Identification of Autoantigens and Their Potential Post-translational Modification in EGPA and Severe Eosinophilic Asthma
Conditions: Churg-Strauss Syndrome, Eosinophilic Asthma, Eosinophilic Esophagitis, Eosinophilic Pneumonia
Sponsor: Queen Mary University of London

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 1 of 25 
 
Identification of autoantigens and their potential post-translational 
modification in EGPA and Severe Eosinophilic Asthma. 
 
JRMO Research Protocol for Research Studies  
 
 
 
 
Full Title  
Identification of autoantigens and their potential 
post-translational modification in EGPA and 
Severe Eosinophilic Asthma. 
 
Short Title 
IDentification of EGPA Autoantigens (IDEA) 
 
Sponsor 
Queen Mary University of London 
 
Contact person: 
 
Tumi Kaminskas 
Research 
Governance 
and 
Performance 
Manager 
Joint Research Management Office 
 
Research Services 
Dept W 
69-89 Mile End Road, 
 
London 
 
E1 4UJ  
 
Phone: 020 7882 7275 
 
Email: research.governance@qmul.ac.uk 
 
IRAS Number 
274097 
 
 
 
 
 
REC Reference 
20/PR/0004 
 
Chief Investigator 
Dr Myles Lewis, 
 
Senior Lecturer and Consultant Rheumatologist, 
 
Queen Mary University of London and Barts 
Health NHS Trust, UK 
 
William Harvey Research Institute,  
Charterhouse Square, 
London EC1M 6BQ, UK 
 
myles.lewis@qmul.ac.uk 
 
 
+44(0) 20 7882 3305 
 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 2 of 25 
 
 
 
List of sites 
Patient and Healthy Control Recruitment: 
 
  St Bartholomew’s Hospital,  
 
  Mile End Hospital and  
 
 
  Royal London Hospital, 
 
  Barts Health NHS Trust 
Royal Free London NHS Trust 
 
 
Research Laboratories: 
 
  William Harvey Research Institute, 
 
  Queen Mary University of London 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 3 of 25 
 
 
 
 
1. Contents 
 
1. 
Contents ............................................................................................................ 3 
2. 
Glossary ............................................................................................................ 5 
3. 
Signature page .................................................................................................. 6 
4. 
Summary and synopsis ..................................................................................... 7 
5. 
Introduction ........................................................................................................ 8 
5.1. 
Background ................................................................................................. 8 
5.2. 
Rationale ..................................................................................................... 9 
5.3. 
Risks / benefits ............................................................................................ 9 
6. 
Study objectives .............................................................................................. 10 
6.1. 
Study Outcomes / Endpoints ..................................................................... 10 
7. 
Study population .............................................................................................. 11 
7.1. 
Inclusion criteria ........................................................................................ 13 
7.2. 
Exclusion criteria ....................................................................................... 13 
8. 
Study design .................................................................................................... 14 
9. 
Study procedures ............................................................................................ 16 
10. 
Statistical considerations ............................................................................. 17 
10.1. 
Sample size ........................................................................................... 17 
10.2. 
Method of analysis ................................................................................. 17 
11. 
Ethics........................................................................................................... 17 
11.1. 
Annual Safety Reporting ........................................................................ 18 
12. 
Public involvement ....................................................................................... 18 
13. 
Data handling and record keeping ............................................................... 18 
13.1. 
Data management ................................................................................. 18 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 4 of 25 
13.2. 
Source data ........................................................................................... 18 
13.3. 
Confidentiality ........................................................................................ 19 
13.4. 
Record Retention and Archiving ............................................................ 19 
14. 
Laboratories................................................................................................. 20 
14.1. 
Study Workflow ...................................................................................... 22 
15. 
Safety reporting ........................................................................................... 22 
16. 
Monitoring and auditing ............................................................................... 22 
17. 
Study committees ........................................................................................ 23 
18. 
Finance and funding .................................................................................... 23 
19. 
Insurance and indemnity .............................................................................. 23 
20. 
Dissemination of research findings .............................................................. 24 
21. 
References .................................................................................................. 24 
 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 5 of 25 
2. Glossary 
 
 
ANCA  
 
Anti-Neutrophil Cytoplasmic Antibodies 
AUC 
 
 
Area Under the Curve 
CI 
 
 
Chief Investigator 
COPD  
 
Chronic Obstructive Pulmonary Disease 
CRF 
 
 
Case Report Form 
EGPA  
 
Eosinophilic Granulomatous PolyAngiitis 
ELISA  
 
Enzyme-Linked ImmunoSorbent Assay  
FeNO  
 
Fractional exhaled Nitric Oxide 
ICF 
 
 
Informed Consent Form 
IL 
 
 
Interleukin 
ILC 
 
 
Innate Lymphoid Cell 
JRMO  
 
Joint Research Management Office 
NHS REC 
 
National Health Service Research Ethics Committee 
NHS R&D 
 
National Health Service Research & Development   
Participant 
 
An individual who takes part in a clinical trial 
PBMC  
 
Peripheral blood mononuclear cells 
PI 
 
 
Principal Investigator 
PIS 
 
 
Participant Information Sheet  
QMUL  
 
Queen Mary University of London 
REC 
 
 
Research Ethics Committee 
ROC  
 
Receiver Operating Characteristic 
SAE 
 
 
Serious Adverse Event 
 
 
 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 6 of 25 
3. Signature page 
 
 
Chief Investigator Agreement 
 
The study, as detailed within this Research Protocol, will be conducted in accordance 
with the principles of Good Clinical Practice, the UK Policy Framework for Health and 
Social Care Research, and the Declaration of Helsinki and any other applicable 
regulations. I agree to take responsibility for the statistical analysis and oversight of 
this study. 
 
 
Chief Investigator Name: 
Dr Myles Lewis 
 
Signature: 
 
 
Date: 
20th June 2022 
 
 
 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 7 of 25 
4. Summary and synopsis 
 
Short title 
IDentification of EGPA Autoantigens (IDEA) 
Methodology 
Laboratory-based research study using blood and 
sputum samples from healthy controls and patients with 
EGPA, Severe Eosinophilic Asthma and other 
eosinophilic diseases. 
Objectives / aims 
To determine the presence, or not, of auto-antibodies to 
self-peptides in severe eosinophilic asthma and EGPA. 
Number of 
participants 
120 participants (90 patients and 30 healthy controls) 
Inclusion and 
exclusion criteria 
Major Inclusion Criteria: 
Patients: Patients with Severe Eosinophilic Asthma 
and/or EGPA and/or eosinophilic COPD and/or 
eosinophilic oesophagitis, age 18 yo and over, and able 
to give consent. 
Healthy Controls: age 18 yo and over, and able to give 
consent. 
Exclusion criteria: 
Pregnancy; anaemia; rituximab, plasmapheresis or IVIG 
infusion ever 
Study duration 
30 months (18 months recruitment) 
 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 8 of 25 
5. Introduction 
 
 
5.1. Background 
 
The pivotal importance of eosinophils in the pathology of severe asthma and 
eosinophilic granulomatous polyangiitis (EGPA) is increasingly evident given the 
success of anti-eosinophil biologic therapies.  The source of eosinophil stimulating IL-
5 in severe asthma is now thought to be ILC2 cells.  However the persistent chronicity 
of the airways inflammation in the absence of an exogenous stimulus in most patients 
is unexplained and of major research importance.  This suggests the presence of an 
endogenous stimulus of persistent auto-reactive airways inflammation - an auto-
immune hypothesis for severe asthma pathology - which with molecular spread could 
then potentially lead to the systemic disease of EGPA (Mukherjee and Nair 2018). 
Auto-antibody responses have been reported in patients with asthma (Mukherjee and 
Nair 2018).  For example Lott and colleagues have detected auto-antibodies to type V 
collagen in asthmatic individuals (Lott, Sehra et al. 2013).  Similarly Liu and colleagues 
have reported auto-antibodies to type V collagen in asthmatic patients, as well auto-
antibodies to other proteins such as Activin A receptor, with clinical correlates to 
markers of asthma severity (Liu, Subramanian et al. 2012).  However auto-antibodies 
within the pulmonary compartment, as detected in sputum, may be more important 
than auto-antibodies in serum (Qin, Long et al. 2019).  In particular auto-antibodies to 
eosinophil peroxidase in sputum is a feature of severe eosinophilic asthma 
(Mukherjee, Bulir et al. 2018). 
Anti-neutrophil cytoplasmic antibodies (ANCA), especially anti-myeloperoxidase 
antibodies, are present in a proportion of patients with EGPA but absent in a similar 
proportion.  Similar perinuclear ANCA is also present in inflammatory bowel disease.  
These issues raise the question of whether ANCA in EGPA is an epiphenomenon or 
pathological. 
Previous research at our centre and with collaborators has shown that post-
translational modification of potential auto-antigens is a key step in breaking self-
tolerance and development of autoimmune disease (Ryan, Nissim et al. 2014) 
(Eggleton, Nissim et al. 2013).  For example we have shown auto-antibodies to 
oxidised type II collagen are much more frequent than those to native type II collagen 
in rheumatoid arthritis (Nissim, Winyard et al. 2005).   Similarly in type 1 diabetes 
mellitus auto-antibodies to insulin that has undergone oxidative post-translational 
modification are significantly more common than those to native insulin (Strollo, Vinci 
et al. 2015). 
Oxidative stress, implicated in the formation of oxidised auto-antigens, is a major 
feature of uncontrolled asthma inflammation (Mak, Ho et al. 2013).  However, to date 
the potential role of modified auto-antigens in the airways has not been studied in 
severe eosinophilic asthma, EGPA and other related diseases.   

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 9 of 25 
In this project we will look for auto-antibodies to relevant proteins both in native form 
and importantly in post-translationally modified forms.  Potential modified auto-
antigens are eosinophil proteins (analogous to the cytoplasmic neutrophil proteins 
identified in vasculitides such as Granulomatosis with Polyangiitis (formerly known as 
Wegener’s) and alternatively structural proteins such as collagen V. As well as 
advancing our understanding of asthma pathology, identifying a serum auto-antibody 
that could then be used as a clinical blood test, analogous to anti-CCP antibodies in 
rheumatoid arthritis, may revolutionise diagnosis of severe eosinophilic asthma and 
EGPA.  There is a considerable burden of undiagnosed severe eosinophilic asthma in 
part due to difficulties in definitive diagnosis and a diagnostic blood test would help 
diagnose these patients, allowing them to receive necessary treatment. 
We have previous experience of identifying novel autoantigens in Systemic Lupus 
Erythematosus (SLE) (Lewis et al. 2018), using an advanced protein microarray 
developed by Oxford Gene Technology, now known as Sengenics. This protein 
microarray technology benefits from using correctly folded proteins with preserved 
native epitopes, and the technology permits on chip modification of proteins such as 
citrullination or oxidation.  
 
 
5.2. Rationale 
 
We hypothesise the presence of auto-antibodies to self-peptides in severe eosinophilic 
asthma and EGPA, underpinning persistent airways inflammation in patients with 
severe eosinophilic asthma refractory to standard therapies. In particular we 
hypothesise the auto-antigens will be to eosinophil proteins and/or basement 
membrane proteins that are post-translationally modified. 
In this study we will collect blood and sputum samples from well-phenotyped patients 
with EGPA, severe eosinophilic asthma and other eosinophilic diseases.  The 
presence or not of auto-antibodies to relevant self-peptides, in native and post-
translationally modified form, will be determined with appropriate assays. 
 
5.3. Risks / benefits 
 
Previous research has shown the profound benefit of translational research and 
potential for such studies to advance our understanding of disease leading to new 
treatments.  The only participant involvement in this study is the consent process, the 
blood donation (venipuncture and collection of up to 50 mls blood in to sterile 
containers using standard phlebotomy practice at the recruiting NHS Trust), and 
sputum donation in patients producing sputum.  As such risks are low and potential 
benefits high.   
 
 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 10 of 25 
6. Study objectives 
 
We will approach the research question with parallel agnostic and targeted 
approaches.  
In the agnostic approach the presence of auto-antibodies in patient serum and sputum 
to inactive and activated eosinophils, with and without post-translational modification, 
will be examined by indirect immunofluorescence.   
In the targeted approach we will examine by ELISA the presence/absence of 
antibodies to pre-selected candidate eosinophil and base membrane proteins both in 
native form and post-translationally modified.  Proteins to examine will be chosen 
based on literature review (e.g. eosinophil peroxidase and collagen V) and eosinophil-
specific proteins identified by FANTOM5 geneset analysis (FANTOM Consortium et 
al. 2014). 
Both blood and sputum samples from highly-characterised patients with severe 
eosinophilic asthma and/or EGPA will be examined given the possibility of 
compartment-specific immune responses. 
Once candidate auto-antigens have been identified in the selected group of patients 
with severe eosinophilic asthma and EGPA we will then examine their prevalence in 
serum samples from a wider selection of patients with eosinophilic airways diseases 
including mild-to-moderate asthma, severe eosinophilic asthma, EGPA, nasal 
polyposis and eosinophilic COPD as well as healthy controls.  Length of disease, 
atopy, presence/absence nasal polyps, gender, age will be examined as co-variates.  
Correlations with highest blood eosinophil counts, requirement for oral corticosteroids 
and presence of other auto-antibodies, e.g. anti-MPO ANCA, will be examined.  In 
particular we will look for the presence of novel autoantibodies in specific patient 
subsets: i) ANCA negative, ii) ANCA positive by immunofluorescence but negative for 
anti-MPO and anti-PR3 antibodies, iii) ANA positive but ANCA and ENA negative; 
since patients in all three groups may have novel, as yet undetermined autoantibodies. 
ROC AUC analyses will be conducted to ascertain the predictive value of blood auto-
antibodies for diagnosis of eosinophilic airways disease. 
 
6.1. Study Outcomes / Endpoints 
 
Primary objectives 
- The presence of serum and/or sputum auto-antibodies to eosinophil and/or basement 
membrane proteins will be established in severe eosinophilic asthma and EGPA 
cohorts, and patients with other asthma phenotypes and related eosinophilic diseases.   
- Whether auto-antibodies to post-translationally modified self-antigens are more 
prevalent than to native self-antigens will be examined, and which post-translational 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 11 of 25 
modifications are important to be studied (including particulate matter air pollution as 
a potential combined stimulus of eosinophil activation and protein modification). 
Primary outcomes 
- Correlations of auto-antibodies to clinical variables such as spirometry (FEV1 % 
predicted), pattern of organ involvement, age of onset, requirement for maintenance 
of oral corticosteroids, eosinophil count, highest recorded eosinophil count and FeNO 
will be studied. 
- Predictive ability to diagnose severe eosinophilic asthma / EGPA of relevant auto-
antibodies will be examined by ROC AUC analysis. 
Secondary outcomes 
- Exploratory Outcomes: Further experimental assays may be conducted to examine 
immunological mechanisms underpinning associations of auto-antibodies to disease. 
 
 
 
7. Study population 
 
90 patients under the care of the Barts Health NHS Trust and Royal Free London NHS 
Trust with severe eosinophilic asthma, EGPA, eosinophilic COPD and other related 
pathologies will be recruited.   
At least 60 patients will have severe eosinophilic asthma and/or EGPA, and will be 
recruited from the regional North Central and East London Severe Asthma Service, 
the Royal Free London NHS Trust and from the Barts Arthritis Centre, with multi-
disciplinary consensus diagnoses of severe eosinophilic asthma and/or EGPA 
respectively.  Patients will be phenotyped with blood leucocyte differential, lung 
function tests and FeNO as appropriate in conjunction with detailed medical history. 
Up to a further 30 patients with milder asthma, other vasculitides, eosinophilic COPD, 
and/or eosinophilic oesophagitis will be recruited from other clinics at Barts Health NHS 
Trust and the Royal Free London NHS Trust.   
In a future extension of this research our intention is to verify positive findings with 
samples from a replication sample of patients with severe eosinophilic asthma from 
the wider network of the North Central and East London Severe Asthma Service 
(principally through our satellite clinic at Royal Free London NHS Trust).  These 
samples will be collected under a separate ethics application. 
30 healthy controls will be recruited from volunteers at Queen Mary University of 
London via poster advertising.  Healthy controls will also be recruited from Bart’s 
Health NHS Trust and the Royal Free London NHS Trust.   
 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 12 of 25 
 
 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 13 of 25 
 
 
7.1. Inclusion criteria 
 
 
Informed consent  
 
Age 18 years and over 
 
 
Patients:  
 
 
- Severe Eosinophilic Asthma  
 
 
 
(with multi-disciplinary diagnosis as per ERS/ATS Criteria, 
 
 
 
blood eosinophils ≥ 0.3 x109/L on inhaled corticosteroids); or  
 
 
EGPA  
 
 
 
(as per American College of Rheumatology (ACR) Criteria); or 
 
 
Eosinophilic COPD  
 
 
 
(post-bronchodilator FEV1/FVC < 70% predicted, 
 
 
 
absence of bronchodilator reversibility, 
 
 
 
> 20 pack year smoking history, 
 
 
 
no medical history of current asthma, 
 
 
 
blood eosinophils ≥ 0.3 x109/L); or 
 
 
Eosinophilic oesophagitis  
 
 
 
(with diagnostic histology); or 
 
 
Granulomatosis with Polyangiitis (GPA, formerly called Wegener’s) 
 
 
 
(as per American College of Rheumatology (ACR) Criteria) 
 
 
Healthy Controls:  
 
 - No acute medical illness.  
 
 - No diagnosed chronic respiratory disease, allergy or infective   
condition, including no history of asthma.   
 
 - No history of EGPA or other vasculitis,  
 
- No systemic immunosuppressive medication. 
 
 
 
7.2. Exclusion criteria 
 
 
Any inclusion criteria not met 
 
Known Pregnancy 
 
Anaemia 
 
Donation of more than 240mls blood in the last sixteen weeks (four months) 
to any other research study or as a donation to the National Blood 
Transfusion Service 
 
Rituximab, plasmapharesis or polyclonal immunoglobulin infusion (ever) 
 
 
 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 14 of 25 
 
8. Study design 
 
Study Visit (Patients) 
This is a single study visit encompassing patient recruitment, consent, data collection 
and provision of a peripheral blood sample with/without concurrent sputum sample, 
with no follow-up visits. 
Patients will be recruited via Outpatient Clinics conducted at St Bartholomew’s (Bart’s) 
Hospital, The Royal London Hospital, and Mile End Hospital (all Bart’s Health NHS 
Trust), Royal Free London NHS Trust, as well as from inpatients at these hospitals 
under the care of the Respiratory Medicine and Rheumatology teams (including those 
under other services needing inpatient respiratory medicine or rheumatology review). 
Potential patients will be identified by their clinical team and invited to participate.  If 
interested they will be given a Participant Information Sheet (PIS).  Informed Consent 
and Screening will be undertaken by a GCP-trained member of the clinical team or 
GCP-trained researcher.  An anonymised data collection case report form (CRF) will 
be completed by a member of the clinical team and a linked (anonymised) blood 
sample collected by a staff member trained in phlebotomy.  If the patient is able to 
spontaneously produce sputum, and there is no clinical concern about TB infection, 
then they will be asked to cough a sample in to a sterile sample pot. 
The linked anonymised CRF and samples will then be taken to the research 
laboratories at QMUL for immune cell analysis.  Laboratory immunology studies will be 
conducted by as per protocol (Section 14) by a dedicated Post-Doctoral Research 
Assistant. 
 
Study Visit (Healthy Controls) 
There is a single study visit encompassing healthy control recruitment, consent, data 
collection and taking of blood sample, with no follow-up visits. 
Healthy controls will be recruited at QMUL, Bart’s Health and Royal Free sites via 
poster advertisement. 
Healthy controls will be identified by the research team and invited to participate.  If 
interested they will be given a Participant Information Sheet (PIS).  Informed Consent 
and Screening will be undertaken by a GCP-trained researcher. A limited anonymised 
data collection case report form (CRF) will be completed by a member of the research 
team and a linked (anonymised) blood sample collected by a staff member trained in 
phlebotomy. The linked anonymised CRF and blood sample will then be transferred to 
the research laboratories at QMUL for immune cell analysis. 
 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 15 of 25 
 
 
 
 
 
 
 
Figure 1: Study design graphic 
 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 16 of 25 
 
 
9. Study procedures 
 
Invitation to Participate: 
 
Invitation to Participate will be by members of their clinical team for Patients and 
by members of the research team for Healthy Controls.  Before participants are 
invited to participate, capacity to process samples from that type of participant 
will be discussed with the research team. Invited participants will be given a copy 
of the Participant Information Sheet (PIS) at invitation to participate. 
Informed Consent Procedures and Screening: 
 
Potential participants interested in the study will be given time to read the PIS 
and ask questions from a GCP-trained member of the research team who may 
or may not be a member of the clinical team treating the participant.  After 
questions have been answered, potential participants will be asked to complete 
the written informed Consent Form. Consenting participants will then be briefly 
screened to check they fulfil inclusion/exclusion criteria. 
Data Collection (CRF): 
 
A member of the clinical team (for patient participants) and the research team 
(for healthy control participants) will complete the case report form (CRF) which 
contains only pseudoanonymised information but with a study code to link the 
patient to the samples (and to Consent Form for governance purposes). 
Sample Collection: 
 
A peripheral blood sample (up to 50ml total volume) will be collected using 
aseptic technique into vacutainers or anti-coagulated syringe by staff trained in 
phlebotomy techniques and securely transported to the research laboratories for 
analysis. 
 
If the patient is able to spontaneously produce sputum, and there is no clinical 
concern about TB infection, then they will be asked to cough a sample in to a 
sterile sample pot. 
 
Samples will be analysed as per Section 14. 
Subject Withdrawal: 
 
 In the event that a participant asks to withdraw from the study before the end of 
the Study Visit then the CRF and samples will be securely destroyed and no 
recording / analysis of data from that patient undertaken. In the event a 
participant asks to withdraw from the study after the first Study Visit has been 
completed the research team will be asked to withdraw any unreported linked 
data from that participant from the study. 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 17 of 25 
Recruitment Time-Frame 
Recruitment will be completed within 21 months.  Laboratory analysis of collected 
samples will continue up to 30 months.  Residual samples may be stored for future 
ethically approved studies. 
 
End of Study Definition  
The end of the study is at completion of laboratory sample analysis for the last 
participant recruited.  Statistical analysis of data may continue after the End of Study. 
 
 
10. 
Statistical considerations 
 
10.1. Sample size 
As a pilot laboratory research study with aim of revealing novel associations, a formal 
power calculation is not possible, since no previous data is available.  The sample size 
is based on previous experience with similar projects and feasibility. The data from this 
study will be used to calculate sample sizes for future larger scale studies. 
10.2. Method of analysis 
For the major body of translational laboratory experiments appropriate parametric and 
non-parametric tests will be conducted using GraphPad Prism.  For experiments 
assessing associations of detected auto-antibodies to clinical variables, linear 
regression analyses will be undertaken in R, and ROC AUC analysis of capacity of 
serum auto-antibodies to detect severe eosinophilic diseases undertaken. 
 
 
11. 
Ethics 
 
The Chief Investigator will ensure that the study is carried out in accordance with the 
ethical principles of the UK policy framework for health and social care research (2017) 
and its subsequent amendments as applicable, as well as applicable legal and 
regulatory requirements.  
The research team has no financial or other competing interests for this study. 
Human genome (gDNA) analysis will not be conducted as part of this research project. 
Clinically defined immunodeficiencies will not be tested for as part of the immunological 
analyses. 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 18 of 25 
The CRF and samples for immunological analysis will be anonymised so donors are 
not known to the research team or anyone reviewing the analyses and results. 
Invitation to participate and consent will be undertaken on the same day.  Potential 
participants will be given as long as they require to review the PIS and ask questions 
before deciding whether to participate. The study clinical procedure (taking of a blood 
sample of 50ml maximum volume) carries minimal risk. Donor decisions to participate 
or not in the study will not affect their health or clinical care.  We therefore feel this is 
an appropriate period for informed consent for this low-risk study.  Requiring potential 
volunteers to come back on a second occasion to give informed consent and samples 
would not facilitate ethical recruitment of patients, many of whom need to travel 
significant distances to Barts Health/Royal Free Hospitals for appointments and many 
of whom have significant chronic ill health. 
11.1. Annual Safety Reporting 
The CI will send an Annual Progress Report to the REC and the sponsor using the 
HRA template on the anniversary of the REC “favourable opinion”. 
 
 
 
12. 
Public involvement 
 
There has been no specific public involvement in the development of this protocol for 
exploratory translational laboratory research.  However this research is commensurate 
with the research strategies suggested by patient advocacy groups such as Asthma 
UK and the European Asthma Research and Innovation Partnership (EARIP) asthma 
research roadmap which themselves had extensive public and patient involvement. 
 
 
 
13. 
Data handling and record keeping 
 
13.1. Data management 
The personal data of each patient will be stored in hard copies using paper-based 
consent forms, paper CRFs and associated data in a secure locked filling cabinet in a 
locked room. Only members of the research team, monitors and/or regulatory bodies 
will have access to the physical hard copy of this data and are authorised to do so. 
13.2. Source data 
 
Case Report Form (Patients) data variable list: 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 19 of 25 
1. Age 
2. Gender 
3. Ethnicity (optional) 
4. Occupational history 
5. Confirmation participant meets Inclusion and Exclusion criteria (Yes/No) 
6. Smoking history 
7. Diagnosed medical condition(s) and basis for diagnoses with focus on 
diagnostic criteria for Severe Eosinophilic Asthma, EGPA 
8. Suspected medical conditions(s) 
9. Immunosuppressive medication(s) dose regimes and history 
10. Previous treatment history, especially corticosteroid history 
11. Full blood count (haematology) 
12. Most recent and previous FeNO breath tests, if done 
13. Auto-immune serology results 
14. Spirometry and gas transfer tests, if done 
15. Highest previous blood eosinophil count, ANCA serology 
(immunofluorescence, anti-PR3 and anti-MPO antibody titre) and trend 
16. Investigations for target organ involvement in patients with suspected 
vasculitis including histology 
17. Clinical disease activity measures (for example Birmingham Vasculitis 
Activity Score (BVAS), Asthma Control Questionnaire (ACQ)-6) 
 
Case Report Form (Healthy Control) data variable list: 
1. Age 
2. Gender 
3. Ethnicity (optional) 
4. Occupational history 
5. Confirmation participant meets Inclusion and Exclusion criteria (Yes/No) 
6. Smoking history 
 
13.3. Confidentiality 
Information related to participants will be kept confidential and managed in 
accordance with the Data Protection Act, the General Data Protection Regulation 
(GDPR), NHS Caldecott Principles, UK policy framework for health and social care 
research (2017), and the conditions of Research Ethics Committee Approval. 
Each patient will be assigned a unique study number. Patients’ initials and date of 
birth in the format dd/mm/yy will be used as a means of confirming patient identity for 
audit checks. This information will be kept in an encrypted screening log, which will 
be updated throughout the study and stored on a secure server. Only patient study 
IDs will be used on paper CRFs and samples (pseudoanonymisation).  The subject 
will be identified by a study specific subject number. The patient’s name will NOT be 
included in any CRF or sample. 
13.4. Record Retention and Archiving 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 20 of 25 
When the research trial is complete, records will be kept for a further 5 years.  
 
 
14. 
Laboratories  
 
Samples will be analysed at the EMR laboratory, Queen Mary University of London: 
EMR Laboratory (QMUL) 
Dr. Rebecca Hands (Laboratory Manager) 
Centre for Experimental Medicine and Rheumatology 
2nd floor, John Vane Science Centre, William Harvey Research Institute Barts and the 
London School of Medicine and Dentistry 
Queen Mary University of London, Charterhouse Square 
London, EC1M 6BQ 
Samples will be processed on day of receipt of the samples (within 24 hours of 
sampling). Following processing, all samples will be stored immediately in -80C 
freezers or subject to analysis as described below within 48 hours. 
- Samples will be analysed in the QMUL laboratories at William Harvey Research 
Institute as follows to investigate the major Study Outcomes: 
INDIRECT IMMUNOFLUORESCENCE ASSAYS 
Eosinophils will be cultured in preparatory experiments from the EoL-1 eosinophil cell 
line and in definitive experiments isolated from patients with eosinophilia in the context 
of severe asthma.  Slides of cell smears will then be prepared of inactive eosinophils 
and those activated to undergo piecemeal degranulation and cytolysis with eosinophil 
extracellular trap formation.  Cell smears will be examined unmodified and after post-
translational modification of proteins.  Fixed slides will then be incubated with dilutions 
of serum and sol phase sputum supernatant from patients and healthy controls, and 
incubated with fluorescein-conjugated anti-human immunoglobulin antibody before 
being examined by fluorescent microscopy and fluorescence intensity measured.  
Confocal microscopy and complementary fluorescein-conjugated antibody markers 
will be used to examine characteristics of target antigens for patient serum auto-
antibodies. 
TARGET PROTEIN ASSAYS 
Eosinophil proteins such as those previously identified as of importance (e.g. 
eosinophil-peroxidase, EPX) and also novel candidate proteins specific to eosinophils 
(including ARFIP1, BCL2A1, PPCDC, SLC12A6 etc) as identified by FANTOM5 
geneset analysis will be purchased or synthesised in Expi-293 cells, by obtaining 
tagged clones from Origene and developing stable expressing cell lines. Protein 
synthesis be confirmed by western blot. Proteins will be used to coat ELISA plates 
with/without prior post-translational modification. Presence of donor serum antibodies 
in serum and in sputum will then be examined by ELISA using appropriate enzyme-

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 21 of 25 
linked secondary antibodies. Similar extracellular matrix proteins such as collagen V 
will be investigated.  
POST-TRANSLATIONAL MODIFICATION OF PEPTIDES 
Eosinophil suspensions / slides and commercially sourced / synthesised target 
proteins (e.g. eosinophil peroxidase and collagen V) will be modified as follows: 
Oxidative modification – by hydroxyl radical exposure with hypochlorous acid using our 
established methodology (Strollo, Vinci et al. 2015).  Citrullination – by recombinant 
peptidylarginine deiminase (PAD).   
Ambient pollutants and nanopollutants are known to be able to induce both oxidative 
stress and protein citrullination as well as potentially direct eosinophil activation.  Given 
the known association between asthma and ambient pollution we will additionally 
examine potential serum antibodies to ambient pollution particulate matter 
stimulated/modified eosinophil and collagen proteins. 
 
 
- Samples may also be analysed to address associated Exploratory Outcomes, to 
examine 
immunological 
mechanisms 
underpinning 
auto-antibody 
disease 
associations.  Applicable experimental techniques to include: 
Blood and sputum leukocyte phenotyping ex vivo by flow-cytometry (FACS) analysis 
of 
circulating 
immune 
cell 
subsets 
(including 
myeloid-lineage 
cells, 
conventional/unconventional lymphocyte populations, and granulocyte populations).   
In vitro culture of blood and sputum leukocytes (whole sample cultures, fractionated 
peripheral blood mononuclear cells, and magnetically separated / FACS-sorted 
leukocyte subsets) to assess responses to a variety of subset-specific agonists and 
stimuli.  Positive and negative controls to be included as appropriate.  Key mediators 
of these responses may be identified using small molecule inhibitors, blocking 
antibodies, recombinant cytokines/proteins, essential supplements/chelators, and 
other equivalent culture additives according to cell type and function of interest.  
Responses to be assessed using multicolour flow-cytometry methods, advanced 
imaging techniques (INCell analysis, immunofluorescence and confocal microscopy, 
ImageStream), quantification of cytokine/chemokine/antibody release by ELISA / 
multiplex bead array, and measurement of gene expression by PCR / RNA-seq 
methodology (mRNA but not gDNA).  To include B-ELISpot (B lymphocyte enzyme-
linked immune absorbent spot) assays to further examine auto-antigen specific 
immune responses. 
Isolated proteins may be examined by immunoblotting, proteomics, mass 
spectrometry, surface plasmon resonance / microscale thermophoresis, and other 
equivalent binding studies. 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 22 of 25 
 
14.1. Study Workflow 
 
Blood samples will be collected in up to 4 Serum Separator Tube (SST) vacutainer 
tubes and up to 4 Acid Citrate Dextose (ACD) vacutainer tubes. 
Serum samples from SST tubes will be aliquoted and stored at -80°C pending analyses 
as above. 
Peripheral blood samples from ACD tubes will be used to store peripheral blood 
mononuclear cells (PBMC) (with/without sorting of eosinophils and/or other cell 
subsets such as neutrophils prior to freezing) as viably-frozen cells in freezing medium 
or in RNA preservation buffer. 
Sputum will be processed to isolate sputum plugs from saliva contamination, diluted 
with buffered saline before centrifugation to separate supernatant and cellular 
components.  Aliquots of supernatant will be stored at -80°C pending analyses as 
above.  The cellular fraction will be treated with dithiothreitol, filtered and final cell 
suspension stored at -80°C for further analysis or in RNA preservation buffer. 
 
 
15. 
Safety reporting 
 
The only patient contact in this study is the consent process and the sample donation 
(venipuncture and collection of blood into sterile containers using standard phlebotomy 
practice at the recruiting NHS Trust, with/without collection of spontaneously produced 
sputum).  As such Adverse Events (AE) and Serious Adverse Events (SAE) are 
extremely unlikely.  In the event of an AE or SAE then this will reported to the CI by 
any research team members, and escalated as appropriate by the CI. 
Notification and Reporting of Serious Adverse Events  
Serious Adverse Event (SAEs) that are considered to be ‘related’ and ‘unexpected’ will 
be reported to the sponsor within 24 hours of learning of the event and to the Main 
REC within 15 days in line with the required timeframe.  
 
 
16. 
Monitoring and auditing 
 
The Sponsor or delegate retains the right to audit any study, study site or central 
facility. In addition, any part of the study may be audited by the funders where 
applicable. 
 
As this is exploratory laboratory research, no formal monitoring plan is planned. 
 
 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 23 of 25 
 
17. 
Study committees 
 
There is no trial committee for this study.  Members of the research team are as 
follows: 
Chief Investigator (CI) 
Dr Myles Lewis 
 
Senior Clinical Lecturer and  
 
Consultant Rheumatologist, 
 
William Harvey Research Institute, 
 
Queen Mary University of London, UK 
 
Co-Investigator 
Dr Paul E Pfeffer 
 
Consultant Respiratory Physician and  
 
Hon Senior Lecturer, 
 
Barts Health NHS Trust and Queen Mary University of 
London, UK 
 
Other Collaborating 
Prof Ahuva Nissim, 
Investigators 
Professor in Antibody and Therapeutic Engineering, 
 
William Harvey Research Institute, 
 
Queen Mary University of London, UK 
 
 
 
 
 
 
18. 
Finance and funding 
 
This research study has been designed by Dr Paul Pfeffer (p.pfeffer@qmul.ac.uk), Dr 
Myles Lewis (myles.lewis@qmul.ac.uk) & Prof Ahuva Nissim (a.nissim@qmul.ac.uk) 
at Barts Health NHS Trust and Queen Mary University of London.  It is being funded 
by GlaxoSmithKine (GSK) through its Supported Studies Programme.  None of the 
doctors or investigators involved in the study will receive any direct payment dependent 
on recruiting participants for this study.   
 
 
 
 
19. 
Insurance and indemnity 
 
The insurance that Queen Mary University of London has in place provides cover for 
the design and management of the study as well as "No Fault Compensation" for 
participants, which provides an indemnity to participants for negligent and non-
negligent harm. 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 24 of 25 
 
 
20. 
Dissemination of research findings 
 
Results will be presented at national and international meetings, and published in 
international peer-reviewed scientific journals.  
 
 
 
21. 
References 
 
Eggleton, P., A. Nissim, B. J. Ryan, M. Whiteman and P. G. Winyard (2013). 
"Detection and isolation of human serum autoantibodies that recognize 
oxidatively modified autoantigens." Free Radic Biol Med 57: 79-91. 
 
 
FANTOM Consortium, Forrest, A.R., Kawaji, H., Rehli, M., Baillie, J.K., de 
Hoon, M.J., Haberle, V., Lassmann, T., Kulakovskiy, I.V., Lizio, M., et al. 
(2014). "A promoter-level mammalian expression atlas." Nature 507: 462-
470. 
 
Lewis MJ, McAndrew MB, Wheeler C, Workman N, Agashe P, Koopmann J, 
Uddin E, Morris DL, Zou L, Stark R, Anson J, Cope AP, Vyse TJ. 
"Autoantibodies targeting TLR and SMAD pathways define new subgroups in 
Systemic Lupus Erythematosus." J Autoimmun 2018; 91: 1-12. 
 
Liu, M., V. Subramanian, C. Christie, M. Castro and T. Mohanakumar (2012). 
"Immune responses to self-antigens in asthma patients: clinical and 
immunopathological implications." Hum Immunol 73(5): 511-516. 
 
Lott, J. M., S. Sehra, P. Mehrotra, E. A. Mickler, A. J. Fisher, W. Zhang, R. G. 
Presson, M. F. Busk, S. Goenka, S. J. Gunst, M. H. Kaplan, D. S. Wilkes and S. 
E. Wenzel (2013). "Type V Collagen–induced Tolerance Prevents Airway 
Hyperresponsiveness." Am J Respir Crit Care Med 187(4): 454-457. 
 
Mak, J. C., S. P. Ho, A. S. Ho, B. K. Law, A. H. Cheung, J. C. Ho, M. S. Ip and 
M. M. Chan-Yeung (2013). "Sustained elevation of systemic oxidative stress 
and inflammation in exacerbation and remission of asthma." ISRN Allergy 
2013: 561831. 
 
Mukherjee, M., D. C. Bulir, K. Radford, M. Kjarsgaard, C. M. Huang, E. A. 
Jacobsen, S. I. Ochkur, A. Catuneanu, H. Lamothe-Kipnes, J. Mahony, J. J. 
Lee, P. Lacy and P. K. Nair (2018). "Sputum autoantibodies in patients with 
severe eosinophilic asthma." J Allergy Clin Immunol 141(4): 1269-1279. 

 
 
 
 
Protocol version 5.0 dated 20th June 2022  
IDentification of EGPA Autoantigens (IDEA) 
 
Page 25 of 25 
 
Mukherjee, M. and P. Nair (2018). "Autoimmune Responses in Severe 
Asthma." Allergy Asthma Immunol Res 10(5): 428-447. 
 
Nissim, A., P. G. Winyard, V. Corrigall, R. Fatah, D. Perrett, G. Panayi and Y. 
Chernajovsky (2005). "Generation of neoantigenic epitopes after 
posttranslational modification of type II collagen by factors present within the 
inflamed joint." Arthritis Rheum 52(12): 3829-3838. 
 
Qin, R., F. Long, X. Xiao, J. Xiao, Z. Zheng, M. Feng, R. Huang, T. Peng and J. 
Li (2019). "Sputum Autoantibodies Are More Relevant in Autoimmune 
Responses in Asthma than Are Serum Autoantibodies." Allergy Asthma 
Immunol Res 11(3): 406-421. 
 
Ryan, B. J., A. Nissim and P. G. Winyard (2014). "Oxidative post-translational 
modifications and their involvement in the pathogenesis of autoimmune 
diseases." Redox Biol 2: 715-724. 
 
Strollo, R., C. Vinci, M. H. Arshad, D. Perrett, C. Tiberti, F. Chiarelli, N. Napoli, 
P. Pozzilli and A. Nissim (2015). "Antibodies to post-translationally modified 
insulin in type 1 diabetes." Diabetologia 58: 2851-2860. 
 
 
 
 
This protocol is based on JRMO Protocol template for Research Studies; 
version 2.0, September 2019.