Exploratory Investigation of Changes in Gene Transcription and Immunophenotypes Following Mepolizumab Treatment for Asthma
Conditions: Eosinophilic Asthma
Sponsor: Mayo Clinic

Study Protocol and Statistical Analysis Plan 
 
Study Title: Exploratory Investigation of Changes in Gene Transcription and 
Immunophenotypes Following Mepolizumab Treatment for Asthma 
 
NCT number: NCT05002621 
 
Document Date: December 11, 2025 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Study Protocol and Statistical Analysis Plan 
 
Study Population 
Study subjects: This study involved twenty adult (≥18yo) individuals with a diagnosis of severe 
eosinophilic asthma (SEA) who have been prescribed mepolizumab during their course of 
routine clinical care. Children (<18yo) will be excluded from this study. 
 
Inclusion criteria: 
1. Diagnosis of SEA by a health care provider 
2. History of an absolute eosinophil count ≥ 300/mm^3 
3. Prescription for mepolizumab provided during course of routine clinical care but mepolizumab 
not yet started 
 
Exclusion criteria: 
1. Age <18 years old 
2. Pregnancy 
3. Currently using or have used within 3 months of the initial baseline visit any biologic or 
immunomodulatory therapy with the exception of #3 
4. Currently using or any prior use of rituximab 
5. History of upper/lower respiratory tract infection or asthma exacerbation within the previous 
   four weeks of the first baseline visit 
6. Any prior history of malignancy, autoimmune disease, or immune deficiency 
7. Any other significant medical issue as determined by the principal investigator 
 
Methods 
Each study subject had sequential whole blood samples obtained at four time points. The first 
time point was prior to receiving the initial dose of mepolizumab. Thereafter, blood samples 
were obtained at 4 weeks, 8 weeks, and 12 weeks post-initiation of mepolizumab (prior to each 
subsequent dose of mepolizumab). Blood obtained was isolated for PBMCs to undergo 
immunophenotyping by mass cytometry (CyTOF) and to undergo RNA sequencing. 

 
Data Collection 
In addition to usual demographical variables and record of meeting inclusion criteria, each 
research subject had the following primary data collected: 
 
1. 
Differentially expressed gene transcripts identified by RNAseq from PBMCs 
2. 
CyTOF mass cytometry (50 test immune phenotype panel including cell surface 
markers, transcription factors, and cytokines) performed on PBMCs isolated from peripheral 
blood 
3. 
Laboratory values in accordance with clinical guidelines and standard of care such as 
CBC with differential, liver function studies, etc. 
4. 
Record review of allergy testing, pulmonary function testing, etc. in accordance with 
clinical guidelines and standard of care 
5. 
Record review of cllinical course and associated therapies such as corticosteroids, 
leukotriene modifiers, etc 
 
Study Endpoints 
Subject participation ended after the fourth blood sample was obtained. Primary analysis was 
descriptive of data collected. Secondary analysis involved associations between changes in 
gene transcripts and immunophenotypes and the respective clinical correlates. 
 
Data analysis 
Our institution sequencing core has developed MAP-RSeq - a comprehensive computational 
pipeline for secondary analysis of RNA- Sequencing data. MAP-RSeq uses a variety of freely 
available bioinformatics tools along with in-house developed methods. The main goal of the 
MAP-RSeq workflow is to align, assess and provide multiple genomic features from 
transcriptomic sequencing data for further tertiary analysis. 
 
For CyTOF data, FCS files were exported and analyzed using Cytobank (Cytobank, Inc., 
Mountain View, CA). Doublets and debris will be excluded using standard methods. Non-
hierarchical clustering was performed using viSNE. Expression levels were displayed as median 
intensities in all viSNE plots.