Efficacy of Mepolizumab in Severe Asthmatics (MESILICO)
Efficacy of Mepolizumab in Severe Asthmatics (MESILICO)
Interleukin (IL)-5 is the main cytokine responsible for the activation of eosinophils, hence
 therapeutic strategies have been investigated and developed for clinical use. Biologics
 targeting IL-5 and its receptor (first mepolizumab and subsequently, reslizumab and
 benralizumab), have been recently approved and used as add-on therapy for severe eosinophilic
 asthma resulting in a reduction in the circulating eosinophil count, improvement in lung
 function and exacerbation reduction in patients with severe asthma.

 Response to biologic therapies in severe asthma is variable, with patients being either
 non-responders, responders or super-responders. There is currently no explanation for this
 broad variation in response. It is important to examine whether these patients have distinct
 characteristics that could help the treating physician in making the correct diagnosis in
 clinical practice.

 Aim of this clinical study is to evaluate the efficacy of mepolizumab, a humanized IL-5
 antagonist monoclonal antibody in patients with late-onset severe eosinophilic asthma with
 fixed obstruction and to identify the characteristics of non-responders and super-responders
 under mepolizumab treatment. This study is considered as non-interventional and every
 procedure included is happening in a clinical routine for the diagnosis and phenotyping of
 the asthmatic patients.

 Hypothesis includes the efficacy of mepolizumab treatment in late-onset severe eosinophilic
 asthmatic patients with fixed obstruction and relation to clinical and inflammatory
 biomarkers.

 Patients will be collected from the outpatient clinics of bronchial asthma from each site
 included (8 in number) which cover the whole population of Greece.

 Overall, this is a prospective multicenter study including eight Pulmonary Clinics. Five
 Pulmonary University Clinics, two of National Health System and one Army General Hospital in
 Thessaloniki. The study will include a screening period of up to 2 weeks to assess
 eligibility and obtain written informed consent, a mepolizumab treatment period of 52 weeks,
 once every 4 weeks, including follow up visits every 3 months during treatment. The study
 population will consist of 45 patients with late-onset severe eosinophilic asthma and fixed
 obstruction receiving mepolizumab, aged 20 and above.
Scientific rationale Interleukin (IL)-5 is the main cytokine responsible for the activation
 of eosinophils, hence therapeutic strategies have been investigated and developed for
 clinical use. Biologics targeting IL-5 and its receptor (first mepolizumab and subsequently,
 reslizumab and benralizumab), have been recently approved and used as add-on therapy for
 severe eosinophilic asthma resulting in a reduction in the circulating eosinophil count,
 improvement in lung function and exacerbation reduction in patients with severe asthma.

 Response to biologic therapies in severe asthma is variable, with patients being either
 non-responders, responders or super-responders. There is currently no explanation for this
 broad variation in response. It is important to examine whether these patients have distinct
 characteristics that could help the treating physician in making the correct diagnosis in
 clinical practice.

 Aim of this clinical study is to evaluate the efficacy of mepolizumab, a humanized IL-5
 antagonist monoclonal antibody in patients with late-onset severe eosinophilic asthma with
 fixed obstruction and to identify the characteristics of non-responders and super-responders
 under mepolizumab treatment. This study is considered as non-interventional and every
 procedure included is happening in a clinical routine for the diagnosis and phenotyping of
 the asthmatic patients.

 Several methods are used to investigate airway inflammation: direct measurements (like
 bronchial biopsies or bronchoalveolar lavages) and indirect methods (like symptom assessment,
 blood sample analysis and lung function tests). The direct techniques have the advantage of
 reliably assessing the airway inflammation, but they are invasive and not feasible at large
 scale because of patient discomfort and the risk incurred. As for the indirect methods, they
 poorly correlate with the direct assessment of airway inflammation.

 In this study, among patients' clinical characteristics spirometry, biomarkers of
 inflammation (blood eosinophils, FeNO, IgE levels), impulse oscillometry (IOS) and the
 thickening of basement membrane via bronchoscopy will be evaluated. It is suggested that
 late-onset severe eosinophilic asthmatic patients with fixed obstruction who do not respond
 to the usual treatment (ICS/LABA/LAMA) appear with increased smooth muscle mass. However, the
 precise effects of increased airway smooth muscle mass on airway narrowing are not known and
 may vary with disease severity. In addition, biomarkers of inflammation will also be
 evaluated before and after 52-week mepolizumab treatment in biological fluids (serum, sputum,
 sputum supernatant and bronchial samples). Biomarkers of inflammation are very important to
 identify responders to non-responders.

 More specific, IOS has been introduced as an alternative technique to assess lung function
 with particular application to asthma. This technique is noninvasive, easily performed during
 tidal breathing and requires only minimal patient cooperation.

 Sputum collection is another way to sample cells from the airways and allows the direct
 assessment of airway inflammation. Furthermore, examples with sputum supernatant include the
 analysis of mediators and the assessment of the chemotactic activity of the sample for
 eosinophils. The sputum cells can also be analyzed by flow cytometry which allows, among
 others, immunophenotyping and sorting cells. Furthermore, sputum cells can be cultured, and
 their mediator production can be measured in vitro. It is worth noting that in this case, the
 mediator content is different from what can be found in sputum supernatant. Indeed, mediators
 in sputum supernatant may be influenced by secretions from airway resident cells and by
 plasma exudation, contrary to the sputum cell culture model. Finally, immunocytochemistry and
 in situ hybridization can also be performed using sputum cells.

 Although phenotypes, blood eosinophils, and serum IgE levels have been proposed for use as a
 reference, there is a dissociation between the blood immune-cell level and the airway
 epithelium immune reaction, as confirmed in previous studies. Airway epithelium
 immunohistochemistry staining for targeted immune cells has been used to determine various
 types of airway inflammation; however, this technique is rarely used in a clinical setting.
 Previous studies have revealed the relative safety of performing bronchoscopy biopsies for
 patients with severe asthma. Among the sampling techniques used for tissue diagnosis,
 including nasal biopsies, nasal or bronchial brushing, and bronchoalveolar lavage,
 bronchoscopy-guided bronchial epithelium sampling provides more accurate information about
 the epithelial and inflammatory cells in the tissue context. It is thus a powerful tool for
 selecting the most suitable biologics in difficult clinical conditions.

 Besides, asthmatic disease is characterized by a chronic mucosal inflammatory process, which
 results in irreversible changes of the bronchial wall, known today as "bronchial remodeling".
 Glandular and smooth muscle fibers hyperplasia and/or hypertrophy, goblet cells hyperplasia,
 and variable thickening of basement membrane (BM) present under the respiratory epithelium
 are part of these morphological changes. the changes generated at the epithelium-connective
 interface account for an "adaptive response" to inflammatory stress and sporadic
 bronchoconstriction. However, current data on BM reactivity in asthmatic patient are still
 incomplete for an accurate assessment of its involvement in pathogenesis and specifically in
 bronchial wall remodeling, mainly as collagen deposits in lamina reticularis are not
 correlated to the degree of disease severity.

 Moreover, it is increasingly evident that severe asthma is not a single disease, as evidenced
 by the variety of clinical presentations, physiologic characteristics, and outcomes seen in
 patients with asthma. To better understand this heterogeneity, the concept of asthma
 phenotyping and endotyping has emerged. Phenotyping integrates both biological and clinical
 features, from molecular, cellular, morphologic, and functional to patient-oriented
 characteristics with the goal to improve therapy. Ultimately, these phenotypes evolve into
 asthma "endotypes," which combine clinical characteristics with identifiable mechanistic
 pathways. Biomarkers, defined as characteristics that can be objectively measured and serve
 as an indicator of underlying biological processes or pathogenesis, are crucial in defining
 phenotypes and endotypes. In asthma, genetic polymorphisms, measures of airway physiology,
 and levels of inflammatory mediators in urine, blood, sputum, tissue, exhaled gas, and breath
 condensate have all been studied as potential markers to improve and objectify asthma
 diagnosis and management. Developing such tools will allow us to phenotype and endotype the
 various clinical patterns described in asthma, with the ultimate goal of tailoring therapy
 based on a specific biomarker profile. Biomarkers can then be used to help better understand
 the pharmacologic response to an intervention and adjust therapy accordingly.

 Biomarkers have been critical for studies of disease pathogenesis and the development of new
 therapies in severe asthma. In particular, biomarkers of type 2 inflammation have proven
 valuable for endotyping and targeting new biological agents. Because of these successes in
 understanding and marking type 2 inflammation, lack of knowledge regarding non-type 2
 inflammatory mechanisms in asthma will soon be the major obstacle to the development of new
 treatments and management strategies in severe asthma. Other potential biomarkers include
 innate lymphoid cells, IL-33 or thymic stromal lymphopoietin.

 Recent publications confirmed crucial regulatory role of miRNAs in the pathomechanism of
 asthma. Some single miRNAs or their sets hold the promise for their use as asthma biomarkers
 facilitating diagnosis or prediction of treatment outcomes. They are also possible target of
 future therapies. The studies in this field are lacking though. Recently it has been shown
 that most of the involved miRNAs increase secretion of Th2 cytokines, decrease secretion of
 Th1 cytokines, promote differentiation of T cells towards Th2 or play a role in hyperplasia
 and hypertrophy of bronchial smooth muscle cells. The profiles of miRNAs correlate with
 clinical characteristics, including lung function, phenotype and severity of asthma.

 Most industry sponsored biologic clinical trials have effectively used the above approach by
 selecting study subjects that resemble the severe asthma clinical phenotypes of SARP
 (high-intensity controller medication regimens, frequent exacerbations, and low lung
 function), but then require an elevated biomarker (in blood, exhaled gas, or sputum) that
 should identify patients most likely to be responders to a specific immunomodulator within
 those clinical phenotypes. The biologics in recently published clinical trials target type 2
 inflammatory pathways and restrict participation to subjects with elevated type 2 biomarkers
 (blood or sputum eosinophils or FeNO). Post hoc responder analyses to "group" biomarkers to
 better understand which patients have the greatest benefit from a biologic immunomodulator
 will lead to an understanding that concurrent elevations in more than 1 type 2 inflammatory
 biomarker may further identify "high responder" patients.

 Hypothesis includes the efficacy of mepolizumab treatment in late-onset severe eosinophilic
 asthmatic patients with fixed obstruction and relation to clinical and inflammatory
 biomarkers.

 Research questions Is there any improvement in exacerbation rate in late-onset severe
 eosinophilic asthmatic patients under mepolizumab treatment during their 1st year of
 treatment and what are the characteristics of non-responders and super-responders? Is there
 any improvement in smooth muscle cells remodeling? Are there any biomarkers to predict
 response to mepolizumab treatment in late-onset severe eosinophilic asthmatic patients? Is
 there any improvement in respiratory parameters? Is there any improvement in asthma control
 and patients' quality of life? Objectives

 The primary objectives of this study are:

 1. To demonstrate the effect of mepolizumab treatment in exacerbation rate in late-onset
 severe eosinophilic asthmatic patients with fixed obstruction.

 2. To identify characteristics of non-responders and super-responders.

 The key secondary objectives of this study are:

 1. Any improvement in remodeling (thickening of bronchial smooth muscle cells).

 2. Any possible biomarkers of response.

 3. Any improvement in respiratory parameters in asthma control and in patients' quality of
 life during mepolizumab treatment in late-onset severe eosinophilic asthmatic patients
 with fixed obstruction.

 Study design and Methods Patients will be collected from the outpatient clinics of bronchial
 asthma from each site (8 in number) which cover the population of Greece. All these Clinics
 are included to reinforce the recruitment as well as to include bronchoscopist experts with
 international impact. Due to the current situation concerning Covid-19 pandemic, the
 investigators assume that this clinical study does not interfere with the workload in our
 Clinics.

 Overall, this is a prospective multicenter study including eight Pulmonary Clinics. Five
 Pulmonary University Clinics, two of National Health System and one Army General Hospital.

 The study will include a screening period of up to 2 weeks to assess eligibility and obtain
 written informed consent, a mepolizumab treatment period of 52 weeks, once every 4 weeks,
 including follow up visits every 3 months during treatment.

 The study population will consist of 45 patients with late-onset severe eosinophilic asthma
 and fixed obstruction receiving mepolizumab aged 20 and above.

 Study participation, assessment and follow up:

 Screening: Investigators will screen selected subjects to the participated clinics (Severe
 Asthma outpatient clinics). Subjects will come in to sign consent. Clinical assessment and
 procedures and structural interviews will be performed during screening. Subjects that are
 eligible/able to complete the screening Visit may do so, on the same day, and if necessary,
 they will complete their baseline assessment within 2 weeks. Subjects with an ongoing asthma
 exacerbation should have their screening and treatment initiation visit delayed until the
 investigator considers the subject has returned to their baseline asthma status. If the
 2-week screening period has elapsed then the subject should be considered a screening
 failure. Subjects will remain on their current maintenance therapy throughout screening
 periods. Eligibility of the patients will be based on the criteria listed in the next
 sections.

 Asthma exacerbation history for the last 12 months will be reported at screening visit. Also,
 exacerbations will be evaluated at each visit as reported.

 Vital signs will be performed at screening visit, at baseline and every 3 months.
 Measurements will include systolic and diastolic blood pressure, pulse rate and body
 temperature.

 Height in centimeters (cm) and body weight in kilograms (kg) will be measured at screening
 visits. Body Mass Index (BMI) will be calculated as the weight in kg divided by the height in
 meters squared

 Laboratory evaluation (Hematology, Blood chemistry, Urinalysis):

 Venous blood and urine will be collected at baseline. Hematology: Hemoglobin, hematocrit, red
 blood cell count, white blood cell count with differential, and platelet will be measured in
 the laboratory of each site's hospital. Basic clinical chemistry and urinalysis will be
 evaluated. Collection of urine for female subjects will also evaluate pregnancy.

 IgE levels will be evaluated at screening visit using immunoCAP tests and will be reported as
 IU/mL.

 The following Questionnaires will be administered: 1) the Asthma Control Questionnaire
 (ACQ-5) and Asthma Control Test (ACT) to assess current asthma control; 2) the Asthma Quality
 of Life Questionnaire (AQLQ+12) to assess quality of life and psychological morbidity. To
 further asses sleeping quality 1) Athens Insomnia Scale (AIS), 2) Epworth Sleepiness Scale
 (ESS), 3) St. George's Respiratory Questionnaire (SGRQ) and 4) WHO (Five) Well-Being Index
 (WHO-5), 5) Fatigue Severity Scale (FSS).

 Exhaled nitric oxide (NO) will be measured at baseline and every 3 months according to the
 American Thoracic Society Guidelines and as specified by the manufacturer. FeNO levels will
 be reported as parts per billion. Measurements will be made before spirometry is performed.

 Venous blood samples will be retrieved between 8 to 10 am, at baseline and every 3 months
 during the 52-week period with mepolizumab treatment. Absolute blood eosinophil count will be
 reported as cells/μl.

 Spirometry: Spirometry A will be conducted, using the site's own equipment at the visits
 (baseline and every 3 months). All clinic visits must occur in the morning. During the
 treatment period the spirometry will meet the current ATS/ERS guidelines and the spirometer
 must produce a printout of all data generated, which should be stored in the subject's notes.
 The spirometer will be calibrated in accordance with the manufacturer's instructions.
 Spirometry must be performed at the same time (±1 hour) of the Visit 0 spirometry. Subjects
 should try to withhold short-acting beta-2-agonists (SABAs) for ≥4 hours and LABAs for ≥15
 hours prior to clinic visit, if possible (GINA 2019). Assessments to be recorded will include
 FEV1, FVC, FEV1/FVC%, PEF and FEF25-75. Post-bronchodilator measurements will be taken at
 every visit, using 200-400mcg albuterol or equivalent.

 Spirometry B will be conducted, using the site's own equipment at the visits (baseline, after
 6 months and after treatment at 52 weeks timepoint). Spirometry B includes diffusion,
 pulmonary volumes and capacities.

 Impulse oscillometry (optional):

 Impulse Osillometry (IOS) will be conducted at baseline and after 52 weeks of treatment. IOS
 includes resistance at 5 Hz-resistance at 20 Hz (R5-R20), a measure reflecting respiratory
 resistance of small to midsized conductive and peripheral airways, AX, a measure reflecting
 distensibility of the peripheral lungs (namely the parenchyma and small peripheral airways),
 and the respiratory system reactance (X5), a measure reflecting inertance and elasticity or
 capacitance (including small peripheral airways).

 Therapy with biologics:

 Upon completion of the screening visits the participant may initiate treatment with biologic
 therapy (mepolizumab) within a month. During therapy subjects will be under the care of the
 pulmonologist who had referred them to the research group for taking part into the study.
 These pulmonologists will be termed as 'associate pulmonologists. Treatment will be provided
 as per product manufacturer instructions.

 Associate pulmonologists providing biologic treatment during the study will require to sign
 an agreement to conduct the study in compliance with the protocol, to acknowledge that he/she
 is responsible for medical conduct and for study conduct, and to ensure that the associate
 (and their colleagues and employees) assisting in the conduct of the study are informed about
 their obligations. Mechanisms will be in place to ensure that associate staff receives the
 appropriate information and support by the research group throughout the study.

 Follow up during biologic therapy: At initiation and at every visit for biologic therapy
 administration collection of information will be performed by the associate pulmonologist,
 and with support by the research group if requested by the associate pulmonologist e.g.,
 researchers via telephone or face-to-face contact will interview the patient. Furthermore,
 compliance will be recorded form the physician based on patient's oral confirmation. Date
 form of drug administration will be recorded at every visit, as well as immediate and delayed
 adverse effects.

 Safety monitoring:

 Recorded adverse events (AEs) will be monitored and collated as required in study reports. An
 adverse event is the appearance or worsening of any undesirable sign, symptom, or medical
 condition occurring after starting the study entry. Medical conditions/diseases present
 before starting study drug are only considered adverse events if they worsen after starting
 study drug. Abnormal laboratory values or test results constitute adverse events only if they
 induce clinical signs or symptoms, are considered clinically significant, or require therapy.
 Also, adverse events related to Covid-19 are included. If a partner pulmonologist or research
 staff becomes aware of an AE associated with a product, it can be reported to the
 manufacturer according to their local standard practice for spontaneous AE reporting. A copy
 of the report should also be sent to the study coordinator. Serious adverse events (SAE) will
 be reported in the eCRF pages and within 24 hours all the investigators will have the
 responsibility to inform the Central Committee in Thessaloniki. Then the Central Committee
 will inform the scientific board and the Greek Medical Agency. Also, medical and scientific
 judgment will be exercised by the principal investigator of each center with the Central
 Committee, in deciding whether the subject should stop the treatment or not.

 Pregnancy report:

 Initially, female patients at screening will have to do a pregnancy test. During treatment,
 if a female subject reports a pregnancy to us, she will be asked to withdraw from the study
 (to prevent bias) Then, she will be asked if she would like to keep getting the treatment
 outside of this protocol and be monitored at each center's outpatient clinic.

 Pre-screening and Screening Failures:

 Subjects will be assigned a study number at the time of signing the consent. Subjects who
 signed but do not progress to the screening visits assessment will be deemed a pre-screen
 failure. No data will be captured in the database for these subjects.

 Subjects that complete at least one of the two screening visits, but do not initiate biologic
 therapy will be designated as screening failures. Information to be collected for
 pre-screening and screening failures will be detailed in the database completion.

 Withdrawal Visit:

 If a participant decides to withdraw from the study during follow-up (for any reason other
 than loss to follow up), they may be asked to attend a withdrawal visit (if deemed necessary
 by investigator and subject to consent). During the visit, the participant may be asked
 questions about their current conditions as part of clinical information collection such as:
 asthma and nasal symptoms, exacerbations and the progression of treatment being received.

 Blood samples' collection and processing:

 Blood samples will be collected at baseline visit, at 6 months and after treatment
 completion. After collecting the blood samples, they will be left undisturbed for about half
 an hour for complete clot formation. The sample will then be centrifuged to separate the
 serum from the clot. After centrifugation the serum will be stored at -20° C in eppendorf
 tubes till the analysis was done. Blood phenotyping will also be evaluated by flow cytometry.

 Induced sputum (optional):

 Induced sputum will be performed at baseline before treatment initiation and after completion
 of treatment according to the ERS Task Force report where a detailed expert consensus-based
 recommendation for induced sputum processing is suggested.

 At the visits, sputum induction will be performed after the routine spirometric assessments
 have been completed.

 The induced sputum will be processed within two hours of the end of the induction procedure
 at the research site. Sputum cells and cell suspension supernatant will be stored at -80ºC
 for analysis. After collecting the induced sputum, the supernatant of the sample will be
 stored at -20° C in eppendorf tubes till the analysis was done. Following these procedures,
 laboratory evaluation of the sputum is next according to guidelines.

 Bronchoscopy, bronchial biopsies and washing samples' collection and processing (optional):
 At the visits, endoscopy with bronchial biopsies, bronchial washing sample (Visit 0b) will be
 performed upon patient's written consent. In specific, a separate inform consent will be
 included to be signed from each patient. Bronchoscopy will be performed at two time-points
 (baseline before treatment initiation and after the completion of treatment). Flexible
 bronchoscopy and diagnostic techniques were performed under monitored anesthesia care as
 recommended by the American Thoracic Society and the British Thoracic Society.

 Tissue samples will be fixed in inflation or immersion using formaldehyde or glutaraldehyde
 and embedded in paraffin. Airways will be systematically sampled or obtained during
 diagnostic dissection at the discretion of the pathologist. Consecutive sections of 0.5, 5
 and 30 μm thickness were cut from each block and stained using the Masson's trichrome
 technique, haematoxylin and eosin, and haematoxylin, respectively. Immunohistochemical
 staining will be done done with the following mAbs: anti-mast cell tryptase clone AA1 (Dako
 UK), anti-alpha smooth muscle actin clone 1A4 (Dako UK), anti-eosinophil major basic protein
 clone BMK-13 (Monosan, Netherlands), anti-neutrophil elastase clone NP57 (Dako UK), and
 anti-endothelium clone EN4 (Monosan, Netherlands) or appropriate isotype controls were used.

 The endobronchial biopsies will be assessed by a single observer blinded to the clinical
 characteristics (ZEN 2012 image analysis software for light microscopy, Carl Zeiss AG,
 Germany) and will be expressed as the mean of measurements undertaken from a minimum of two
 sections either from independent biopsies or as non-contiguous tissue sections at least 20μm
 apart from the same biopsy. Epithelial integrity will be assessed by measuring the lengths of
 intact and denuded epithelium. These will be expressed as percentage of all the reticular
 basement membrane (RBM) length. Vascularity will be measured using the Chalkley count, a
 surrogate of both vessel density and vascular area. A Chalkley eyepiece graticule (NG52
 Chalkley Point Array, Pyser-SGI Ltd, UK) will be used at x200 to measure Chalkley counts in
 four non-overlapping vascular hotspots (6). Airway smooth muscle content will be determined
 as the proportion of the total area. Inflammatory cells were expressed as the number of
 nucleated cells/area of lamina propria.

 Bronchial washing samples will be immediately transported to the laboratory, to be stored at
 -80 degrees. Examination of bronchial washing fluids will be performed at a later stage to
 determine several biomarkers.

 Analysis of biomarkers of inflammation with Flow Cytometry Analysis (FACS) or ELIZA:

 Biomarkers included before and after treatment with mepolizumab in two or three timepoints,
 (at baseline, at 6 months treatment and after treatment completion visit): lymphocyte
 subsets, Th2/Th17 cytokines, chemokines, microRNAs expression to be quantitated by flow
 cytometry or ELISA) in biological fluids (blood, serum, bronchial washing, sputum and sputum
 supernatant).

 Panel kits and appropriate antibodies will be tested by Flow Cytometer Analysis using a BD
 FACS Calibur system (BD Biosciences, San Jose, CA), according to the manufacturer's
 recommendations and instructions.

 ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for
 detecting and quantifying substances such as peptides, proteins, antibodies and hormones.
 ELISA kits will be analyzed using VICTOR X3 spectrophotometer according to the manufacturer's
 recommendations and instructions.

 Allergy testing (If applicable) The patients' atopic status will be evaluated (if not
 available) at screening visit by the skin prick test (SPT) to a standard panel of
 aeroallergens for detecting total IgE antibodies against various common inhalant allergens.

 Database An electronic data collection platform will be the method used at baseline every 3
 months until 1 year follow up for this database. During treatment visits paper
 forms/questionnaires will be completed by the associate pulmonologist. Each site will entry
 specific data in an electronic database. Each patient is uniquely identified by a Subject
 number which is composed by the site number assigned by the responsible monitoring site and a
 sequential number assigned by the investigator. Upon signing the informed consent form, the
 patient is assigned the next sequential number by the investigator. The site must contact the
 central research personnel and provide the requested identifying information for the patient
 as registered into the electronic database. The treatment visit forms will also be sent to
 the central research personnel. The authorized study personnel will be responsible for data
 collection, editing, and also protecting the data being collected at their own site and the
 database is backed up on a daily basis. Quality review/control will be the joint
 responsibility of all individuals involved and the central research personnel [data
 custodian].

 Sample size calculation G power analysis Sample size is calculated using the G*Power software
 (Die Heinrich-Heine-Universität Düsseldorf, Germany). The difference in annual exacerbation
 rate of the patients with late onset severe eosinophilic asthmatics before and after
 Mepolizumab treatment with is the primary endpoint of the study. In the DREAM study (one of
 the only two studies that has reported results similar to our primary endpoint so far) it was
 reported that the annual exacerbation rate of patients with severe Asthma declined from 3.6
 to 1.33 after the initiation of treatment with Mepolizumab. Using this data and a two-sided
 test, with a power at 0.95 and a level of statistical significance at 0.05, the investigators
 calculated an effect size of 0.556, which indicated a total sample size of 45 participants.

 Statistical Plan Analysis Statistical analysis will be performed using the SPSS (version 21.0
 IBM SPSS statistical software, Armonk, NY, USA). Categorical variables will be stated as
 numbers (n) and percentages (%), continuous variables as mean ± SD if normally distributed,
 or median and interquartile ranges if otherwise. Where necessary variables without a normal
 distribution will be transformed. P < 0.05 was accepted as statistically significant.
 Shapiro-Wilk test will be performed in order to separate parametric from non-parametric
 variables. Paired-Samples T Test for parametric or Wilcoxon Signed Ranks test for
 non-parametric variables will be used to detect differences in the variables measured before
 and after Mepolizumab treatment.

 There is no comparator arm as every patient is control to himself. There are baseline
 characteristics (clinical and laboratory biomarkers) that are going to be measured at
 baseline, every 3 months and after 52-week treatment completion. Study endpoints include the
 analysis of all those biomarkers as the identification of the characteristics of
 non-responders and super-responders. Also, secondary endpoints include the evaluation of the
 biomarkers mentioned in the methods as assessments. Statistical analysis will be performed
 initially with the number of patients that have completed the 52-week period of treatment.
 Secondly, patients that have not completed the 52-week treatment will be analyzed separately
 and finally these two groups are going to be compared to each other.

 More specifically, with univariate analysis (two-sided independent sample tests) the
 investigators will test for associations/correlations between all baseline outcomes compared
 with the ones after mepolizumab treatments (every 3 months until 1 year of treatment):
 biomarkers in sputum and in blood, clinical outcomes. Similarly, univariate associations
 between outcomes and personal/demographic characteristics will be tested for identifying
 potential confounders. Significant associations will be then tested with adjusted regression
 models. Associations/correlations will be evaluated separately at baseline clinical
 characteristics and every 3 months follow up during treatment as well as at baseline and
 after 1 year of mepolizumab treatment. Besides, subgrouping analysis will be performed for
 each biomarker for the identification of biomarker level to treatment response.

 Paired-Samples T Test for parametric or Wilcoxon Signed Ranks test for non-parametric
 variables will be used to detect differences in the variables measured before and after
 Mepolizumab treatment. In specific, levels of biomarkers at baseline will be compared to
 levels after 1-year of mepolizumab treatment.

 Overall response to treatment in one year (from baseline to 1-year post treatment initiation)
 for each outcome will be measured with paired test of dependent samples (parametric and
 non-parametric paired tests) in each patient. Then response to treatment will be defined as
 the change (absolute and % change) of the outcome in one year. The investigators will assess
 predictors influencing response to treatment (change) such as biomarkers by using multiple
 regression models for each clinicopathological outcome. For outcomes with more than 2
 observations per subject during the study period the investigators will use mixed models
 using the repeated measurements of the outcome. All tests will be 2-sided with a statistical
 significance level at 5%.
Inclusion Criteria:

 Key inclusion criteria

 -  Written informed consent must be obtained at screening visit, before any assessment
 will be performed. Subjects should be able to provide informed written consent (study
 participation informed consent form): Able to give written informed consent prior to
 participation in the study, which will include the ability to comply with the
 requirements and restrictions listed in the consent form. Subjects must be able to
 read, comprehend, and write at a level sufficient to complete study related materials.

 -  Confirmed asthma diagnosis and severity and treatment requirements.

 -  Fixed airway obstruction (FAO) where post-bronchodilator treatment ratio of forced
 expiratory volume in 1 second [FEV1] to forced vital capacity [FVC] is below < 70%.

 -  Confirmed optimized management skills (inhaler technique, education, adherence)

 -  Triggers and relevant co-morbidity have been assessed and managed Triggers such as
 smoking, beta-blockers, aspirin/NSAIDs, allergen exposure; Comorbidities such as
 rhinitis, obesity, GERD, OSA, VCD, depression/anxiety.

 -  Severe asthmatics with blood eosinophils ≥150cells/ul at screening visit or
 ≥300cells/ul the last 12 months.

 -  Patients with late-onset severe according to ERS/ATS guidelines eosinophilic asthma
 and fixed obstruction under the treatment of high dose of ICS+LABA±LAMA (late-onset
 asthma determined as age at diagnosis from 20 years and above)

 -  Patients with late-onset severe eosinophilic asthma with history ≥ 1 exacerbation the
 previous year under the treatment of high dose of ICS+LABA±LAMA.

 -  Asthma Exacerbation: Subjects with an ongoing asthma exacerbation should have their
 screening and treatment initiation visit delayed until the investigator considers the
 subject has returned to their baseline asthma status. If the 4-week screening period
 has elapsed then the subject should be considered a screening failure. An exacerbation
 is defined as worsening of asthma requiring the use of systemic corticosteroids and/or
 emergency department visit, or hospitalization. For subjects on maintenance oral
 corticosteroids, an exacerbation requiring oral corticosteroids was defined as the use
 of oral/systemic corticosteroids at least double the existing dose for at least 3
 days.

 -  Maintenance Asthma Therapy: No changes in the dose or regimen of baseline ICS and/or
 additional controller medication during the screening period (except for treatment of
 an exacerbation).

 -  Meet requirements for biologic therapy with mepolizumab.

 Exclusion Criteria:

 -  Asthma exacerbation, within 6 weeks prior to screening that required hospitalization
 or emergency room visit.

 -  Prior use of other biologics (including but not limited to Omalizumab, Reslizumab,
 Dupilumab, Benralizumab etc., for asthma or any other indications) that has potential
 to interfere/ affect disease progression, in the previous 6 months.

 -  Pregnant or nursing women, or women of child-bearing potential.

 -  History of malignancy of any organ system or any other serious co-morbidities defined
 by the treating physician.

 -  Patients with a history of conditions other than asthma that could result in elevated
 eosinophils (e.g. hypereosinophilic syndromes, Churg-Strauss Syndrome, eosinophilic
 esophagitis).