Effects of anti‐IL5 biological treatments on blood IgE levels in severe asthmatic patients: A real‐life multicentre study (BIONIGE)

Abstract Background Mepolizumab and benralizumab are clinically effective biological treatments for severe eosinophilic asthmatic patients by hampering eosinophilic inflammation. The effects of these compound on the immunoglobulin (Ig)E T2 component are virtually unknown. Objectives To evaluate the change in total IgE levels at 4 ± 2 months after initiation of the mepolizumab (primary outcome) or benralizumab. When available, the changes of blood inflammatory cell counts, lung function and asthma control test (ACT) were also assessed and correlated with changes in total IgE levels. Methods Observational, retrospective, multicentre, cohort study. Severe eosinophilic atopic asthmatic patients treated with mepolizumab or benralizumab were included in the analysis. Results Three‐month treatment (on average) with mepolizumab (n = 104) or benralizumab (n = 82) resulted in significantly higher reduction of blood eosinophil and basophil levels in patients treated with benralizumab compared to mepolizumab. Mepolizumab did not significantly modified the levels of blood total IgE during the study period, whereas benralizumab significantly reduced (−35%, p < 0.001) total blood IgE levels. In patients treated with benralizumab the reduction of blood total Ig‐E levels correlated with the reduction of blood basophils (but not eosinophils) and weakly with the improvement of asthma control. Conclusion Benralizumab but not mepolizumab, treatment led to a significant reduction of circulating IgE level. The study provides different and specific mechanisms of action for anti‐IL5‐pathway treatments.

Mepolizumab did not significantly modified the levels of blood total IgE during the study period, whereas benralizumab significantly reduced (−35%, p < 0.001) total blood IgE levels. In patients treated with benralizumab the reduction of blood total Ig-E levels correlated with the reduction of blood basophils (but not eosinophils) and weakly with the improvement of asthma control.

Conclusion:
Benralizumab but not mepolizumab, treatment led to a significant reduction of circulating IgE level. The study provides different and specific mechanisms of action for anti-IL5-pathway treatments.

K E Y W O R D S
basophils, benralizumab, eosinophils, Ig-E, mepolizumab, severe asthma

| INTRODUCTION
The management of severe asthma is a major unmet medical need in respiratory medicine. 1 Severe asthmatic patients are not clinically controlled despite optimised treatment or require maximal treatment to achieve asthma control. 2,3 Major advances have been made in the past few years in the management of severe asthma given the development of targeted biological therapies. 4,5 Mepolizumab and benralizumab are humanised monoclonal antibodies directed against interleukin (IL)-5 and a subunit of the IL-5 receptor (IL-5R), respectively. 6,7 They inhibit eosinophil-driven inflammation by blocking IL-5 or its receptor. 6,7 Clinically, these compounds can improve the quality of life, reduce the need for oral corticosteroid use, and reduce the exacerbation rate in patients with severe asthma with elevated blood eosinophil counts. [8][9][10][11][12][13] Anti-IL-5 and anti-IL-5R monoclonal antibodies share several mechanisms of action; however, targeting IL-5 or its cellular receptor (IL-5R) can interfere with different inflammatory pathways. 6,7 Understanding the specific mechanisms of action of biological treatments can help clinicians select the most appropriate treatment for the right patient.
The effects of these differences on other key effector and regulatory molecules of the T2-immune response, such as immunoglobulin (Ig) E, are virtually unknown. IgE is a pleiotropic molecule that acts as a key inflammatory molecule in the T2-inflammatory cascade as well as a modulatory molecule of innate and adaptive immune systems. 14 Augmentation of the total IgE levels is common in asthma irrespective of the underlying atopic status. 15 Targeting blood circulating IgE has proven effective in improving clinical outcomes under different conditions characterised by high levels of blood IgE, including severe atopic asthma. [16][17][18] Thus, it is worth investigating whether anti-IL-5 or anti-IL-5R treatments impact blood IgE levels.
To investigate the effects of treatments interfering with the IL-5 pathways on IgE levels, we performed a multicentre study primarily aimed at evaluating the effect of anti-IL-5 mepolizumab on circulating total IgE levels. To assess the specificity of the effect, a cohort of patients treated with the anti-IL-5 receptor monoclonal antibody benralizumab was also evaluated. The analyses were conducted in patients with severe asthma receiving mepolizumab or benralizumab, for which the following information was available: (i) positive atopic status. Atopy was defined as a positive detection of specific IgE (RAST test) to any allergen (including house dust mites, dogs, cats, and seasonal pollen, such as grasses, birch, cypress, hazel, Alder, and Alternaria); (ii) total IgE levels in stable conditions within 6 months prior to the beginning of the anti-IL5/Ra treatments and a second assessment at 4 � 2 months after the initiation of the biologic treatments. Total IgE values should be obtained under stable conditions, that is, free from any exacerbations for at least 8 weeks. Within 6 months before the initiation of treatment with biologics, IgE assessment closer to the date of treatment initiation was considered. The first IgE assessment was performed 2-6 months from the date of initiation of treatment with biologics. Only patients with repeated measurements of blood IgE levels within the study timeframe were screened and included in the analysis. Measurements of blood inflammatory cell counts, fractional nitric oxide (NO) concentration in exhaled breath (FeNO), and clinical outcomes were also considered if performed within the same timeframes and with the same temporal criteria. Patients receiving chronic oral corticosteroids (defined as therapy for at least 6 continuous months before enrolment 13 ) were excluded from the analysis.

| Study design
This is a real-life (Phase IV), observational, retrospective, multicentre, and cohort study. Eight centres participated in the study. All centres had dedicated clinics for severe asthma. A list of centres is provided in Appendix. The study conformed to the Declaration of Helsinki and was approved by the institutional ethics committee. Informed written consent was obtained from all participants. The study was registered at ClinicalTrials.gov (identifier NCT04181190).

| Aims and measurements
The study was designed to primarily evaluate the changes in total IgE levels (kU/l) in atopic patients with severe eosinophilic asthma at 4 � 2 months after initiation of the anti-IL-5 mepolizumab treatment.
To assess the specificity of the intervention, we also investigated the changes in total IgE levels (kU/l) in a similar population of atopic patients with severe eosinophilic asthma at 4 � 2 months after initiation of the anti-IL5R monoclonal antibody benralizumab treatment. We further assessed blood inflammatory cell counts before monoclonal antibody treatment and 4 � 2 months after the initiation of each of the two treatments. When available during the study timeframe, lung function, fractional nitric oxide (NO) concentration in exhaled breath (FeNO) and asthma control test (ACT) measurements 19 at baseline before initiation of monoclonal antibody treatments and at 4 � 2 months after initiation of treatment were also collected for each of the two biologics.

| Statistical analysis
A formal calculation of the sample size for this study was not possible, as no clinical study has specifically assessed the effect of anti-IL-5 monoclonal antibodies on circulating total IgE levels in patients with severe eosinophilic allergic asthma (the primary outcome of this study). Therefore, we estimated the sample size considering (a) mean values of total baseline IgE described in a population of patients with severe asthma 20 and (b) an expected 35% reduction in the total IgE level that has been reported with other biologic treatments. 21 Based on these assumptions, we calculated that 104 patients on mepolizumab therapy would be sufficient to address the primary outcome of this study (alpha 0.05, power 0.8). Assessment of clinical outcomes of mepolizumab and benralizumab treatments only had a descriptive value; the comparison of effectiveness between treatments was beyond the statistical power of the present study.
Data are shown as the mean � SD unless otherwise specified.
The standardised difference between means (for continuous variables) and between prevalence (for dichotomous variables) was calculated to compare the baseline characteristics of the mepolizumab-and benralizumab-treated groups. 22,23 The changes in biomarkers and inflammatory cell counts were assessed using the paired t-test or Wilcoxon test, as appropriate. Comparisons between groups were assessed using the unpaired t-test or Mann-Whitney U test, as appropriate. Correlation coefficients were calculated using the Spearman's rank method. p-values of 0.05 or less were considered to indicate statistical significance. Post hoc Holm-Bonferroni correction was applied for multiple testing.

| Study population
The medical records of 165 patients with severe eosinophilic asthma treated with mepolizumab with repeated measurements within the study timeframe of blood IgE levels were screened to obtain a cohort of 104 patients fulfilling the inclusion criteria of the analysis.
A total of 28 patients were excluded from the analysis because they were non-atopic. Another 33 patients (10 non-atopic and 23 atopic patients) were excluded because of chronic oral corticosteroid treatment. Eighty-two atopic severe eosinophilic asthmatic patients treated with benralizumab who fulfilled the inclusion/exclusion criteria were also included in the specificity analysis. The patient characteristics are shown in Table 1 and in the online supportive information. No statistically significant differences were found between the two groups at baseline before the biological treatments.
To further evaluate the match between study groups, standardised differences in baseline clinical, functional, and biological variables were calculated (Table S1 in Supporting Information S1). Standardised differences between groups were laid within a 10% window (standardised difference <0.1) that met the criterion of a negligible difference. 22,23 Further information is available in Supporting Information S1. The durations of biologic treatments before assessing the IgE change were similar in the two groups of patients (117 � 4 days and 108 � 4 days for mepolizumab and benralizumab, respectively, p > 0.05). No major side effects were documented in patients treated with mepolizumab or benralizumab (see also Supporting Information S1).

| IgE levels
The measurements of total IgE levels were available for all patients included in the study both at baseline (within 6 months from the beginning of biological treatments) and at 4 � 2 months after initiation of treatment. Similar levels of total IgE were found in the two CONTOLI ET AL.  Note: Demographic and clinical characteristics refer to the assessment performed within 6 months from the beginning of biological treatments. Data are presented as mean (SD) unless otherwise indicated (ACT: asthma control test score; BMI: body mass index; FEV1: forced expiratory volume in the 1st second).
F I G U R E 1 Blood total IgE levels at baseline (before biologic treatments) and on-treatment in severe eosinophilic asthmatic patients treated with mepolizumab or benralizumab Information S1). These analyses provided results similar to those obtained when considering the entire population (detailed in Supporting Information S1).
In mepolizumab-treated patients (in whom overall, a nonsignificant 2% increase in total IgE levels was found compared to baseline), an increase in blood IgE levels was found in 28 patients (27%), a stability in 38 patients (37%), and a reduction in 36 patients (35%). Conversely, a reduction in blood IgE levels was found in the vast majority (92%) of patients treated with benralizumab, with only eight patients showing an increase of at least 35% compared to baseline.

| Blood inflammatory cell counts and FeNO measurements
Blood sample tests performed before and after initiation of monoclonal antibody treatments were available for 94% (n = 98) of patients treated with mepolizumab and for 88% (n = 72) of patients treated with benralizumab. Similar levels of total blood leukocytes, blood lymphocytes, blood eosinophils, and blood basophils were found at baseline in the two patient groups (Table 1). No treatment effect was observed on blood lymphocyte ( Figure 2A) and neutrophil ( Figure 2B) levels. Both treatments resulted in significant reductions in blood eosinophil counts ( Figure 2C). On-treatment blood eosinophil levels were significantly lower in patients receiving benralizumab than in those receiving mepolizumab (p < 0.05; Figure 2C).
Paired before and after measurements of FeNO were available for more than one-third of the patients [36 patients (35%) treated with mepolizumab and 28 patients (34%) treated with benralizumab].
In line with a recent publication, 24 we found that benralizumab but not mepolizumab treatment resulted in a significantly reduction in FeNO levels (p < 0.05, Figure 2E).

| Correlations between IgE levels and blood inflammatory cell counts
No correlations were found in both groups of patients between IgE levels and blood eosinophil ( Figure 3A,B) or basophil ( Figure 3C,D) counts, both at baseline ( Figure 3A-D) and on treatment (data not shown).
A significant positive correlation was found in patients treated with benralizumab (but not in patients treated with mepolizumab) between the on-treatment reduction of IgE levels and the reduction of blood basophil levels (p < 0.01; r = 0.34; Figure 3E) but not of eosinophil counts ( Figure 3F). This was confirmed by Holm-Bonferroni correction for multiple comparisons (adjusted p-value 0.016).
Pre-bronchodilator FEV1 significantly improved in both groups ( Figure 4A). No correlations were found between changes in lung function and change in blood IgE levels. The ACT scores before and after monoclonal antibody treatment were available for 57% (n = 60) of patients treated with mepolizumab and for 47% (n = 39) of patients treated with benralizumab (121 � 4 vs. 120 � 5 days of treatment, respectively; p > 0.05). The ACT scores were clinically improved in both groups ( Figure 4B). In patients treated with benralizumab, we found a weak but statistically significant negative correlation between the on-treatment change in ACT score with change in IgE levels (p < 0.05; r = −0.32; Figure 4C). This was confirmed by Holm-Bonferroni correction for multiple comparisons (adjusted p-value 0.048).

| DISCUSSION
In this retrospective, multicentre study, we found that similar durations of mepolizumab and benralizumab treatments (approximately 3 months) resulted in different effects on total blood IgE levels. While mepolizumab administration did not significantly modify the levels of total IgE in the blood during the study period, benralizumab administration led to a significant reduction in total blood IgE levels.
Interestingly, and in line with recently published preliminary data, 25 we found that benralizumab strongly reduced blood basophil counts in the study population. In patients treated with benralizumab, the data suggest a correlation between the reduction in total blood IgE levels with the reduction of blood basophils (but not eosinophils) and with the improvement of asthma control. These latter correlations require assessment in properly designed longitudinal studies.
Mepolizumab and benralizumab are effective treatments that can reduce the need for systemic corticosteroid treatment and the exacerbation rate in patients with eosinophilic severe asthma. [8][9][10][11][12][13] The clinical and immunological profiles of patients with severe asthma are rarely black and white, and there is an overlap of indications for different biological treatments in these patients. 2