Effect of Anti-IL5 and Anti-IgE on Alternative Functions of Eosinophils in Severe Asthma
Conditions: Severe Asthma
Sponsor: University of Liege

January 2nd 2023  
 
Effect of Biologicals on Alternative Functions of Eosinophils in Severe Asthma 
EudraCT: NCT04520165  
Scientific Background and goal 
Refractory asthma still represents between 3 to 10% of asthmatic patients. These patients 
represent a high burden in terms of healthcare costs. Biotherapies have been developed within 
the last years to reduce the use of oral corticosteroids (OCS), which are responsible for long-
term and costly side effects. Monoclonal antibodies directed against interleukin (IL)-5 or its 
receptor (IL-5R) are approved to treat severe eosinophilic asthma with tangible improvements 
of the patient conditions. But it appears that the patients do not respond the same way to 
biotherapies.  
In addition to the “super-response”, a new concept of asthma “remission” has recently emerged 
as a therapeutic target to achieve after 12 months of treatment and was defined according to 
results of randomized control trials (RCT) as obtaining asthma control, no exacerbation, no 
treatment with OCS while there is no consensus on the importance of improvement in lung 
function and reduction in type-2 (T2) inflammation in the current definition of remission. 
Post-hoc analysis of RCT looked at the predictive factors of response to benralizumab or 
mepolizumab in vast cohorts of patients and observed that high blood eosinophil level was 
linked to a better improvement in terms of reduction of exacerbation rate. Even if blood 
eosinophils help to predict a general response to anti IL-5/ anti IL-5R, biomarkers reflecting 
local inflammation, such as those measured in induced sputum, have a better potential to predict 
the intensity of response to biologics as they reflect what really happens in the bronchi. So are 
there biomarkers of remission after therapy targeting IL-5 in the sputum of severe eosinophilic 
asthmatics? There are, to our knowledge, no studies analyzing inflammatory mediators as 
predictors of response after one year of anti-IL-5 treatment directly in the sputum itself in severe 
eosinophilic asthmatics. The goal of this paper is to analyze the role of mediators of the T2 
cascade in the sputum as predictors of remission.  
 
 

Study design and methods 
Patients 
Fifty-two severe asthmatics initiated with an anti-IL-5 (51 with Mepolizumab and one with 
Reslizumab) were recruited from our asthma clinic. This observational study was performed at 
the CHU of Liege, Belgium, between 2014 and 2021. Inclusion criteria included a diagnosis of 
asthma defined by the Global Initiative for Asthma (http://ginasthma.org/), severe asthma was 
defined according to European Respiratory Society (ERS)/ American Thoracic Society (ATS) 
criteria. The treatment was stable for all patients at the time of the sampling and the time 
between baseline and the next evaluation was in average one year. Remission was defined as 
patients combining after the instauration of the biotherapy: no oral corticosteroids, no 
exacerbation, ACQ lower than 1.5 and/or asthma control test (ACT) greater than 19 and, a FEV1 
 80% predicted and/or an improvement of FEV1  10% and a blood eosinophil count lower 
than 300 cells/l.  
Sub-analyses were also performed to assess the predictive values of sputum mediators in terms 
of response for some specific parameters alone, such as: an improvement of ACT greater than 
19 post-treatment, a decrease of ACQ lower than 1.5 post-treatment, a decrease of sputum 
eosinophil under 3% or by 50%, stopping OCS post-treatment, no more exacerbations post-
treatment and improvement of FEV1 of at least 10% post-treatment.  
A diagnosis of nasal polyps by an Ear-Nose-Throat specialist and a diagnosis of atopic 
dermatitis or urticaria done by a dermatologist were reported. 
This study was approved by the Ethics committee of CHU Liege (2005/181) and all subjects 
gave written informed consent for participation. The study ClinicalTrials.gov Identifier is 
clintrial.org EudraCT: NCT04520165. 
 
Study design 
This study aims to investigate the airway expression of mediators of the T2 cascade as 
predictors of remission after anti-IL-5 treatment in a cohort of patients with severe eosinophilic 
asthma. To detect a difference between groups with a power of 70-80% and a significance level 
of 
5%, 
the 
sample 
size 
was 
estimated 
as 
10-13 
patients 
per 
group 
(https://www.sealedenvelope.com/). These calculations were based on a significant difference 
of sputum IL-5 obtained as preliminary results. Usually, in the different studies, even if there is 
no consensus on the remission definition in the asthma context, there is mention of a percentage 
around 20% of patients exhibiting a good response to anti-IL-5. Consequently, with a cohort of 
52 patients, we expected a sample size of at least 10 patients in the remission group. 

 
Respiratory function 
 
FeNO was measured using NiOX (Aerocrine, Solna, Sweden) at a flow rate of 50 mL/s. 
Spirometry was performed before and after bronchodilation according to the ATS/ERS standard 
criteria.  
Blood samples 
Blood samples of patients were analyzed by the routine laboratory of the University Hospital 
of Liege for leukocyte count, c reactive protein (CRP) and fibrinogen levels. 
Sputum induction and processing. 
The sputum was induced and processed as previously described and the sputum supernatant 
was collected before the addition of dithiothreitol (DTT), which could impact the 
measurements. Cell viability was determined by trypan blue exclusion and the differential cell 
count was performed by counting 500 non-squamous cells on cytospins stained with May-
Grünwald-Giemsa. 
Sputum inflammatory mediators measurement 
Eosinophil peroxidase (EPX) was measured by classic ELISA (Mybiosource, CA, USA). 
Detection limit was 5.4 ng/ml. Immunoglobulin E (IgE) levels were measured with Human IgE 
ELISA kit from Abcam (Amsterdam, Netherlands). The detection limit was 30.5 pg/ml. Finally,  
interleukin (IL)-3, IL-4, IL-5, IL-13, IL-25, IL-33, granulocyte-macrophage colony-stimulating 
factor (GM-CSF), thymic stromal lymphopoietin (TSLP) and eotaxin-1 were measured by 
multiplex electrochemiluminescent assays (Meso Scale Discovery). This technic was already 
used with success in sputum supernatant in the past and spiking recovery were optimal.  
 
Statistical analysis 
Categorical variables are presented as numbers and percentages, continuous variables are 
presented as mean ± standard deviation or as median (interquartile range P25-p75) when 
appropriate. 
The normality of continuous variables was assessed by Shapiro-Wilk test (normal distribution 
was found for age, BMI, ACQ, AQLQ, pulmonary function parameters, fibrinogen level, and 
sputum neutrophil percentage). To investigate baseline characteristics of patients remission 
group, Chi-square or Fisher exact test was used for categorical variables and Student t test or 
Mann-Whitey U test was performed for continuous variables when appropriate.   

Univariate logistic regression was considered to model the association between patient 
remission status and demographic, clinical and biological variables. Results of logistic 
regression were presented using odds ratios and corresponding 95% confidence interval.  
All tests were performed 2-sided and p<0.05 was considered significant. All statistical analyses 
were performed using SAS 9.4 (SAS Institute, Cary, NC), except for ROC curve calculation, 
GraphPad Prism 7 (GraphPad Software San Diego, CA, USA) was used. The p-values 
associated with the ROC curves tested the null hypothesis that the area under the curve equals 
0.50.