S95021, a novel selective and pan-neutralizing anti interferon alpha (IFN-α) monoclonal antibody as a candidate treatment for selected autoimmune rheumatic diseases

Abstract

Increased interferon-α (IFN-α) production is a critical component in the pathophysiology of systemic lupus erythematosus (SLE) and other rheumatic autoimmune diseases. Herein, we report the characterization of S95021, a fully human IgG1 anti-IFN-α monoclonal antibody (mAb) as a novel therapeutic candidate for targeted patient populations. S95021 was expressed in CHOZN GS-/- cells, purified by chromatography and characterized by using electrophoresis, size exclusion chromatography and liquid chromatography-mass spectrometry. High purity S95021 was obtained as a monomeric entity comprising different charge variants mainly due to N-glycosylation. Surface plasmon resonance kinetics experiments showed strong association rates with all IFN-α subtypes and estimated KDs below picomolar values. Pan-IFN-α-binding properties were confirmed by immunoprecipitation assays and neutralization capacity with reporter HEK-Blue IFN-α/β cells. S95021 was IFN-α-selective and exhibited superior potency and broader neutralization profile when compared with the benchmark anti-IFN-α mAbs rontalizumab and sifalimumab. STAT-1 phosphorylation and the type I IFN gene signature induced in human peripheral blood mononuclear cells by recombinant IFN-α subtypes or plasmas from selected autoimmune patients were efficiently reduced by S95021 in a dose-dependent manner. Together, our results show that S95021 is a new potent, selective and pan IFN-α-neutralizing mAb. It is currently further evaluated as a valid therapeutic candidate in selected autoimmune diseases in which the IFN-α pro-inflammatory pathway is dysregulated.

Keywords: Autoimmune diseases; Biological therapy; Interferon-alpha; Primary Sjögren syndrome; Systemic lupus erythematosus.

Fig. 1

Characterization of S95021. (A) SDS-PAGE of purified S95021 mAb under reducing (R) and non-reducing (NR) conditions. The position of the standard mass markers is depicted on the right side of the gel. (B) SEC-UV-MALS chromatogram of S95021. Continuous line: UV chromatogram at 280 ​nm; dotted line: MALS-based mass estimation obtained using ASTRA (Y axis). (C) cIEF electropherogram of S95021. Measured isoelectric points are indicated above peaks (10.17 pI marker is indicated as M). (D-F) LC-MS mass measurement and N-glycan identification of S95021. (D) RP-UHPLC-UV chromatogram of S95021 following reduction. Peaks corresponding to LC and HC are depicted with dotted lines. (E) Deconvoluted MS spectrum of LC glycoforms with main glycoforms indicated. (F) Deconvoluted MS spectrum of HC glycoforms. The relative content of N-glycans is reported in Supplementary Table 3.

Fig. 2

Binding analysis by SPR of IFN-α subtypes flowed over captured S95021. (A) Binding in single dose assay of the 12 IFN-α subtypes at 250 pM over captured S95021. (B) Binding analysis of IFN-α1 and IFN-α8 flowed over captured S95021. IFN-α1, IFN-α8 and the negative control IFN-γ were injected in single cycle kinetics binding cycles, as 2-fold dilution series with top at 750 ​nM.

Fig. 3

Binding activity of S95021, rontalizumab and sifalimumab to IFN-α subtypes using luciferase based immunoprecipitation system (LIPS). LIPS assay was carried out with NanoLuc-IFN fusion proteins and serial dilutions of monoclonal antibodies. Dose-response curves used for effective concentration 50 (EC₅₀) calculation are depicted for each IFN-α subtype. EC₅₀ values are reported in Supplementary Table 5.

Fig. 4

Neutralization of the effect of IFN-α subtypes by S95021, rontalizumab and sifalimumab in reporter HEK-Blue^TM IFN-α/β cells. IFN-α subtypes were incubated with serial dilutions of monoclonal antibodies and tested for bioactivity on reporter cells. Dose-response curves used for inhibitory concentration 50 (IC₅₀) calculation are depicted for each IFN-α subtype. IC₅₀ values are reported in Supplementary Table 6.

Fig. 5

IFN-α subtypes induce P-STAT1 at different levels in immune cell populations. (A) Representative FACS plots showing the dose-dependent induction of STAT1 phosphorylation by IFN-α subtypes in human healthy PBMCs gated in CD14⁺ monocytes. (B) Percentage of P-STAT1 positive cells in human heathy PBMCs gated in CD14⁺ monocytes after stimulation with IFN-α subtypes. (C) Percentage of P-STAT1 positive cells in human heathy PBMCs (gated in CD14⁺ monocytes, CD20⁺ B cells, CD3⁺ T cells and CD16⁺CD56⁺ NK cells) after stimulation with IFN-α16. Values in (B) and (C) are means and SEM from experiments completed with 4 individual donors. Significance was calculated using Mann-Whitney test with ∗p ​< ​0.05; ns, not significant.

Fig. 6

S95021 is more potent than benchmark antibodies in neutralizing P-STAT1 after IFN-α stimulation in control healthy PBMCs. P-STAT1 analysis was assessed in human healthy PBMCs gated in CD14⁺ monocytes. (A) Dose-dependent neutralization of STAT1 phosphorylation by S95021, rontalizumab or sifalimumab after IFN-α16 stimulation. (B) Relative intrinsic neutralization potency of S95021 (1 ​μg/mL), sifalimumab (10 ​μg/mL) or rontalizumab (10 ​μg/mL) on STAT1 phosphorylation induced by IFN-α subtypes. (C) Dose-dependent neutralization of STAT1 phosphorylation by S95021, rontalizumab or sifalimumab after incubation with plasma from an individual SLE donor. Values in (A) and (B) are means and SEM from experiments completed with 5 and 3 individual donors, respectively. Significance was calculated using Bonferroni-Holms adjustment on group after a two-way Anova compared with the condition S95021. ∗∗p ​< ​0.01 and ∗p ​< ​0.05.

Fig. 7

S95021 is more potent than benchmark antibodies in neutralizing the type I IFN gene signature induced by IFN-α16 in control healthy PBMCs. (A) Induction of the 18 IFN gene signature by 1000 IU/mL IFN-α16. (B) Effect of S95021, rontalizumab or sifalimumab (each mAb at 10 ​μg/mL) on the 18 IFN gene signature after stimulation of PBMCs with 1000 IU/mL IFN-β or IFN-γ compared with untreated condition. (C) Heatmap showing the differential expression and dose-dependent neutralization of individual genes of the 18 IFN gene signature by S95021, rontalizumab or sifalimumab after stimulation with 1000 IU/mL IFN-α16 (left panel). The percentage of neutralization of the IFN gene signature in comparison with the untreated condition is shown on the right panel. Values in (A, B and C) are means and SEM from experiments completed with 5 individual donors. Significance was calculated using (A, B) Mann-Whitney test and (C) Bonferroni-Holms adjustment on group after a two-way Anova compared with the condition S95021. ∗∗∗p ​< ​0.001; ∗∗p ​< ​0.01; ∗p ​< ​0.05 (C) ns; not significant.

Fig. 8

S95021 is more potent than benchmark antibodies in neutralizing the type I IFN gene signature induced by plasma from SLE patients in control healthy PBMCs. Heatmap showing the differential expression and dose-dependent neutralization of individual genes of the 18 IFN gene signature by S95021, rontalizumab or sifalimumab after stimulation with plasma from SLE donors. The heatmap is representative of 6 individual SLE donors.