Introduction

Evidence from basic research and clinical trials demonstrates that the interleukin (IL) 17 immune axis exerts distinct biological effects dependent on the tissue or disease context.1 IL-17-producing T cells (Th17) and innate immune cells (including neutrophils, monocytes and macrophages) play key protective roles in the immune response to various microbial pathogens.2 However, IL-17-driven responses are responsible for tissue damage linked to infection-associated immunopathology and can result in the development of immune-mediated inflammatory diseases (IMIDs),3 4 such as psoriasis, psoriatic arthritis (PsA), rheumatoid arthritis (RA), inflammatory bowel disease (IBD) and ankylosing spondylitis (AS).5 Dysregulation of IL-17A (and in a less extent, IL-17F and IL-17A/F) production and/or binding to its receptor(s) have been associated with IMID pathology,6 making this complex an attractive target for therapeutic interventions.7 Indeed, secukinumab, ixekizumab (anti-IL-17A antibodies) and bimekizumab (anti-IL-17A/F antibody) are already effective in treating plaque psoriasis, PsA and AS.7 Despite the potent blockade of cytokine signalling offered by these biological therapies, many patients have only partial or transient responses associated with various side effects. Therefore, identifying potential novel therapeutic targets or optimising those already available is urgently needed and will likely have a significant clinical benefit.5 8

IL-17 is composed of six family members, of which IL-17A and IL-17F are predominantly involved in driving inflammatory responses.4 Convincing evidence indicates that IL-17A/F use their C-terminal sequence to bind the heterodimeric receptor IL-17RA and IL-17RC.9 On binding, this receptor complex recruits the ubiquitin ligase Act-1 (via the SEF/IL-17R domain),10 which in turn recruits tumour necrosis factor α (TNF-α) receptor-associated factor 6 (TRAF6), leading to the activation of nuclear factor kappa B (NFκB) and the mitogen-activated protein kinase pathways. Activation of these pathways generates a plethora of inflammatory mediators, such as IL-1α/β, IL-6, IL-8 and TNF-α,1 11 12 which contribute to pathological processes in various IMIDs. Identification of the key active amino acids in the C-terminal sequence of IL-17A/F could, therefore, be critical in generating a more biologically active neutralising antibody with reduced off-target effects.13

Exploring murine and human IL-17A/F protein sequences, we have identified an essential 20-mer IL-17-derived peptide (nIL-17) that is responsible for the bioactivity of IL-17A, IL-17F and/or IL-17A/F heterodimer, mimicking a range of actions elicited by the full-length cytokines. Specifically, we demonstrate that nIL-17 activates IL-17RA/C-dependent intracellular signalling to induce activation of NIH-3T3 mouse embryonic fibroblast cells and human dermal blood endothelial cells (HDBECs) leading to increased cytokine, chemokine and adhesion molecules expression. Additionally, nIL-17 promoted leucocyte recruitment to pre-inflamed tissues in vivo (air pouch model) and in vitro (to inflamed endothelium). Subsequently, we developed a monoclonal neutralising antibody (Ab-IPL-IL-17) targeting nIL-17, which effectively reversed the actions of nIL-17 leading to reductions in chemokine, cytokine and adhesion molecule levels on target cells, as well as reducing the inflammatory infiltrate. Finally, we compared the therapeutic efficacy of Ab-IPL-IL-17 with reference anti-IL-17 antibodies in preclinical models of IMIDs, specifically arthritis and IBD. Crucially, Ab-IPL-IL-17 exhibited significantly more neutralising activity limiting inflammation and disease progression, with lower immunogenicity and adverse haematological side effects when compared with reference antibodies. Future studies and clinical trials will need to address the varying requirements of Ab-IPL-IL-17 as an alternative biological therapy for treating patients with IMIDs.

ResultsCharacterising the bioactive sequence in IL-17A and IL-17F

To identify the bioactive portion within IL-17A and IL-17F, we designed a series of peptides (online supplemental figure S1A–C), of different lengths, which mimic the C-terminal region of IL-17A/F, considered essential in the interaction with the receptor counterpart. These truncated peptides would meet the affinity/receptor interaction requirements of both murine and human IL-17A/F for their cognate receptors.3 4 Subsequently, we assessed the ability of these peptides to mimic the actions of native IL-17A, IL-17F and IL-17A/F heterodimer to induce IL-6 production from a murine embryonic fibroblast cell line NIH-3T3.14 Fibroblasts are a major cellular target for IL-17A, leading to the generation of several inflammatory cytokines (eg, IL-6 and IL-8), which drive the local inflammatory response.9 We found that only peptide 2 (named nIL-17) was able to promote IL-6 release to a greater extent when compared with both IL-17A and IL-17F at similar molar concentrations (from 0.610 nM to 0.725 nM) (figure 1A, online supplemental figure S1D). nIL-17A also displayed similar biological activity to the recombinant full-length native IL-17A/F heterodimer (figure 1A).

Figure 1

Biological characterisation of a novel IL-17-derived peptide (nIL-17). (A) Amino acids sequence of nIL-17 was obtained after a study of primary structures of both mouse/human IL-17A and IL-17F. (B) To assess the biological activity of nIL-17 peptide, IL-6 production was evaluated in NIH-3T3 cell supernatants following 24 hours of incubation in the presence of either IL-17A protein (50 ng/mL), IL-17F protein (50 ng/mL), IL-17A/F heterodimer (50 ng/mL), nIL-17 (50 ng/mL), nIL-17 (50 ng/mL) with terminal NH2 sequence, denatured (–DN) form or ‘scrambled’ (–SC) sequence (both at 50 ng/mL). (C)–(D) Whole cell lysates from NIH-3T3 cells stimulated with IL-17 or nIL-17 (50 ng/mL) were analysed, by western blot, for IL-17RA (~120 kDa), IL-17RC (~110 kDa), Act-1 (~72 kDa), NFκB (~65 kDa) and actin (~42 kDa) expression. Representative western blot images are shown from three pooled experiments with similar results. (E) To evaluate the binding interaction of nIL-17 with IL-17RA and IL-17RC, biotinylated IL-17 and nIL-17 (0–750 ng/mL) were co-incubated for 30 min with IL-17RA-Fc or IL-17RC-Fc prior to fluorescence being measured. (B)–(E) Data are presented as mean±SD of n=3 independent experiments. (F)–(G) Macrophages, derived from primary human CD14+ monocytes, were stimulated with LPS and IFN-γ (M1 stimuli) for 16 hours. Following differentiation, M1 macrophages were treated with IL-17 vehicle, IL-17 or nIL-17 (100 ng/mL) for 24 hours. Supernatants from all experimental conditions were assayed by ELISA for (F) IL-6 and (G) TNF-α. (H) Transwell chemotaxis assay was employed to determine the chemotactic activity of nIL-17. M199 media (final volume: 700 µL) was added to the bottom well of a Transwell-24 permeable support with 3.0 µm pores with IL-17 (10–500 ng/mL), nIL-17 (10–500 ng/mL) or fMLP (10−6 M as positive control). Neutrophils were added to the top chamber, which had a confluent stimulated (TNF-α and IFN-γ) HDBEC monolayer. (H) After 2 hours of incubation at 37°C, neutrophils were collected from the bottom of the wells and quantified using flow cytometry. (F)–(H) Data are presented as mean±SD of n=3 healthy donors. Statistical analysis was performed using the one-way analysis of variance test followed by Bonferroni. #p≤0.05, ##p≤0.01, ###p≤0.001, ####p≤0.0001 vs vehicle group; *p≤0.05, **p≤0.01, ***p≤0.001 vs IL-17s group. fMLP, formyl-methionyl-leucyl-phenylalanine; HDBEC, human dermal blood endothelial cell; IFN-γ, interferon gamma; IL-17, interleukin 17; LPS, lipopolysaccharide; NFκB, nuclear factor kappa B; TNF-α, tumour necrosis factor α.

Furthermore, our in vitro cytotoxic examination revealed a safe profile for nIL-17 in tested concentration on NIH-3T3 cell lines (online supplemental figure S2). Moreover, modifications through the replacement of carboxy (–CO2H) with amide (–CONH2) group at C-terminal (nIL-17A-NH2), removal of any tertiary structure by denaturing the peptide (nIL-17A-DN) or scrambling the amino acid sequence (nIL-17A-SC) had no effect on IL-6 production by NIH-3T3 (figure 1B). These data demonstrate for the first time that a 20-mer sequence from both murine and human IL-17A/F is responsible for IL-17s biological activity.

IL-17A, and to a lesser extent IL-17F and the heterodimer IL-17A/F, binding to the IL-17 receptor complex (IL-17RA/RC) leads to the recruitment of Act-1/TRAF6 and, ultimately, activation of inflammatory transcription factors via NFκB to induce gene expression.15 Indeed, we found that nIL-17 further amplified the expression of both Act-1 and NFκB, but not IL-17RA or IL-17RC, when compared with full-length native IL-17A protein (figure 1C,D; online supplemental figure S3). A similar observation was reported in mouse embryonic fibroblasts (NIH-3T3) and mouse macrophages (J774A.1) where both IL-17RA and IL-17RC expression remained unchanged following treatment with IL-17A.14 16 To confirm peptide–receptor interactions, biotinylated native IL-17A and IL-17F protein or nIL-17A were incubated with either mouse or human IL-17RA or IL-17RC and binding was assessed. nIL-17A displayed similar binding profiles to both receptors as was seen with full-length IL-17A/F (figure 1E; online supplemental figure S4, respectively).

To gain a better understanding of how nIL-17 binds IL-17RA and IL-17RC receptors at an atomic level, molecular docking studies were performed. The 3D structure of nIL-17 peptide was predicted using the PEP-FOLD4 computational tool17 and experimentally analysed by circular dichroism (online supplemental figure S5-I-A). Using the BeStSel method,18 we selected a structural model of nIL-17 that consisted of two β-strands followed by a short α-helix (online supplemental figure S5-I-B; online supplemental auxiliary table 1). This model showed the closest structural similarity to the receptor-binding region of human IL-17A. The three-dimensional (3D) structures of IL-17RA and IL-17RC proteins were obtained from the crystal structure of the two receptors in complex with human IL-17A (protein data bank id: 7ZAN), also considering the similarity between IL-17RC isoform 1 used in our biological tests and isoform 2 present in the 3D structure (online supplemental auxiliary table 2). Subsequent docking analysis predicted that nIL-17 interacts with IL-17RA and IL-17RC in a manner similar to that of the C-terminal region of IL-17A, whose sequence it mimics (online supplemental figure S5-II). In particular, nIL-17 interacts with the IL-17RA binding pocket between two type III fibronectin domains, with D1 binding the N-terminal region of nIL-17 and D2 interacting mainly with the C-terminal α-helix (online supplemental figure S5-III). While the predicted binding of nIL-17 was mostly superimposable with IL-17A homodimer, there were significant differences in the positioning of nIL-17 N-terminal region and in the interaction with the receptor amino acids (online supplemental figure S5-III-B,C). The predicted binding mode of nIL-17 with IL-17RC was similar to that seen for IL-17RA, except no interactions between the N-terminal region of nIL-17 and the D1 domain were predicted (online supplemental figure S5-IV). The higher affinity of nIL-17 for IL-17RA compared with the other peptides and the IL-17A C-terminal region may be attributable to the multiple interactions formed with the D2 domain that anchors the peptide to the receptor. This binding mode may be possible due to the peculiar structural conformation of nIL-17, permitting closure of the binding site between the two type III fibronectin domains (online supplemental auxiliary figure 1), thereby improving the biological activity of nIL-17 compared with other peptides (online supplemental figure S1). Conversely, the limited interaction between nIL-17 and IL-17RC may in part be due to the lack of closure of the binding cavity (online supplemental auxiliary figure 2), which may explain the differential binding activity of nIL-17 toward the two receptors. Collectively, these data support our hypothesis that the bioactive region within both IL-17A and IL-17F meets the affinity/receptor interaction requirements of both murine and human cognate receptors.

Figure 2

nIL-17 promotes leucocyte recruitment in vivo and migration in vitro. To evaluate the pro-inflammatory activity of nIL-17, we used a subchronic model of inflammation, the dorsal air pouch. (A) Mice were treated with IL-17 vehicle (0.5% CMC), IL-17 (1 µg/pouch) or nIL-17 (1 µg/pouch). (B) Total CD45+ leucocyte numbers were quantified by flow cytometry. (B) Data are presented as means±SD of n=7 mice per group. (C)–(E) Inflammatory supernatants obtained from pouch cavities were assayed using a Proteome Profiler Cytokine Array. (F) Densitometric analysis is presented as a heat map with dots indicating the most significant modulated cyto-chemokines mediators. (F) Data are presented as means±SD.D. of positive spots from three independent experiments run each with n=7 mice per group pooled. (G)–(I) To determine the impact of nIL-17 on leucocyte adhesion and transmigration on HDBEC, a static migration assay was used. HDBECs were treated with IL-17 vehicle (HCl 4 mM PBS), IL-17 (100 ng/mL) or nIL-17 (100 ng/mL), alone or in combination with TNF-α (100 U/mL) for 24 hours. (G) Representative images of the static adhesion assay are shown (200 µm magnification). PBMCs were added for 20 min on stimulated HDBEC, followed by washing to remove all non-adherent cells. Phase bright PBMCs were considered (H) adherent (red arrow), whereas phase-dark were quantified as (I) transmigrated (% of adherent cells) (orange arrow). (J)–(K) VCAM-1 and ICAM-1expression on HDBECs was quantified by flow cytometry. (H)–(K) Data are presented as means±SD of n=3 independent healthy donors. Statistical analysis was conducted by one or two-way analysis of variance test followed by Bonferroni’s correction for multiple comparisons. #p≤0.05, ##p≤0.01, ###p≤0.001, ####p≤0.0001 vs vehicle group; ****p≤0.0001 vs IL-17 group; +++p≤0.001 vs TNF-α group. CMC, carboxymethyl cellulose; HDBECs, human dermal blood endothelial cells; ICAM-1, intercellular adhesion molecule-1; IL-17, interleukin 17; PBMCs, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; TNF-α, tumour necrosis factor α; VCAM-1, vascular cell adhesion molecule-1.

Table 1

Haematological parameters of vehicle, Ab-IPL-IL-17, MAB421 and secukinumab-treated mice

nIL-17 is a potent activator of inflammatory response

IL-17A, and in part IL-17F, can modulate a variety of leucocyte functions, having a broad and wide-ranging impact on inflammatory responses.19 In the context of the myeloid lineage, it has previously been shown that treatment with IL-17A can amplify the production of inflammatory cytokines from human M1 macrophages.20 In agreement with this study, nIL-17 significantly increased IL-6 and TNF-α release from M1 macrophages to a similar degree as seen with native full-length IL-17A (figure 1F,G). In line with previous studies,21 this response was specific for M1 macrophages, and not seen in M0 or M2 macrophages (online supplemental figure S6A,B). Furthermore, there were no intrinsic differences in IL-17RA or IL-17RC expression following IL-17A or nIL-17 treatment on any of the macrophage subsets (online supplemental figure S6C–E). These data demonstrate that nIL-17 retains a similar inflammatory amplification activity as that observed with the native full-length protein.

In the context of inflammatory cell recruitment, IL-17A, but not IL-17F, can directly function as a chemotactic agent for neutrophils promoting their entry into inflamed tissues.22 Moreover, IL-17A acts synergistically with TNF-α to increase the expression of neutrophil capture receptors (E-selectin and P-selectin) and presentation of neutrophil specific chemokines (chemokine C–X–C motif ligand (CXCL) 1, 2, 8) by HDBEC.23 We, therefore, assessed the chemotactic potential of nIL-17 to drive neutrophil migration through inflamed endothelial cells (ECs) (figure 1H). Notably, we found that nIL-17 significantly increased neutrophil migration in a concentration-dependent manner, unlike native IL-17A (figure 1H). Interestingly, 500 ng/mL nIL-17 had a greater chemotactic capacity to drive neutrophil migration when compared with native IL-17A at the same concentration (figure 1H).

To validate these findings in vivo, we used the myeloid-driven subchronic model of inflammation, the mouse dorsal air pouch (figure 2A), where we previously demonstrated that IL-17A preferentially increases the recruitment of pro-inflammatory Ly6Chi monocytes and Gr1+ neutrophils in acute and chronic inflammatory settings.16 24 25 Native murine IL-17A or nIL-17 were administered on day 6 following establishment of the air pouch. We observed a significant increase in CD45+ leucocytes recruited in response to IL-17A, which was further exacerbated (~48%) in the presence of nIL-17 (figure 2B). Moreover, in the presence of a commercially available neutralising IL-17A monoclonal antibody (MAB421), this effect was lost (online supplemental figure S7). Such effects have been previously described in models of endotoxin-induced lung inflammation.26 Interestingly, blocking the neutrophil (IL-8/keratinocyte-derived cytokine (KC)) or monocyte (JE/monocyte chemoattractant protein-1 (MCP-1)) chemokines simultaneously with administrating nIL-17 significantly impaired leucocyte recruitment to the air pouch (online supplemental figure S7), indicating that nIL-17 drives neutrophil and monocyte infiltration via indirect release of these chemoattractants.16 Indeed, previous reports have shown that IL-17A-driven neutrophil transmigration (through TNF-α-stimulated murine ECs or resting human lung microvascular ECs in vitro) was completely abolished when chemokine receptor (CXCR)2-/- neutrophils were perfused over the cultures23 or when cultures were treated with neutralising antibodies against CXCL8/IL-8.27 We further corroborated this idea through proteome analysis of cyto-chemokines released locally in response to either IL-17A or nIL-17 (figure 2C–F). Importantly, nIL-17 augments the amount of several pro-inflammatory mediators, such as IL-8/KC, MCP-1/JE, soluble intercellular adhesion molecule 1 (sICAM1),28 when compared with native IL-17A (figure 2F). Others have reported similar IL-17-induced increases in inflammatory mediators (KC, JE, interferon gamma (IFN-γ), IL-1α, sICAM1, macrophage inflammatory proteins and IL-6) within a variety of tissues, including pouch cavities, brain, blood and aorta.16 29 30 Taken together, these data reinforce our earlier observations that nIL-17 is a more potent pro-inflammatory stimulus than native full-length IL-17A protein and confirm that this peptide truly represents the most biological active sequence of this cytokine.

nIL-17 amplifies EC activation to support leucocyte trafficking

IL-17 receptors are expressed by both haematopoietic cells of the immune system and by stromal cells, such as HDBEC.27 31 IL-17A synergistically amplifies HDBEC response to TNF-α, further increasing expression of the adhesion molecules ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1).23 27 Given this, the effects we describe above for nIL-17 could be due to a direct effect on the leukocytes or the HDBEC alone or on both cell types. To address this, we analysed the adhesion (phase bright) and transmigration (phase dark) of peripheral blood mononuclear cells (PBMC) across inflamed ECs following the addition of IL-17A or nIL-17 (figure 2G). As we have previously published,32 TNF-α stimulation enhanced PBMC adhesion, but this was not further amplified in the presence of either IL-17A protein or nIL-17 peptide (figure 2H). In agreement with our findings, IL-17 did not enhance absolute numbers of adherent leucocytes (neutrophils) to TNF-α stimulated endothelium in vitro or in vivo, but rather altered cellular behaviour, increasing the number undergoing transmigration.23 Like earlier findings, nIL-17 exacerbated PBMC migration through inflamed endothelium to the same extent as seen with full-length IL-17A (figure 2I). PBMC capture and firm adhesion are mediated through E-selectin and VCAM-1 expressed by the inflamed endothelium,32 with VCAM-1 levels remaining unaffected by the addition of either IL-17A protein or peptide in combination with TNF-α when compared with TNF-α alone (figure 2J) in agreement with previous publications.23 Of note, neither IL-17A protein nor peptide induced VCAM-1 expression in the absence of TNF-α. By contrast, PBMC transmigration is dependent on β2-integrins binding endothelial ICAM-1,33 with the protein for the latter being synergistically elevated by both IL-17A or nIL-17 (figure 2K). No differences were observed in IL-17RA or RC expression on ECs under any conditions used (online supplemental figure S8A,B). These findings demonstrate that nIL-17 enhances endothelium activation in response to inflammation, to further amplify leucocyte migration.

nIL-17 specific antibody (Ab-IPL-IL-17) displays potent neutralising activity

Given that nIL-17 clearly demonstrates a more prominent inflammatory activity than full-length IL-17A/F, we embarked on generating a novel IL-17 neutralising antibody: Ab-IPL-IL-17 targeting the nIL-17 sequence (IT patent no.102022000016722). Ab-IPL-IL-17 significantly decreased IL-6 production from (commercially available) IL-17A homodimer and nIL-17-stimulated NIH-3T3 cells in a concentration-dependent manner (figure 3A) without any cytotoxic effect for all tested concentrations on murine embryonic fibroblast cell lines (online supplemental figure S9); reduced binding affinity of IL-17A to IL-17RA or RC (figure 3B); blocked the production of IL-6 and TNF-α from IL-17A-treated M1 macrophages (figure 3C,D, respectively) and decreased IL-17A-induced neutrophil migration (figure 3E). In vivo, Ab-IPL-IL-17 simultaneously administered with IL-17A reduced influx of total CD45+ leucocytes into the inflamed air pouch (figure 3F), with a corresponding reduction observed in several cyto-chemokines (figure 3G). Similar observations were made in vitro, where pretreatment with Ab-IPL-IL-17 significantly reduced both PBMC and peripheral blood lymphocyte adhesion to and transmigration through IL-17 + TNF-α treated endothelium (figure 3H–J and online supplemental figure S10, respectively), which was mirrored by decreased HDBEC expression of VCAM-1 and ICAM-1 (figure 3K,L).

Figure 3

Biological characterisation of a novel IL-17 neutralising antibody (Ab-IPL-IL-17). (A) To assess the biological activity of Ab-IPL-IL-17, IL-6 production was evaluated in NIH-3T3 cell supernatants following 24 hours treatment with IL-17 (50 ng/mL) or nIL-17 (50 ng/mL) alone or in combination with Ab-IPL-IL-17 (75–750 ng/mL). (B) To analyse the neutralisation effect of Ab-IPL-IL-17 on IL-17/IL-17Rs interactions, biotinylated IL-17 (EC50 concentrations) and Ab-IPL-IL-17 (0–750 ng/mL) complex was co-incubated for 30 min with IL-17RA-Fc or IL-17RC-Fc prior to fluorescence being measured. (A)–(B) Data are presented as mean±SD of n=3 independent experiments. (C)–(D) Macrophages, derived from primary human CD14+ monocytes, were stimulated with LPS and IFN-γ (M1-stimuli) over 16 hours. Following differentiation, cells were treated with IL-17 vehicle, IL-17 (100 ng/mL) alone or in combination with Ab-IPL-IL-17 (10 µg/mL) for 24 hours. Supernatants from all experimental conditions were assayed by ELISA for (C) IL-6 and (D) TNF-α. (E) For the transwell chemotaxis assay, neutrophils were added to the top chamber, which had a confluent stimulated (TNF-α and IFN-γ) HDBEC monolayer. (E) Chemotactic migration to IL-17 (500 ng/mL) alone or in combination with Ab-IPL-IL-17 (10 µg/mL) was quantified using flow cytometry. (C)–(E) Data are presented as means±SD of n=3 independent healthy donors. (F) For in vivo experiment, mice were treated with IL-17 vehicle (0.5% CMC), IL-17 (1 µg/pouch) alone or in co-administration with Ab-IPL-IL-17 (10 µg/mL), and thereafter total CD45+ leucocyte numbers were quantified by flow cytometry. (F) Data are presented as means±SD of n=7 mice per group. (G) Inflammatory supernatants obtained from the pouch cavities were assayed using a Proteome Profiler Cytokine Array. Densitometric analyses are presented as a heat map indicating the most significant modulated cyto-chemokines mediators. (G) Data are presented as means±SD of positive spots of three separate independent experiments run each with n=7 mice per group pooled. (H)–(J) HDBECs were treated with IL-17 vehicle (HCl 4 mM PBS), IL-17 (100 ng/mL) plus TNF-α (100 U/mL) alone or in combination with Ab-IPL-IL-17 (10 µg/mL) for 24 hours. Phase bright PBMCs were considered (H) adherent (red arrow), whereas phase-dark were quantified as (I) transmigrated (% of adherent cells) (orange arrow). (J) Representative images of the static adhesion assay are shown (200 µm magnification). (K)–(L) VCAM-1 and ICAM-1 expression on HDBECs was quantified by flow cytometry. (H)–(L) Data are presented as means±SD of n=3 independent healthy donors. Statistical analysis was conducted by one or two-way analysis of variance test followed by Bonferroni’s for multiple comparisons. ##p≤0.01, ###p≤0.001, ####p≤0.0001 vs vehicle group; *p≤0.05, **p≤0.01, ****p≤0.0001 vs IL-17 group; §§p≤0.01, §§§p≤0.001, §§§§p≤0.0001 vs nIL-17 group; +p≤0.05, ++p≤0.01, ++++p≤0.0001 vs IL-17+TNF-α group. CMC, carboxymethyl cellulose; HDBECs, human dermal blood endothelial cells; ICAM-1, intercellular adhesion molecule-1; IFN-γ, interferon gamma; IL-17, interleukin-17; LPS, lipopolysaccharide; PBMCs, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; TNF-α, tumour necrosis factor α; VCAM-1, vascular cell adhesion molecule-1.

Ab-IPL-IL-17 maintains activity in the absence of off-target immunogenic effects as seen with secukinumab

Current anti-IL-17A therapies (secukinumab and ixekizumab) are associated with unwanted off-target immunogenic effects, lymphocytosis and thrombocytopenia.34 As such, numerous clinical trials are currently investigating new biologic therapies targeting IL-17 biology/function to improve clinical outcomes in patients with IMIDs.35 Here, we evaluated in vivo the neutralising potential of Ab-IPL-IL-17 on IL-17A, IL-17F and IL-17A/F heterodimer production. Ab-IPL-IL-17 was able to significantly reduce the plasma concentration of IL-17A and IL-17F to similar levels as seen with secukinumab (figure 4A,B). Notably, Ab-IPL-IL-17 significantly reduced the plasma concentration of IL-17A/F heterodimer to a similar extent as the reference anti-IL-17A/F antibody bimekizumab (figure 4C). To assess immunogenicity under homeostatic conditions, we administered a single dose of Ab-IPL-IL-17 and measured total immunoglobulin G (IgG) and IgG1 levels over 21 days, comparing levels with the reference anti-IL-17 antibody (MAB421) and the current gold standard clinical therapies secukinumab and bimekizumab (figure 4D,E). A significant increase in total IgG and IgG1 was observed at 72 hours and remained elevated until day 14 with secukinumab, but no increase was observed in mice injected with Ab-IPL-IL-17 (figure 4D,E) or bimekizumab treatment. Whole blood analysis revealed that secukinumab and MAB421 increased total circulating lymphocyte numbers 72 hours post-injection/administration, which remained significantly elevated up to 7 days when compared with vehicle control (figure 4F). Furthermore, secukinumab induced thrombocytopenia as early as 24 hours and platelet numbers remained significantly reduced at 72 hours and 7 days post administration (figure 4G). Strikingly, Ab-IPL-IL-17 (and the selective IL-17A/F neutralising antibody bimekizumab) had no effect on total lymphocyte or platelet numbers at any time point assessed (figure 4F,G). No changes were observed for other haematological parameters (table 1). Collectively, these data show that Ab-IPL-IL-17 retains strong neutralising activity without triggering unwanted immunogenic response, making it an attractive clinical therapy.

Figure 4

Ab-IPL-IL-17 displays a protective profile in murine preclinical models of immune-mediated inflammatory diseases. To assess the neutralising activity of Ab-IPL-IL-17, CD-1 mice were injected i.p. with 100 µg/mouse of Ab-IPL-IL-17, MAB421, secukinumab or bimekizumab as positive controls. After 30 min, an i.p. injection of 10 µg/mouse of IL-17A, IL-17F or IL-17A/F heterodimer was administered. After 2hours, blood was collected by intracardiac puncture and serum levels of (A) IL-17A, (B) IL-17F or (C) IL-17A/F were quantified by ELISA. (D)–(G) For the evaluation of immunogenic effects, CD-1 mice were injected i.p. with 100 µg of IgG1 isotype antibody (vehicle) or IL-17 neutralising antibodies (secukinumab, bimekizumab, MAB421 or Ab-IPL-IL-17). In the selected time-point (2 hours, 24 hours, 72 hours, 7 days, 14 days and 21 days), (D) total IgG, (E) IgG1, (F) lymphocytes and (G) platelets levels were determined by ELISA and haematological blood count test, respectively. Data are presented as mean±SD for n=5 mice per group. Statistical analysis was conducted by one or two-way analysis of variance test followed by Bonferroni’s for multiple comparisons. *p≤0.05, **p≤0.01, ***p≤0.001 vs IL-17 group; #p≤0.05, ##p≤0.01, ###p≤0.001, ####p≤0.0001 vs vehicle group (in red refers to secukinumab and in light blue refers to MAB421, respectively). IgG, immunoglobuin G; IL-17, interleukin 17; i.p., intraperitoneal.

Ab-IPL-IL-17 reduces pathological symptoms of arthritis and IBD

Secukinumab and ixekizumab are current therapies for PsA and AS; therefore, we investigated the clinical efficacy of Ab-IPL-IL-17 in preclinical murine models of arthritis and ex vivo analysis from tissues of patients with RA or IBD. Excitingly, we found that therapeutic administration of Ab-IPL-IL-17 significantly reduced joint swelling in the murine antigen-induced arthritis (AIA) model (figure 5A,B). Indeed, treating AIA with commercially available neutralising antibodies to IL-17 has been previously reported to reduce clinical symptoms of arthritis36 and neutrophil accumulation within the joint.37 It is important to note that Ab-IPL-IL-17 was as effective at halting disease progression and triggering resolution as the gold-standard current treatment for RA, infliximab (figure 5A,B) with a significant reduction in infiltrating neutrophils (figure 5C). and monocytes (figure 5D) observed.