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| Name | Class |
|---|---|
| GlaxoSmithKline | INDUSTRY |
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Two parts: A:Case-control study including 15 healthy adult donors and 15 severe adult eosinophilic asthmatics selected for treatment with mepolizumab. B: A longitudinal cohort study,where the same patients once on mepolizumab treatment are followed over time (0, 4, 16 and 32 weeks). SCOPE: response to mepolizumab in severe adult eosinophilic asthma.
INCLUSION CRITERIA: Male or female, 18-75 years-old, with severe eosinophilic asthma. EXCLUSION CRITERIA: Smoking history, recent exacerbations, other pulmonary or systemic disease with eosinophilia, malignancy, pregnancy, obesity (BMI >35). OBJECTIVES: General objective: Discovery of predictive/prognostic biomarkers of response to mepolizumab using flow cytometry, transcriptomic, and proteomic technologies. OTHER OBJECTIVES: 1.-To identify changes in surface markers of eosinophils and eosinophil subpopulations in response to treatment with mepolizumab using flow cytometry techniques. 2.-Transcriptomic analysis to identify mRNAs within the eosinophil transcriptome displaying enhanced or reduced levels in response to treatment with mepolizumab.3.-Proteomic profiling to identify proteins with differential abundance within the eosinophils in response to treatment with mepolizumab.4.-Check whether late-onset severe eosinophilic asthmatics display elevated levels of IGF-1, IGF-BP3, IGF-ALS in serum samples, if the response of mepolizumab depends on the levels of this markers, and if treatment with this biological reduces the concentration in serum of these IGF-family members. 5.-Identify proteins with differential abundance within the deep serum proteome of patients with SEA in response to treatment with mepolizumab by means of non-targeted proteomic analysis.
MEASUREMENTS: Flow cytometry assays with multimarker panels 1 (regulatory), 2 (activation), and 3 eosinophil subsets. Clinical, hematological, biochemical and flow cytometry data generated at times T4, T16 and T32. Total RNA extraction from eosinophil lysates, assay of quality and quantity of RNA, and storage at -80ºC. Evaluation of the levels of 770 human protein-coding mRNAs linked to the recruitment, activation, and effector functions of myeloid cells by means of a direct multiplexed molecular measurement platform named nCounter® NanoString) in combination with a pre-made "nCounter® Human Myeloid Innate Immunity Panel (v2)". Perform retrotranscription and qPCR analyses of those mRNAs in eosinophils displaying the greatest abundance changes in response to mepolizumab treatment according to the nCounter® study. In addition, some additional mRNAs not included in the "nanoString Myeloid Innate Immunity" panel, such as FOXP3 (regulatory function), CRLF2, ST2, or IL-7R (cytokine receptors; activation), will be analysed. HPRT1 gene will be used as a house-keeping gene in this set of RTqPCR experiments. Perform SWATH-MS analysis in samples from 15 healthy donors and 15 patients (T0, T4, T16, T32) ("information-dependent acquisition" method or IDA; "Targeted label-free proteomics") in eosinophil homogenates. High abundant serum protein depletion using two protocols (P1: affinity chromatography, and P2: DTT precipitation) and SWATH-MS analysis of medium-low abundant serum proteome in samples from 15 healthy donors and 15 patients (T0, T4, T16, T32) ("information-dependent acquisition" method or IDA; "Targeted label-free proteomics").
Hypotheses
Hº1. The levels of certain surface molecules on eosinophils or the presence or absence of certain proteins in the proteome of this leukocyte subset prior mepolizumab treatment can be used as predictive/prognostic markers of response to this biological.
Hº2. Mepolizumab alters the abundance of several surface or intracellular proteins in eosinophils as an outcome related to changes in their activation status, migratory ability, regulatory/effector function, or subset composition.
Hº3. Late-onset severe eosinophilic asthmatics have elevations in the serum concentration of different members of the IGF family (IGF-1, IGF-BP3, IGF-ALS) and mepolizumab treatment reduces these levels and behaves as a response-biomarker along with the number of eosinophils and clinical exacerbations.
H°4. Mepolizumab alters the abundance of several proteins in the medium-low abundance serum proteome of patients with SEA. Therefore, these proteins could be used as predictive/prognostic markers of response to this biological and could provide a better understanding of both eosinophilic and non-eosinophilic-related biological functions of IL-5 in SEA.
Objectives or research questions
OB (Aspirational objective): Discovery of predictive/prognostic biomarkers of response to mepolizumab using flow cytometry, transcriptomic, and proteomic technologies.
OB1.- To identify changes in surface markers of eosinophils and eosinophil subpopulations in response to treatment with mepolizumab using flow cytometry techniques:
OB2.- Transcriptomic analysis to identify mRNAs within the eosinophil transcriptome displaying enhanced or reduced levels in response to treatment with mepolizumab:
OB3.- Proteomic profiling to identify proteins with differential abundance within the eosinophils in response to treatment with mepolizumab:
OB4. Check whether late-onset severe eosinophilic asthmatics display elevated levels of IGF-1, IGF-BP3, IGF-ALS in serum samples, if the response of mepolizumab depends on the levels of these markers, and if treatment with this biological reduces the concentration in serum of these IGF-family members:
OB5.- Identify proteins with differential abundance within the low abundant serum proteome of patients with severe eosinophilic asthma in response to treatment with mepolizumab.
Publication of results:
We expect to present 2-3 communications to Spanish Respiratory Congress (SEPAR) and the European Respiratory Congress (ERS) resulting from the study of the clinical and experimental data. In addition, we expect to publish 3 publications in Q1 journals.
Study population The study population will include healthy controls (i.e., subjects without asthma, allergy, systemic diseases or scheduled for minor surgeries) and severe eosinophilic asthma patients, who will be recruited from different areas of Galicia (Santiago de Compostela, A Coruña, Lugo, Vigo, and Ourense), Spain. Diagnosis of severe eosinophilic asthma patients at screening will be based on several inclusion criteria and exclusion criteria that we describe below.
Inclusion criteria:
Exclusion criteria:
Sample size
Cohort of healthy controls (n=15) only for analysis at T = 0.
Cohort of n=15 subjects with severe eosinophilic asthma that start with mepolizumab therapy with no modification to their currently prescribed medications. Follow-up study visits at 4 (T4), 16 (T16) and 32 (T32) weeks after the original study visit (T=0).
Anticipated rate of enrolment Since this will be a multicentre study, we expect to reach a rate of enrolment of at least 2 severe eosinophilic asthmatics beginning with mepolizumab therapy per month (4 weeks) in each hospital (Total = 8 per month). This means that the 15 subjects should be scheduled to receive mepolizumab along the first 36 weeks of this study. Considering the recruitment and experimental parts, we expect to complete the study in 120 weeks (30 months; ~2.5 years). We also expect that at least 90% of subjects complete this study.
Estimated study start date: June 2021 Estimated study completion date: 2.5 years (30 months)
Study design and methods
This is an observational, longitudinal, prospective, and multicentre study to evaluate both the early response (4 weeks) and late response (16 and 32 weeks) to mepolizumab therapy in severe eosinophilic asthmatics. The study will be headed by Dr. Francisco Javier González Barcala (Pneumology Service at CHUS), the leader of the Translational Research in Airway Diseases (TRIAD) group. Other members of the TRIAD group are Dr. Francisco Javier Salgado Castro, the Project Manager, and Dr. Juan José Nieto Fontarigo, both experts in Immunology, Biochemistry, Proteomics, and Respiratory Diseases. Dra. Marina Blanco Aparicio is responsible for the Asthma Unit at the University Hospital Complex of A Coruña (CHUAC); Dr. Uxío Calvo Álvarez, at the University Hospital Complex of Ferrol (CHUF); Dra. Coral González Fernández at the University Hospital Complex of Ourense (CHUO); Dra. Mar Mosteiro Añón at the University Hospital Complex of Vigo-Alvaro Cunqueiro (CHUVI); and Dolores Corbacho Abelaira at the POVISA Hospital Centre (Vigo). Proteomics experiments will be carried out by Dra. Susana Belén Bravo López at (FIDIS). nCounter® analysis will be carried out by GENVIP group, at FIDIS.
The research project will be minimally invasive (e.g., no bronchoscopic examinations) but the protocol needs to be reviewed and approved by the Ethics Committee of Clinical Research of Galicia, Spain. Only fifteen patients who meet the severe asthma diagnosis criteria, are scheduled to receive mepolizumab, and sign the informed consent will be enrolled in this study. The same protocol will be followed by the different clinical teams. Demographic, as well as clinical, haematological, and biochemical variables will be included in a database. Skin prick test to common allergens and the presence of allergen-specific IgE (ImmunoCAP, Thermo Fisher) will be used to check for allergic sensitization. Lung function parameters (forced expiratory volume in the 1st second (FEV1), forced vital capacity (FVC), and FEV1/FVC ratio) also will be analysed. Spirometry will be performed before and after use of a bronchodilator. The Asthma Control Test (ACT) and the Asthma Quality of Life Questionnaire (AQLQ) questionnaire will be performed. Asthmatics must be in a stable phase of the disease (i.e. absence of exacerbations for at least 4 weeks before sample collection). Exacerbations will be managed in accordance with standard clinical guidelines. Patients (n=15) will receive 100 mg subcutaneous injection of mepolizumab at 4 weeks intervals, and blood and serum samples (2-3 EDTA tubes; 1 SST tube) will be withdrawn at T=0, 4, 16 and 32 weeks, in order to evaluate both early-response (4 weeks) and late-response (16 and 32 weeks) to treatment.
Methods
Tubes: EDTA (complete blood) and SST (serum)
Eosinophils purification
ELISA studies.
Total RNA purification from eosinophils and nCounter nanoString analysis (Discovery-based/hypothesis-generating approach):
RTqPCR studies (Hypothesis-driven approach):
Flow cytometry studies (Hypothesis-driven approach):
EDTA-treated peripheral blood samples from healthy controls (n=15; T0) and mepolizumab-treated patients (n=15; T0, T4, T16, T32).
Label 100 μL/tube of whole peripheral blood (EDTA) with both specific and isotype-matched control antibodies (BD). Red cells lysis with FACSlyse (BD). Analysis with a FACSCalibur flow cytometer (BD). Use FSC/SSC to select granulocytes; then SSC vs CCR3 (FITC) to separate eosinophils from neutrophils. Gate eosinophils:
Analysis of eosinophil proteome (Discovery-based/hypothesis-generating approach):
Analysis of medium-low abundant serum proteome (Discovery-based/hypothesis-generating approach):
Study endpoints:
Demographic data for all individuals enrolled in the study will be obtained at basal. In addition, several data will be collected, including asthma history, lung function parameters, skin prick test, allergen-specific IgE, AQLQ score, ACT score, the number of exacerbations, and consumption of prednisone. During the following visits to the Pneumology service at T0, 4, 16, and 32, patients treated with mepolizumab will be followed up. This includes measurements of lung function (FEV1, FEV1/FVC), biochemical and haematological parameters.
Peripheral blood and serum samples will be collected, and eosinophils will be magnetically purified, at T0, T4, T16, and T32. Flow cytometry, RTqPCR, and proteomic analyses, as well as immunoassays, will be performed. All the experimental variables (e.g., the abundance of eosinophil proteins in proteomic assays, the abundance of serum proteins, eosinophil activation markers, …) will be correlated with clinical parameters (e.g., lung function, asthma control, number of exacerbations) in order to assess the association of these variables with the response to treatment. We will consider a favourable response to mepolizumab if one of the following criteria is met:
To obtain adequate asthma control ACT ≥20 [60], or/ a change of ≥3 points representing a minimally important difference.
To achieve a reduction in the annual rate of exacerbations of 48%. Exacerbation is defined as the increase in symptoms requiring treatment with systemic corticosteroids for ≥3, or an unscheduled medical consultation, similar to that reflected in clinical trials with mepolizumab [20, 61].
Get a 50% reduction in the annual rate of hospital admissions due to asthma exacerbation, similar to that reflected in clinical trials [62].
To achieve a reduction in the median annual dose of systemic corticosteroids of 50% [63].
Study primary endpoints:
Study secondary endpoints:
Statistical plan or data analysis:
Graph Pad Prism will be used to create graphics. IBM SPSS, Statistics 22.0, or R. will be used for the statistical study. During the analyses, we will be assisted by the USC Statistics and Operational Research area (Dr. Rosa María Crujeiras Casais).
Sample size The calculation of sample size (N) has been carried out by using G*Power 3.1.9.4 [64]. During these analyses we calculate N necessary get statistical significance in a F test (ANOVA: Repeated measures, within factors), given α (0.05), power (1-β, 0.95), the number of measurements (T0, T4, T16 and T32), and the effect size (f = 0.4; large effect size, which gives as a more clinically relevant results). The output N was 15, with a critical F= 2.82705.
For clinical, flow cytometry and transcriptomic data. Cross-sectional comparisons between HC and patients in T0 (before treatment) following a normal distribution and having homogeneity of variances will be made by using t-test. For non-normal distributed variables, we will use Mann-Whitney U test. Changes in the different study variables in response to treatment with mepolizumab (longitudinal study; T0, T4, T16, and T32) will be tested using RM-ANOVA. Multivariate analysis (e.g., PCA, unsupervised clustering) as well as functional enrichment analysis will be performed with flow cytometry, and above all, transcriptomic data.
For total proteome characterization and quantitative SWATH analysis We will use ProteinPilotTM 5.0.1 software from ABSciex which have the algorithm ParagonTM for database search and ProgroupTM for data grouping. Data will be searched using a Human specific Uniprot database. False discovery rate will be performed using a non-lineal fitting method displaying only those results that reported a 1% Global false discovery rate or better [65].
Functional analysis will be performed by different open-access software. FunRich (Functional Enrichment analysis tool) for functional enrichment and interaction network analysis (http://funrich.org/index.html). For statistics, FunRich uses hypergeometric test, BH and Bonferroni [66, 67]. We will use DAVID (https://david.ncifcrf.gov/tools.jsp) or GO (http://geneontology.org/page/go-enrichment-analysis) for gene ontology enrichment and for protein-protein interaction, network construction and clustering, we will use String (https://string-db.org/) or Cytoscape 3.7 (https://cytoscape.org/ ) [68].
For SWATH data, MarkerView software will give us a multivariate statistical analysis using principal component analysis (PCA) to compare the data across the samples. The average MS peak area of each protein will be derived from the replicates of the SWATH-MS of each sample followed by Student's t-test analysis using the MarkerView software for comparison among the samples based on the averaged area sums of all the transitions derived for each protein. The t-test will indicate how well each variable distinguishes the two groups, reported as a P-value. For the library, its set of differentially abundant proteins (p-value <0.05) with a 1.5 up-regulated or down-regulated proteins will be selected.
Limitations
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| Label | Type | Description | Intervention Names |
|---|---|---|---|
| Control | Healthy adults | ||
| Severe eosinophilic asthma | Severe uncontrolled asthma according to ERS/ATS criteria and persistent eosinophilia in blood (>300 cells/μL) |
|
| Name | Type | Description | Arm Group Labels | Other Names |
|---|---|---|---|---|
| Mepolizumab 100 MG | Drug | Discovery of predictive/prognostic biomarkers of response to mepolizumab using flow cytometry, transcriptomic, and proteomic technologies. |
|
| Measure | Description | Time Frame |
|---|---|---|
| Measure changes in surface markers of eosinophils and eosinophil subpopulations in response to treatment with mepolizumab using flow cytometry techniques | Flow cytometry assays with multimarker panels 1 (regulatory), 2 (activation), and 3 (eosinophil subsets) | 32 weeks |
| Measure changes in medium-low abundant serum proteins in response to treatment with mepolizumab using LC-MS/MS | Changes in the levels of multiple proteins within the low abundant serum proteome from patients, measured as the ratio to baseline (T=0) at weeks 4 and 32 (SWATH-MS), and ratio to healthy controls (T=0) in patients at T=0. | 32 weeks |
| Measure | Description | Time Frame |
|---|---|---|
| Transcriptomic analysis to identify mRNAs within the eosinophil transcriptome displaying enhanced or reduced levels in response to treatment with mepolizumab. | Evaluation of the levels of 770 human protein-coding mRNAs linked to the recruitment, activation, and effector functions of myeloid cells by means of a direct multiplexed molecular measurement platform named nCounter® NanoString) in combination with a pre-made "nCounter® Human Myeloid Innate Immunity Panel (v2)" |
Not provided
Inclusion Criteria:
Exclusion Criteria:
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Severe uncontrolled asthma according to ERS/ATS criteria Persistent eosinophilia in blood (>300 cells/μL)
| Name | Role | Phone | Extension | |
|---|---|---|---|---|
| FRANCISCO-JAVIER GONZALEZ-BARCALA, MD, PHD | Contact | +34981951173 | francisco.javier.gonzalez.barcala@sergas.es | |
| FRANCISCO-JAVIER SALGADO-CASTRO, PHD | Contact | +34981563100 | 12263 | franciscojavier.salgado@usc.es |
| Name | Affiliation | Role |
|---|---|---|
| FRANCISCO-JAVIER GONZALEZ-BARCALA, MD, PHD | CLINIC UNIVERSITY HOSPITAL | Principal Investigator |
| Facility | Status | City | State | ZIP | Country | Contacts |
|---|---|---|---|---|---|---|
| Complejo Hospitalario Universitario de Ferrol | Recruiting | Ferrol | A Coruña | 15405 | Spain |
| PubMed Identifier | Type | Citation | Retractions |
|---|---|---|---|
| 19483109 | Background | Woodruff PG, Modrek B, Choy DF, Jia G, Abbas AR, Ellwanger A, Koth LL, Arron JR, Fahy JV. T-helper type 2-driven inflammation defines major subphenotypes of asthma. Am J Respir Crit Care Med. 2009 Sep 1;180(5):388-95. doi: 10.1164/rccm.200903-0392OC. Epub 2009 May 29. | |
| 21281866 | Background | Lotvall J, Akdis CA, Bacharier LB, Bjermer L, Casale TB, Custovic A, Lemanske RF Jr, Wardlaw AJ, Wenzel SE, Greenberger PA. Asthma endotypes: a new approach to classification of disease entities within the asthma syndrome. J Allergy Clin Immunol. 2011 Feb;127(2):355-60. doi: 10.1016/j.jaci.2010.11.037. |
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| ID | Type | URL | Comment |
|---|---|---|---|
| FGBMEP202001 | Study Protocol | View IPD |
Individual data are confidential
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| Type | Date | Date Unknown |
|---|---|---|
| Release | Nov 26, 2025 | |
| Reset | Dec 11, 2025 |
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| Release Date | Unrelease Date | Unrelease Date Unknown | Reset Date | MCP Release Number |
|---|---|---|---|---|
| Nov 26, 2025 | Dec 11, 2025 |
| ID | Term |
|---|---|
| D001249 | Asthma |
| D011657 | Pulmonary Eosinophilia |
| ID | Term |
|---|---|
| D001982 | Bronchial Diseases |
| D012140 | Respiratory Tract Diseases |
| D008173 | Lung Diseases, Obstructive |
| D008171 | Lung Diseases |
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| ID | Term |
|---|---|
| C434107 | mepolizumab |
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Whole blood and serum
| 32 weeks |
| Hospital Clínico Universitario de Santiago de Compostela | Not yet recruiting | Santiago de Compostela | A Coruña | 15706 | Spain |
|
| Hospital Povisa - Grupo Ribera Salud | Not yet recruiting | Vigo | Pontevedra | 36211 | Spain |
|
| Complexo Hospitalario Universitario A Coruña | Not yet recruiting | A Coruña | 15006 | Spain |
|
| Complexo Hospitalario Universitario de Ourense | Recruiting | Ourense | 32005 | Spain |
|
| 22561835 | Background | Wenzel SE. Asthma phenotypes: the evolution from clinical to molecular approaches. Nat Med. 2012 May 4;18(5):716-25. doi: 10.1038/nm.2678. |
| 30206782 | Background | Kuruvilla ME, Lee FE, Lee GB. Understanding Asthma Phenotypes, Endotypes, and Mechanisms of Disease. Clin Rev Allergy Immunol. 2019 Apr;56(2):219-233. doi: 10.1007/s12016-018-8712-1. |
| 30578390 | Background | Pavlidis S, Takahashi K, Ng Kee Kwong F, Xie J, Hoda U, Sun K, Elyasigomari V, Agapow P, Loza M, Baribaud F, Chanez P, Fowler SJ, Shaw DE, Fleming LJ, Howarth PH, Sousa AR, Corfield J, Auffray C, De Meulder B, Knowles R, Sterk PJ, Guo Y, Adcock IM, Djukanovic R, Fan Chung K; on behalf of the U-BIOPRED Study Group. "T2-high" in severe asthma related to blood eosinophil, exhaled nitric oxide and serum periostin. Eur Respir J. 2019 Jan 3;53(1):1800938. doi: 10.1183/13993003.00938-2018. Print 2019 Jan. |
| 31047111 | Background | Yancey SW, Bradford ES, Keene ON. Disease burden and efficacy of mepolizumab in patients with severe asthma and blood eosinophil counts of >/=150-300 cells/muL. Respir Med. 2019 May;151:139-141. doi: 10.1016/j.rmed.2019.04.008. Epub 2019 Apr 8. |
| 28862877 | Background | Kelly EA, Esnault S, Liu LY, Evans MD, Johansson MW, Mathur S, Mosher DF, Denlinger LC, Jarjour NN. Mepolizumab Attenuates Airway Eosinophil Numbers, but Not Their Functional Phenotype, in Asthma. Am J Respir Crit Care Med. 2017 Dec 1;196(11):1385-1395. doi: 10.1164/rccm.201611-2234OC. |
| 29514744 | Background | Gonzalez-Barcala FJ, San-Jose ME, Nieto-Fontarigo JJ, Carreira JM, Calvo-Alvarez U, Cruz MJ, Facal D, Garcia-Sanz MT, Valdes-Cuadrado L, Salgado FJ. Association between blood eosinophil count with asthma hospital readmissions. Eur J Intern Med. 2018 Jul;53:34-39. doi: 10.1016/j.ejim.2018.02.034. Epub 2018 Mar 4. |
| 29317355 | Background | Ortea I, Ruiz-Sanchez I, Canete R, Caballero-Villarraso J, Canete MD. Identification of candidate serum biomarkers of childhood-onset growth hormone deficiency using SWATH-MS and feature selection. J Proteomics. 2018 Mar 20;175:105-113. doi: 10.1016/j.jprot.2018.01.003. Epub 2018 Jan 6. |
The study protocol was added to clinicaltrials.gov |
| D012130 |
| Respiratory Hypersensitivity |
| D006969 | Hypersensitivity, Immediate |
| D006967 | Hypersensitivity |
| D007154 | Immune System Diseases |
| D017681 | Hypereosinophilic Syndrome |
| D004802 | Eosinophilia |
| D007960 | Leukocyte Disorders |
| D006402 | Hematologic Diseases |
| D006425 | Hemic and Lymphatic Diseases |