C for 20 min. The vectors were purified using GeneJet PCR purification kit (Thermo Fisher) according to manufacturer’s instructions.

Transfection

RAW 264.7 cell transfections were performed using Lipofectamin 2000 (Invitrogen, Waltham, MA, USA). Briefly, 2.5 μg of linearized plasmids were diluted into 125 μl of serum- and antibiotic-free Opti-MEM (Invitrogen), then combined with 10 μl Lipofectamin 2000 diluted into 125 μl of serum- and antibiotic-free Opti-MEM and allowed to complex at room temperature for 5 min. The DNA/Lipofectamin solution was added to RAW 264.7 cells and incubated at 37°C for 24 h before the replacement by fresh cell medium. After 48 h, selection medium containing either 300 μg/mL Geneticin or 300 μg/mL Geneticin plus 200 μg/mL Hygromycin was used to obtain multiclonal stable cultures. Selection medium was replaced every 48 h until cells became confluent. Cells were removed by pipetting up and down and used for the generation of stably transfected single cell clones by serial dilutions. Wells have been checked for the appearance of single cell clones daily by microscopy. The expression of mouse IL-18Rα and IL-18Rβ was confirmed by flow cytometry (see below). Clones positive for both IL-18Rα and IL-18Rβ were used for subsequent experiments. In addition, RAW 264.7 cells stably transfected with empty vectors were used as controls.

RAW 264.7 cells stimulation assays

Stably transfected RAW 264.7 cells were washed in stimulation medium (DMEM High glucose GlutaMax (Thermo Fisher) supplemented with 10% Fetal Bovine Serum (Pan Biotech, Aidenbach, Germany) and 1% Penicillin/Streptomycin (Thermo Fisher Scientific)). 20000 cells per well in 200 μl cell media were plated in 96-well flat bottom plates and kept overnight at 37°C before stimulation with recombinant murine (rm)IL-18 (5 ng/mL) with or without recombinant mouse IL-18BP isoform d (Creative BioMart, Shirley, USA) (81 ng/mL) with or without anti-IL-18BP antibodies, or 100 ng/mL lipopolysaccharide (LPS) for 24 h. The effect of IL-18 or LPS on stably transfected RAW 264.7 cells was assessed by measuring the levels of TNFα in cell supernatants by ELISA (Thermo Fischer) following the manufacturer’s instructions.

IL-18BP ELISA

Recognition of murine and human IL-18BP by monoclonal anti-IL-18BP antibodies (as described above), and calculation of IL-18BP serum levels in wild-type (WT) mice, were determined with a homemade ELISA, as previously described (16). Briefly, anti-mouse IL-18BP antibodies were coated on 96-well plates (dilution in PBS to 1 μg/mL). Nonspecific binding was blocked with 1% BSA (Sigma-Aldrich, Buchs, Switzerland) in PBS. The range of IL-18BP standard (rmIL-18BPd) was from 20 to 0.156 ng/ml. A goat anti-mouse IL-18BP isoform c–biotinylated Ab (Bio-Techne, R&D Systems, Abingdon, U.K.) was used as detection Ab. The samples and standards were diluted in PBS–1% BSA. Absorbance was measured at 450 nm and normalized to 560 nm using a Ledetect 96-plate reader (Labexim, Lengau, Austria).

Free IL-18 ELISA

Serum free IL-18 levels were determined by a previously described home-made ELISA (20). Briefly, the 96-well plates were coated with recombinant human IL-18BP isoform a (rhIL-18BPa, AB2 Bio, Lausanne, Switzerland) at 5 μg/mL in PBS. Non-specific binding was blocked with 1% BSA (Merck Millipore, Darmstadt, Germany) in PBS 0.1% Tween 20. Tested samples and standards were added to the plate pure, at 1/10 and 1/100 dilutions, and incubated with biotinylated rat monoclonal anti-mouse IL-18 antibody (Medical & Biological Laboratories, Nagoya, Japan) in 0.1 M HEPES (pH 7), 0.35M NaCl, and 0.4% Triton X-100. Absorbance was measured at 450 nm and normalized to 560 nm using a Ledetect 96-plate reader (Labexim).

Free IL-18 levels were measured in the serum of either WT (C57BL/6N) mice pre-incubated with PBS or anti-IL-18BP antibody clones 441 or 445 for 30 min at 37°C, at a molecular weight ratio of antibodies to IL-18BPd of 1:3 (according to serum IL-18BP concentrations measured by ELISA), before the addition of different concentrations of rmIL-18 ranging from 0 to 10000 pg/mL. Serum of IL-18BP KO mice was used as control without the addition of antibodies.

Production of recombinant mIL-18BP

Recombinant murine IL-18BP for biolayer interferometry (BLI) experiments was produced in suspension-adapted HEK293S cells with native glycosylation based on a strategy previously developed for human IL-18BP (17), by transiently transfecting a plasmid encoding for mIL-18BP (UniProt ID: Q9Z0M9) with a caspase-3 cleavable C-terminal His-tag using polyethyleneimine as transfection agent. The cell line was obtained from Prof. N. Callewaert, Unit for Medical Biotechnology, Medical Biotechnology Center, VIB-UGent, Ghent, Belgium. Five days post-transfection, conditioned medium was harvested and clarified via centrifugation and filtered through a 0.22-μm filter prior to chromatographic steps. The standard chromatography running buffer used during protein purification experiments was HBS (20 mM HEPES pH 7.4, 150 mM NaCl) and the IMAC elution buffer was HBS supplemented with 250 mM imidazole. His-tagged mIL-18BP was captured using Immobilized Metal Affinity Chromatography (IMAC) (complete His-tag purification resin, Roche) and polished via size exclusion chromatography (SEC) (Superdex 200 increase 10/300 GL, Cytiva). The His-tag was removed by overnight digestion of the purified protein with in-house caspase-3 at RT. To remove any undigested protein and the protease, the digestion mixture was loaded onto a HisTrap FF column (Cytiva). The flowthrough containing tag-free mIL-18BP was polished on SEC equilibrated on HBS.

Biolayer interferometry experiments

The binding kinetics and dissociation constants of antibody 441 and 445 towards murine IL-18BP were characterized by BLI. Binding assays were performed using an Octet Red 96 machine (Sartorius, Göttingen, Germany) in assay buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 0.1 % (w/v) BSA, 0.05% (v/v) Tween 20) at 25°C. Antibody 441 and 445 were biotinylated using the EZ-Link NHS-PEG4 Biotinylation Kit (Thermo Scientific), and immobilized at 100 nM onto Octet® SA capture biosensors (Sartorius) until an optical shift of 2 nm was achieved. To verify the absence of non-specific binding to the biosensors, non-functionalized biosensors were tested against the different ligand concentrations. After equilibrating the biosensors in assay buffer, the biosensors were dipped in mIL-18BP at concentrations ranging from 12.5 nM to 100 nM for 400 s during the association phase followed by a 1200 s dissociation phase in assay buffer. After subtraction of the references, a 1:1 global model was fitted. Graphs show a representative and the reported kinetic parameters (KD, ka, kd) represent the averages from three technical replicates. Assay design and data acquisition were performed using the BLI Data acquisition software 9.0.0.49 (Sartorius) and data analysis was performed using the Data Analysis software 9.0.0.14 (Sartorius).

Co-immunoprecipitation (co-IP) and serum IL-18BP IP

Adherent HEK293T were transiently transfected with a plasmid encoding for C-terminally His-tagged mIL-18BP and N-terminally V5-tagged mature mIL-18 (UniProt ID: P70380, residues 36-192) in 6-well plates. The secretion signal of RTPμ (Ariscescu et al, 2006) was added to mIL-18 to enable co-secretion with mIL-18BP. Three days post transfection, the conditioned medium was harvested. Dynabeads™ Protein G (Invitrogen) (150 μg, 5 μl) were conjugated with 1 μg of the anti-mIL-18BP antibodies (441-450), after which they were incubated with 250 μl conditioned medium of the HEK293T cells expressing mIL-18BP and mIL-18. The remaining steps were performed according to manufacturer instructions (Invitrogen). Elution from beads was performed with the denaturing buffer containing Laemlli, β-mercaptoethanol and 50 mM glycine pH 2.8. mIL-18BP and mIL-18 were visualized on Western blot using the 6X His Tag Antibody Dylight™ 680 Conjugated (Rockland) and the Anti-V5-HRP Antibody (R961-25, Invitrogen), respectively.

Circulating IL-18BP immunoprecipitation using 441 and 445 antibodies was tested by pulldown experiments using pierce protein A/G magnetic beads (Thermo Scientific, 88802) on plasma from WT and IL-18BP KO mice. Beads were washed and handled according to manufacturer recommendations. Briefly, pre-clearing was performed by the incubation of 100 μl mouse plasma with 20 μl beads 1 h at RT. After discarding the beads, 10 μg of each antibody was added to the plasma for 1 h at 37°C. Next, immunoprecipitation of antibodies was accomplished by adding new pre-washed beads to the samples for 1 h at RT. Beads were then collected and washed twice. Following denaturation and elution, Western-blot was performed, and IL-18BP was detected using our rabbit anti-IL18BP antibody (clone 448 at 1:10000 dilution) and was revealed with a IRDye 800CW goat anti-rabbit (Li-Cor, Bad Homburg, Germany, 926-32211, 1:20000). Images were acquired using the Li-Cor Odyssey XF imaging system (Li-Cor).

Serum levels of IFN-γ, CXCL9 and ferritin

Circulating levels of IFN-γ (Thermo Fisher), CXCL9 (RnD system, Abingdon, U.K.) and ferritin (ALPCO, Salem, NH, USA) were measured by ELISA following the manufacturer’s instructions.

Mouse lines and procedures

C57BL/6N wild-type (WT) mice and Il18bp-/- (IL-18BP KO) mice were previously described (20). Heterozygous Il18bp+/- mice were bred in the conventional area of the animal facility of the Geneva University School of Medicine (Geneva, Switzerland) to obtain homozygous IL-18BP KO and WT mice. IL-18 KO/KI Neongreen reporter mice were generated using CRISPR/Cas9 technology on C57BL/6J ES in the VIB Department of Molecular Biomedical Research (Ghent. Belgium), as previously described (23).

All experiments were performed in 10- to 15-weeks-old, age-matched male and female mice. To induce MAS, a class B phosphorothioate CpG 1826 oligonucleotide (5′-TCCATGACGTTCCTGACGTT-3′; Eurofins Genomics GmbH, Ebersberg, Germany) was injected intraperitoneally (ip) at a dose of 2.5 μg/g of body weight on days 0, 2, and 4. 200 μl of PBS or 200 μg anti-IL-18BP clones 441 or 445 (in 200 μl volume) were injected 2 h before CpG (Eurofins Genomics) injection. One additional injection of 200 μg anti-IL-18BP antibody clones 441 or 445 was performed on day 6. Assessment of clinical readouts were performed up to day 7 when the mice were sacrificed. Blood and organs were collected as previously described (8, 20). All experiments were approved by the Geneva cantonal authority for animal experimentation (licenses GE/92/21, GE/382).

Blood cell counts and Flow cytometry

Blood cell counts were assessed on peripheral blood collected in EDTA-coated vials using a Sysmex blood cell counter (Sysmex, Yverdon, Switzerland).

Selected transfected RAW cells with empty vector, vector encoding the IL-18Rα or IL-18Rβ were detached, washed, and stained with an anti-IL18Rα-PE (P3TUNYA, ebioscience, Waltham, MA, USA) an anti-IL18Rβ (TC30-28E3, BD Biosciences, Fanklin Lakes, NJ, USA), followed by a goat anti-rat-PE as a secondary (Biolegend, San Diego, CA, USA) or an isotype control IgG2a-PE (eBR2a, eBiosciences).

For spleen and blood, single cell suspensions were generated from spleen and blood cells, pelleted by centrifugation, followed by red blood cell lysis. Single cell suspensions from spleen and blood were blocked with FcR blocking reagent (Miltenyi, Bergisch Gladbach, Germany) in 0.5% BSA in PBS, and stained with the following fluorochrome-conjugated antibodies: anti-CD3-PE-CF594 (145-2C11, BD), anti-CD4-BV711 (GK15, BD), anti-CD11b-BV605 (M170, Biolegend), anti-CD19-APC-Fire750 (6D5, Biolegend), anti-CD45-Vioblue (30F11, Miltenyi), anti-MerTK-PE-Cy7 (DS5MMER, eBiosciences), anti-F4/80-PE-Cy7 (BM8, Biolegend), anti-Siglec-H-FITC (551, Biolegend), CD11c-AF700 (N418, Biolegend), NK1.1-AF700 (PK136, BD), anti-HLA-DR-APC (M5/114.15.2, Miltenyi), anti-Ly6C-PerCP-Cy5.5 (HK1.4, Biolegend) and anti-Ly6G-APC-Cy7 (1A8, Biolegend), and analyzed on a LSR II/Fortessa flow cytometer. Data were analyzed using FlowJo V10 (TreeStar, Ashland, OR, USA). All antibodies and secondary reagents were titrated to determine optimal concentrations. Comp-Beads (BD Biosciences) were used for single-color compensation to create multicolor compensation matrices. For gating, fluorescence minus one control were used. The instrument calibration was controlled daily using Cytometer Setup and Tracking beads (BD Biosciences). Gating strategy can be found in the supplementary material (Figure Suppl 1).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA from mouse spleen was extracted using TRIzol (Invitrogen) and RNeasy columns (Qiagen). 500 ng of total RNA was used for reverse transcription with the PrimeScript™ reverse transcriptase (Takarabio, Kusatsu, Japan). Quantitative real-time PCR reactions were performed with a SYBR Select Master Mix and a QuantStudio 6 pro Real-Time PCR System (Thermo Fisher Scientific). Relative mRNA expression was analyzed based on the DDcycle threshold method and normalized to Rpl32 as a housekeeping gene. All primers were purchased from Eurofins (Germany). Primer sequences used are indicated in Table S1.

Statistical analysis

Results are represented as individual values, except for variations from baseline body weights, that are expressed as mean percentage ± SEM. 2way ANOVA test with Dunnett’s correction or Tukey’s correction, One-way ANOVA test with Tukey’s correction and two-tailed Mann–Whitney test have been used to test statistical significance, or a simple linear regression was used as indicated in the figure legends. p-values <0.05 were considered significant. Only statistically significant differences are shown. Outlier, if existing were removed following the use of Grubb’s test. All plots and statistical analyses were performed using GraphPad Prism 10 software (GraphPad Software, La Jolla, CA). Adobe Illustrator (Adobe, San Jose, CA) was used to reformat graphics and figures.

Results

Development of anti-mouse IL-18BPd antibodies and an IL-18 bioassay

Using B cell clones isolated from the peripheral blood of rabbits immunized against mIL-18BP, we obtained 10 monoclonal anti-mIL-18BP antibodies (labeled from 441 to 450) according to a first screen by direct ELISA (data not shown). When these antibodies were tested in a sandwich ELISA as capture antibodies, all of them, but antibody 443, were able to bind recombinant mouse IL-18BPd. In addition, one of these antibodies, clone 447, was also able to recognize hIL-18BPa (Figure 1). Accordingly, the results of pulldown experiments showed that all the antibodies, except from clone 443, were able to bind mouse IL-18BP (Figure 2D). Following evaluation of their ability to bind to mIL-18BPd, we then examined whether these antibodies could interfere with the inhibitory effect of mIL-18BP.

Figure 1. Development of monoclonal antibodies capable of binding to mIL-18BPd.

Figure 1

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Levels of mIL-18BPd (black) or hIL-18BPa (red) were measured using sandwich ELISA in plates coated with nine different anti-IL-18BP clones (441 to 450) as capture antibodies. A biotinylated polyclonal goat anti-mouse IL-18BP isoform c was used for the detection of IL-18BP. The results are presented as optical density values. Recombinant human IL-18BPa was used as control.

Figure 2. Anti-IL-18BP antibodies prevent the binding of IL-18 to IL-18BP.

Figure 2

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(A-C) Stably transfected RAW cells expressing both IL-18Rα and IL-18Rβ. (A) Expression of cell surface IL-18Rα and IL-18Rβ was assessed by flow cytometry on cells transfected with empty vectors (EV, transparent peaks) or vectors encoding IL-18Rα and IL-18Rβ (Trf, full peaks) with antibodies against IL-18Rα (green), IL-18Rβ (red), or an isotype control (grey). (B) Cells transfected with empty vectors or vectors including IL-18Rα and IL-18Rβ (IL-18R) sequences were incubated 24 h with mIL-18 only, mIL-18 together with different concentrations of mIL-18BPd corresponding to different IL-18BPd:IL-18 molecular weight ratios, mIL-18BPd only, LPS, or left unstimulated. (C) IL-18R transfected cells were incubated for 24 h with mIL-18 only, mIL-18 with mIL-18BPd, mIL-18 and mIL-18BPd with different concentrations of anti-IL-18BP antibodies corresponding to anti-IL-18BP antibodies:IL-18BP molecular weight ratios, mIL-18BPd only, anti-IL-18BP antibodies, or unstimulated. Results of B and C represent TNFα levels present in cell supernatants measured by ELISA (D) Table representing the IC50 of each anti-IL-18BP clones. n.d stands for not determined. (E) The conditioned medium of adherent HEK293T cells, transiently transfected with C-terminally His-tagged mIL-18BP and N-terminally V5-tagged mIL-18, was incubated with DynabeadsTM Protein G conjugated with monoclonal anti-mIL-18BP antibodies (Ab441 to Ab450). Co-immunoprecipitation was evaluated by Western blot using an anti-His antibody to detect mIL-18BP and an anti-V5 antibody to detect mIL-18. (B; C) Each data point corresponds to one biological replicate. Data are means ± SEM. *p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. P-values were calculated using One-way ANOVA test with Tukey’s correction.

We created a stably transfected RAW 264.7 cell line with constitutive expression of IL-18Rα/β (Trf). Stably transfected RAW 264.7 cells with an empty vector (EV) were used as control. The cell surface expression of IL-18Rα/β in Trf cells was confirmed by flow cytometry (Figure 2A). To ensure that IL-18Rs were functional, the cells were incubated with different concentrations of mIL-18 (Figure Suppl 2A). We observed that Trf, but not EV, cells responded to IL-18 stimulation with the production of TNFα, and that incubation with mIL-18BP led to a dose-dependent inhibition of the stimulatory effect of IL-18. As expected, both Trf and EV cells responded to LPS stimulation (Figure 2B).

Functional activity of monoclonal anti-IL-18BP antibodies

To examine the effect of anti-IL-18BP antibodies, we used a fixed concentration of IL-18BP that was shown to inhibit most of the stimulatory activity of 5 ng/mL IL-18. IL-18BP and IL-18 were either used alone or after the subsequent addition of antibodies at different molar ratios according to the concentration of IL-18BP. While the antibody clone 441 was devoid of any effect on IL-18BP inhibitory activity, all the other monoclonal antibodies antagonized the effect of IL-18BP leading to significant increased TNFα production as compared to the combination of IL-18 and IL-18BP (Figure 2C). Of note, antibody 444 interfered only modestly with the inhibitory effect of IL-18BP. In contrast, antibodies 445, 447 and 449 were able to exert efficient IL-18BP inhibition even at a low molecular ratio (1:1). The effect of the antibodies on IL-18BP inhibitory activity were used to determine their IC50 values. For antibodies 445, 447 and 449, IC50 values could not be extrapolated due to their high inhibitory activity with the concentrations used in the experiments (Figure 2D). Pulldown experiments were performed using the supernatant of cells expressing both tagged IL-18BP and IL-18. The results showed that antibody 441, and to a lower extent antibody 444, were able to co-immunoprecipitate IL-18, indicating that these antibodies do not interfere, or only weakly, with IL-18BP/IL18 complexes (Figure 2E). Two bands are observed in the gel, that can be explained by different degree of glycosylation as previously shown in mouse and human IL-18BP (15, 17). For the following experiments, we decided to use the antibody clones 445 and 441 considering their opposite biological activity.

We then examined whether the effect of anti-IL-18BP antibody 445 was restricted to preventing the binding of IL-18 to IL-18BP or if it could also act on existing IL-18:IL-18BP complexes. Trf cells were stimulated with mIL-18 alone or together with mIL-18BPd. Anti-IL-18BP antibodies 441 or 445 were added at the same time as IL-18 and IL-18BP on cell cultures (0h), 30 min (0.5 h) or 2 h later. As depicted in Figure 3A, incubation with the antibody 445 reversed the inhibitory activity of IL-18BP even when added later, whereas antibody 441 had no effect. To examine the effect of antibody 445 on endogenous IL-18BP, IL-18 was added into the serum of WT mice at different concentrations without or with the presence of anti-IL-18BP antibodies 441 and 445. IL-18 was then measured using a home-made ELISA that recognizes only free IL-18. Due to the large amount of circulating IL-18BP, we previously showed that detection of free IL-18 is impaired when IL-18 is added into WT mouse serum (20, 21). Our results showed that antibody 445 allows the detection of spiked IL-18 in WT serum to a similar extent as in IL-18BP KO serum, whereas antibody 441 had no effect on the detection of free IL-18 (Figure 3B).

Figure 3. Binding affinity of antibodies 441 and 445 to recombinant mIL-18BP and their relative inhibitory activity against recombinant and endogenous IL-18BP.

Figure 3

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(A) Stably transfected RAW 264.7 cells were unstimulated or stimulated for 24 h with mIL-18 only (5ng/mL), mIL-18 (5 ng/mL) with mIL-18BPd (81 ng/mL) (molecular ratio of IL-18BPd to IL-18 corresponding to 1:3), mIL-18 and mIL-18BPd with anti-IL-18BP antibodies 441 or 445 (molecular ratio of antibodies to mIL-18BPd corresponding to 1:3). Antibodies 441 or 445 were added either at the same time, 0.5 h or 2 h after incubation of the cells with IL-18 and IL-18BPd. TNFα was measured by ELISA. (B) mIL-18 was spiked at concentrations of 0; 0.1; 1 and 10 ng/mL in WT (C57BL/6N) serum without or with antibodies 441 or 445, or in IL-18BP KO serum. Levels of free IL-18 were measured using ELISA. TNFα levels were measured in culture supernatants. (C) BLI traces (blue/red) and 1:1 global fitted model (black) were plotted as the spectral nanometer shift as a function of time upon interaction of mIL-18BP with Ab441 (blue) or Ab445 (red). Biotinylated Ab441 and Ab445 were immobilized on streptavidin biosensors and dipped into wells containing mIL-18BP at concentrations ranging from 12.5 to 100 nM. Graphs show a representative and the reported kinetic parameters (KD, ka, kd) as the averages from three technical replicates. (D) Endogenous IL-18BP was pulldown from plasma of WT or IL-18BP KO mice, using anti-IL-18BP 441 or 445. Pulldown of recombinant IL-18BP in culture medium with both antibodies (Ctrl +) and recombinant mouse IL-18BP (rmIL-18BP) were used as positive controls (A, B). Each data point corresponds to one biological replicate. Data are means ± SEM. *p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (A, B) p-values were calculated using One-way ANOVA test with Tukey’s correction.

We then decided to rule out the possibility that our results comparing antibodies 441 and 445 could be due to differences of IL-18BP binding affinity. Using the biolayer interferometry (BLI) method, we observed that both antibodies 441 and 445 had comparable IL-18BP binding affinity (KD) in the low nanomolar range (Figure 3C). To further examine their ability to bind endogenous IL-18BP, immunoprecipitation studies were performed using the plasma of WT and IL-18BP KO mice as well as recombinant mIL-18BP added into culture media. We observed that both antibodies had the same ability to pull down endogenous IL-18BP (Figure 3D).

Anti-IL-18BP antibody 445 aggravates CpG-induced macrophage activation syndrome

We previously showed that the severity of CpG-induced macrophage activation syndrome (MAS) was markedly increased in IL-18BP KO as compared to WT mice, including more severe cachexia, enlarged spleen and liver size, peripheral blood cytopenia, hyperferritinemia, abnormal liver enzymes, hemophagocytosis and enhanced production of IFN-γ and IFN-γ signature genes (20). We therefore decided to use this model to examine the effect of anti-IL-18BP antibodies. Prior to their use in in vivo studies, both antibodies 441 and 445 were murinized as IgG2a with the addition of a LALAPG mutation in the Fc region to prevent their binding to Fcγ receptors as well as complement activation (22). The activity of these antibodies was validated using the IL-18 bioassay (Figure Suppl 2B). We observed that the manifestations of CpG-induced MAS were significantly aggravated in WT mice injected with antibody 445 as compared to 441 with more pronounced weight loss, splenomegaly, anemia, higher spleen mRNA levels of Ifng, Cxcl9, Ciita, and Il18bp, and serum levels of IFN-γ, CXCL9 and ferritin (Figure 4A-E). As compared to WT mice injected with the anti-IL-18BP antibody 441, the administration of antibody 445 led to a higher number of splenic macrophages and lower numbers of neutrophils, total T cells (both CD4+ and CD8+ T cells), and NK cells (Figure 4F), as well as higher percentages of circulating total DCs and pDCs and lower percentages of Ly6C+ monocytes and total and CD4+ T cells (Figure 4G).

Figure 4. Anti-IL-18BP clone 445 neutralizes IL-18BP activity in the CpG-induced MAS model.

Figure 4

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(A-G) CpG-induced MAS model was induced in WT mice together with the administration of non-blocking anti-IL-18BP clone 441 (blue) or blocking anti-IL-18BP clone 445 (red). (A) Body weight was monitored daily, and the body weight variations are presented from day 0 to day 7. (B) Normalized spleen to body weights at day 7. (C) Blood Red blood cells (RBC) levels were measured at day 7 using the blood numeration system Sysmex. (D) Spleen mRNA levels of Ifng, Il18, Ciita Cxcl9 and Il18bp were measured by RT-qPCR. (E) Serum levels of IFN-γ, CXCL9 and ferritin were determined by ELISA. (F) Normalized levels of myeloid cells, macrophages (Mϕ), dendritic cells (DCs), neutrophils, Ly6C+ monocytes (Mo), B cells, T cells (CD4+ and CD8+) and NK cells from spleen and (G) frequency of DCs, plasmocytoid DCs (pDCs), Ly6C+ monocytes, neutrophils, B cells T cells (CD4+ and CD8+) and NK cells from peripheral blood were measured using flow cytometry (A-G) Each data point corresponds to one animal. Data are means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; **** p < 0.0001. p-values were calculated using (A) 2-way ANOVA test with Tukey’s correction and (B-G) Mann-Whitney test between 441 vs 445.

Specificity of the effects of anti-IL-18BP antibodies 441 and 445

We performed additional experiments to ensure that the effects of antibody 445 were specific and that antibody 441 was devoid of any inhibitory activity. First, both antibodies were administered to naïve WT mice using the same schedule and dosage as in the CpG experiments. As depicted in Figure 5A, body and spleen weights as well as circulating levels of IFN-γ, CXCL9 and ferritin were not different in mice treated with antibodies 445 and 441, respectively. Second, we compared the effect of the administration of antibody 441 and PBS, respectively, in WT mice with CpG-induced MAS. The results showed no difference regarding body and spleen weights and circulating levels of IFN-γ, CXCL9, and ferritin (Figure 5B). Finally, to ensure that both antibodies do not exert any effect in the absence of IL-18 or IL-18BP, antibodies 441 and 445 were injected in IL-18 KO and IL-18BP KO mice with CpG-induced MAS. The results showed that body and spleen weights as well as circulating levels of IFN-γ, CXCL9 and ferritin did not differ in mice treated with antibodies 441 and 445, respectively (Figure 5C, D). Spleen Ifng, Cxcl9, Il18 and Il18bp mRNA levels were consistent with these results (Figure Suppl 3). We examined the effect of antibodies 441 and 445 on splenic immune cell populations (Figure 6A-D) and did not find any difference besides a minor decrease of CD4+ T cell levels in IL-18 KO mice.

Figure 5. Blocking activity of anti-IL-18BP 445 is dependent on the presence of the IL-18/IL-18BP complex.

Figure 5

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The non-blocking anti-IL-18BP clone 441 (blue) or the blocking anti-IL-18BP clone 445 (red) were administered to naïve WT mice and in the CpG-induced MAS model in WT, IL-18 KO and IL-18BP KO mice. Left panels represent the evolution of body and spleen weights, right panels represent the serum levels of IFN-γ, CXCL9 and ferritin. (A) Administration of the non-blocking anti-IL-18BP clone 441 or the blocking anti-IL-18BP clone 445 in naïve WT mice. (B) Administration of antibody 441 or PBS (black curve and white bars) in WT with CpG-induced MAS. (C) Administration of the non-blocking anti-IL-18BP clone 441 or the blocking anti-IL-18BP clone 445 in IL-18 KO mice with CpG-induced MAS. (D) Administration of the non-blocking anti-IL-18BP clone 441 or the blocking anti-IL-18BP clone 445 in IL-18BP KO mice with CpG-induced MAS. Body weight was monitored daily, and the body weight variations are presented from day 0 to day 7, and the spleen was normalized to the body weights at day 7. Serum levels of IFN-γ and CXCL9 and ferritin were determined by ELISA. Each data point corresponds to one animal. Data are means ± SEM.

Figure 6. Splenic immune cell content is not affected by the blocking activity of anti-IL-18BP 445 in absence of the IL-18/IL-18BP complex.

Figure 6

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The non-blocking anti-IL-18BP clone 441 (blue) or the blocking anti-IL-18BP clone 445 (red) were administered to naïve WT mice and in the CpG-induced MAS model in WT, IL-18 KO and IL-18BP KO mice. Normalized levels of splenic myeloid cells, macrophages (Mϕ), dendritic cells (DCs), neutrophils, Ly6C+ monocytes (Mo), B cells, T cells (CD4+ and CD8+) and NK cells at day 7. (A) Administration of the non-blocking anti-IL-18BP clone 441 or the blocking anti-IL-18BP clone 445 in naïve WT mice. (B) Administration of antibody 441 or PBS (white) in WT with CpG-induced MAS. (C) Administration of the non-blocking anti-IL-18BP clone 441 or the blocking anti-IL-18BP clone 445 in IL-18 KO mice with CpG-induced MAS. (D) Administration of the non-blocking anti-IL-18BP clone 441 or the blocking anti-IL-18BP clone 445 in IL-18BP KO mice with CpG-induced MAS. Each data point corresponds to one animal. Data are means ± SEM. *p < 0.05. p-values were calculated using Mann-Whitney test.

Discussion

In this study, we reported the development and characterization of novel anti-mouse IL-18BP antibodies. Two of these antibodies, clones 441 and 445, were further examined and showed distinct activities. Whereas both antibodies were able to bind recombinant mIL-18BP with comparable affinity and pulldown endogenous plasma IL-18BP with the same efficacy, an in vitro bioassay and an in vivo model of systemic inflammation dependent on IL-18 signaling showed that antibody 445 was able to fully antagonize the regulatory activity of IL-18BP in contrast to antibody 441. Antibody 441 did not antagonize the IL-18-IL-18BP complex as demonstrated by its ability to co-immunoprecipitate IL-18.

The biological activity of IL-18 is tightly regulated by a natural soluble decoy receptor, IL-18BP, that binds to IL-18 with high affinity (Kd ~ 100 pM) (17). Circulating IL-18BP is present at high concentrations in humans and mice, thus sequestering excessive IL-18 activity under physiological conditions. In fact, free IL-18 is undetectable under homeostatic physiologic conditions and most inflammatory diseases (4, 8). Extraordinarily elevated levels of IL-18 are present in some specific conditions such as Still’s disease, macrophage activation syndrome and NLRC4 gain-of-function mutations associated with MAS and enterocolitis (7). Elevated IL-18 concentrations in these conditions overwhelm the binding ability of IL-18BP, allowing the presence of free IL-18 in the circulation. Free IL-18 levels correlate with signs of disease severity and these patients respond to therapy with recombinant IL-18BP (7). The CpG-induced MAS model used in our studies recapitulate the findings of patients with MAS (24). We previously showed that injections of CpG led to an aggravated form of MAS in IL-18BP KO mice with the presence of detectable serum free IL-18. Furthermore, clinical and laboratory manifestations correlated with serum levels of free IL-18 and the administration of anti-IL-18Rα antibodies abrogated the severity of MAS (20). The results included in our studies showed that we were able to recapitulate the phenotypic findings of IL-18BP deficient mice by using a neutralizing anti-IL-18BP antibody, without any off-target effects. Nevertheless, as the CpG model of MAS cannot fully recapitulate the complexity of the human disease, caution should be taken with the interpretation of our results. Thus, it will be important to assess the effect of these antibodies in other IL-18 dependent disease models.

Although the antibodies were generated by immunizing rabbits against mouse IL-18BP isoform d, our results demonstrate that antibody 445 is able to neutralize all IL-18BP activity, thus indicating that it binds to both IL-18BPc and d. Importantly, both IL-18BP isoforms share 85% amino acid sequence homology differ by just 25 amino acid in their C-terminal region due alternative use of exon 7 and mRNA splicing (15).

The beneficial effect of targeting cytokine antagonists seems counterintuitive due to the risk of developing severe inflammatory manifestations. However, the equilibrium between agonists and inhibitors is involved in the development of appropriate responses against pathogens. Recent findings have demonstrated that targeting IL-1 receptor antagonist (IL-1Ra) enhances host responses against pathogens leading to a better outcome. In the case of disseminated infection with Candida albicans, splenic and tissue macrophage-derived IL-1Ra secretion impedes neutrophil recruitment and impairs pathogen containment. The ablation of macrophage production of IL-1Ra using cell-specific KO mice or the administration of neutralizing anti-IL-1Ra antibodies improved the neutrophil function and had a beneficial effect on the infection-associated lethality (25). Similarly, enhancement of IL-1 signaling by targeting the macrophage production of IL-1Ra led to a better bacterial control in three models of Mycobacterium tuberculosis infection (26). Depletion of ST2+ regulatory T cells producing IL-1Ra or genetic ablation of IL-1Ra expression by these cells led to increased neutrophils influx and improved survival to influenza (27).

IL-18 plays a central role in host defense via its ability to stimulate the production of IFN-γ by T and NK cells. IL-18 also induces CD8+ T cell- and NK-mediated cytotoxicity (28, 29). Considering these effects and the results of preclinical studies demonstrating that IL-18 exerted anti-tumoral activities, phase I and II clinical trials were conducted in patients with cancer. Overall, the efficacy was very limited although some promising results were reported in a minority of patients (30). It is plausible that these disappointing results were related to the presence of high levels of IL-18BP in response to an IFN-γ-mediated negative feedback mechanism. Therefore, strategies aimed at targeting the inhibitory effect of IL-18BP have emerged to improve the anti-tumoral effect of IL-18. The administration of a mutated form of recombinant IL-18 that does not bind to IL-18BP but retains its ability to bind to IL-18R showed promising effects in models of cancer in mice (18). Recently, it has been demonstrated that the administration of a monoclonal anti-IL-18BP antibody in mice implanted with cancer cells displayed marked beneficial effects by increasing immune responses against cancer cells (19). Most importantly, blockade of IL-18BP was not associated with any safety signals, most likely because these antibodies exerted their effects in the tumor environment without significant leakage of IL-18 in the circulation. Therefore, this strategy emerges as a promising intervention in oncology but may also improve responses to vaccination. Despite these encouraging results, more preclinical data are absolutely required prior to the start of a therapeutic program to ensure that targeting IL-18BP with neutralizing antibodies is safe and will not ultimately lead to uncontrolled systemic hyperinflammation. We previously showed that the administration of IL-36γ, another IL-1 cytokine with Th1 activity, enhanced humoral responses to vaccines in adult mice. Unfortunately, this strategy was toxic in neonate mice due to the occurrence of a massive TNFα-dependent cytokine storm (31).

Supplementary Material

Supplementary Material

Key Points.

  • We generated anti-mouse IL-18BP antibodies with and without neutralizing activities

  • A neutralizing anti-IL-18BP antibody recapitulates the phenotype of IL-18BP KO mice in response to CpG injections

Acknowledgements

We thank Prof. Gaby Palmer for her helpful scientific advice.

Abbreviations

AIFEC

Autoinflammation with infantile enterocolitis

BLI

Biolayer interferometry

DCs

dendritic cells

EV

empty vector

NHS-PEG4

N-hydroxysuccinimide ester-polyethylene glycol 4

IL-18BP

IL-18 binding protein

IL-1Ra

IL-1 receptor antagonist

IP

immunoprecipitation

KI

knock in

KO

knock out

MAS

macrophage activation syndrome

Mϕ

macrophage

Mo

monocyte

NLRC4

NLR family CARD domain-containing protein 4

pDCs

plasmocytoid dendritic cells

RT

room temperature

Trf

transfected

WT

wild type

Footnotes

Contribution

A.H., S.F.D., J.G., P.M., A.D.-B., J.A., S.N.S., and CG designed experiments. A.H., S.F.D., J.G., P.M., M.J., A.C., E.R., J.A. performed experiments. A.H., S.F.D., J.A. and S.N.S. analyzed results. A.H. and C.G. wrote the manuscript, and all authors reviewed and critically edited the manuscript

Grant support

This study was supported by the Swiss National Science Foundation grant No 310030B-201269, the ERA-NET + EJP (Cure-AID) grant No 31ER30_179596, and the Rheumasearch Foundation).

J.A. was supported by a pre-doctoral fellowship from Research Foundation Flanders, Belgium (FWO grant nr. 1S83421N). S.N.S. is supported by the Flanders Institute for Biotechnology (VIB), Belgium (grant nr. C0101).

Conflict-of-interest disclosure: The authors declare no competing financial interests.

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