Beyond the Eosinophil: Understanding the Impact of Eosinophil Depletion on T2 Inflammation. (BEUTI)
Conditions: Asthma
Sponsor: Guy's and St Thomas' NHS Foundation Trust

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 1 of 31 
 
1. Protocol details 
1.1 PROTOCOL TITLE: 
Beyond the Eosinophil: Understanding the impact of eosinophil depletion on T2 Inflammation 
Short Title: BEUTI 
Design: Prospective open label phase IV study (single centre) 
Intervention: Benralizumab 
Target Disease: Severe Asthma 
1.2 Names (titles), roles and contact details of: 
Sponsor 
Name of Sponsoring Organisation/s: King's College London 
Name of Sponsor Representative: Professor. Reza Razavi 
Address: King's College London, 
Room 5.31 James Clerk Maxwell Building,  
London, SE1 8WA 
Telephone: +44 (0)207 8483224 
Fax: 
Email: reza.razavi@kcl.ac.uk 
 
Chief Investigator  
Name: Professor. David Jackson 
Address: King's Centre for Lung Health  
Faculty of Life Sciences & Medicine 
School of Immunology and Microbial Sciences 
5th Floor Tower Wing, Guy’s Campus 
King’s College London 
London SE1 9RT 
Telephone: 
Fax: 
Email: david.jackson@gstt.nhs.uk 
 
 
Name and address of Co-Investigator(s)  
Name: Dr Rocio T Martinez-Nunez 
Address: King's Centre for Lung Health  
Faculty of Life Sciences & Medicine 
School of Immunology and Microbial Sciences 
5th Floor Tower Wing, Guy’s Campus 
King’s College London 
London SE1 9RT 
Telephone: 02071880595 
Fax: 
Email: rocio.martinez_nunez@kcl.ac.uk 
 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 2 of 31 
 
 
Name: Dr Alexandra Nanzer-Kelly 
Address: Guy’s and St Thomas’ NHS Foundation Trust 
Great Maze Pond 
London SE1 9RT 
Telephone: 
Fax: 
Email: alexandra.nanzerkelly@gstt.nhs.uk 
 
Name: Dr Jaideep Dhariwal 
Address: Guy’s and St Thomas’ NHS Foundation Trust 
Great Maze Pond 
London SE1 9RT 
Telephone: 
Fax: 
Email: jaideep.dhariwal@gstt.nhs.uk 
 
Statistician 
Name: Dr Rocio T Martinez-Nunez 
Address: King's Centre for Lung Health  
Faculty of Life Sciences & Medicine 
School of Immunology and Microbial Sciences 
5th Floor Tower Wing, Guy’s Campus 
King’s College London 
London SE1 9RT 
Telephone: 02071880595 
Fax: 
Email: rocio.martinez_nunez@kcl.ac.uk 
 
Laboratories 
Name: King's Centre for Lung Health 
Address: Faculty of Life Sciences & Medicine 
Peter Gorer Department of Immunobiology, 
Asthma, Allergy & Lung Biology, 
School of Immunology and Microbial Sciences, 
5th Floor Tower Wing, Guy’s Campus 
King’s College London 
London SE1 9RT 
 
1.3 Protocol details 
BEUTI STUDY Jackson KCL 
Version number: 1.0 
Final/draft: Final 
Date: 15/12/2022 
IRAS: 310865 
–  

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 3 of 31 
 
2 Signature Page 
 
The Chief Investigator and the R&D (sponsor office) have discussed this protocol. The 
investigators agree to perform the investigations and to abide by this protocol  
The investigator agrees to conduct the trial in compliance with the approved protocol, EU GCP, 
the UK Data Protection Act (1998), the Trust Information Governance Policy (or other local 
equivalent), the Research Governance Framework (2005’ 2nd Edition; as amended), the 
Sponsor’s SOPs, and other regulatory requirements as amended. 
 
Chief investigator 
Professor David Jackson            
 
15/12/2022 
 
Signature 
Date 
Sponsor Representative 
R&D to Add  
 
 
GSTFT 
Signature 
Date 
 
 
 
 
This Protocol template is intended for use with UK sites only. 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 4 of 31 
 
Contents Page 
1. 
Protocol details .......................................................................................................................... 1 
1.1 
PROTOCOL TITLE: ............................................................................................................... 1 
1.2 
Names (titles), roles and contact details of: ........................................................................ 1 
1.3 
Protocol details .................................................................................................................. 2 
2 
Signature Page ........................................................................................................................... 4 
Contents Page ................................................................................................................................... 4 
3 
List of Abbreviations and Definitions .......................................................................................... 7 
4 
Summary/Synopsis .................................................................................................................... 8 
5 
Introduction ............................................................................................................................ 11 
6 
Trial objectives and purpose .................................................................................................... 12 
7 
Study design & Flowchart ........................................................................................................ 14 
7.1 
Study Design .................................................................................................................... 14 
7.2 
Flowchart ......................................................................................................................... 16 
8 
Subject selection...................................................................................................................... 17 
8.1 
Subject inclusion criteria .................................................................................................. 17 
8.2 
Subject exclusion criteria .................................................................................................. 17 
9 
Study procedures ..................................................................................................................... 18 
9.1 
Subject recruitment ....................................................................................................... 117 
9.2 
Screening Procedures (delete as appropriate) .................................................................. 20 
9.3 
Schedule of assessments for each visit ............................................................................. 20 
9.4 
Follow up Procedures ....................................................................................................... 20 
9.5 
End of Study Definition..................................................................................................... 20 
10 
Laboratories (delete as appropriate) .................................................................................... 20 
10.1 
Central/Local Laboratories ............................................................................................... 19 
10.2 
Sample Collection/Labelling/Logging ................................................................................ 19 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 5 of 31 
 
10.3 
Sample Analysis Procedures ............................................................................................. 21 
10.4 
Sample Storage Procedures (if applicable) ........................................................................ 24 
10.5 
Data Recording/Reporting ................................................................................................ 24 
10.6 
Sample Receipt/Chain of Custody/Accountability ............................................................. 25 
10.7 
Sample Transfer to sites outside the Organisation ............................................................ 26 
11 
Assessment of Safety ........................................................................................................... 26 
11.1 
Ethics Reporting ............................................................................................................... 27 
11.2 
Trial Steering Committee (if applicable) ............................................................................ 27 
11.3 
Ethics & Regulatory Approvals .......................................................................................... 27 
12 
Compliance and withdrawal ................................................................................................. 27 
12.1 
1Subject compliance ........................................................................................................ 27 
12.2 
Withdrawal / dropout of subjects ..................................................................................... 27 
12.3 
Protocol Compliance ........................................................................................................ 27 
13 
Data ..................................................................................................................................... 28 
13.1 
Data to be collected ......................................................................................................... 28 
13.2 
Data handling and record keeping .................................................................................... 29 
14 
Statistical considerations ..................................................................................................... 29 
14.1 
Sample size calculation (some pilot/feasibility studies may not require a formal sample size 
calculation) .................................................................................................................................. 29 
14.2 
Statistical analysis ............................................................................................................ 30 
15 
Ethical considerations .......................................................................................................... 31 
16 
Financing and Insurance....................................................................................................... 31 
17 
Reporting and dissemination ............................................................................................... 31 
 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 6 of 31 
 
3 List of Abbreviations and Definitions 
AE 
 
 
Adverse Event 
 
 
AR 
 
 
Adverse Reaction 
ASR 
 
 
Annual Safety Report 
CA 
 
 
Competent Authority 
CI 
 
 
Chief Investigator 
CRF 
 
 
Case Report Form 
CRO  
 
Contract Research Organisation 
DMC  
 
Data Monitoring Committee 
EC 
 
 
European Commission 
GAfREC 
Governance Arrangements for NHS Research Ethics Committees 
ICF 
 
 
Informed Consent Form 
MA 
 
 
Marketing Authorisation 
MS 
 
 
Member State 
Main REC 
 
Main Research Ethics Committee 
NHS R&D 
 
National Health Service Research & Development   
PI 
 
 
Principle Investigator 
QA 
 
 
Quality Assurance 
QC 
 
 
Quality Control 
Participant 
 
An individual who takes part in a clinical trial 
REC 
 
 
Research Ethics Committee 
SAE 
 
 
Serious Adverse Event 
SDV 
 
 
Source Document Verification 
SOP 
 
 
Standard Operating Procedure  
SSA 
 
 
Site Specific Assessment 
TMG  
 
Trial Management Group 
TSC 
 
 
Trial Steering Committee 
 
 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 7 of 31 
 
4 Summary/Synopsis 
 
Title  
 
 
Beyond the Eosinophil: Understanding the impact of eosinophil 
depletion on T2 Inflammation 
Protocol Short Title/Acronym 
 
BEUTI 
Protocol Version number and Date 
 
1.0 7th October, 2022 
Study Phase if not mentioned in title 
 
Prospective open label phase IV study (single centre) 
IRAS Number 
 
310865 
REC Reference 
 
 
Sponsor Reference 
 
 
Study Duration 
 
24 months 
Sponsor name 
 
King’s College London 
Chief Investigator 
 
Professor David Jackson 
Funder 
 
Astra Zeneca 
Medical condition or disease under 
investigation 
 
Severe Asthma 
Purpose of research  
 
Benralizumab is a relatively new treatment that is approved by NICE for 
patients with severe asthma with ongoing eosinophilic inflammation 
that remains poorly controlled despite high dose inhaled corticosteroid 
medication. 
Benralizumab targets a receptor on the surface of eosinophils called IL-
5R leading to the rapid death of these cells and consequently a 
reduction in airways inflammation. In clinical trials, benralizumab has 
been shown to reduce both symptoms and the number of asthma 
attacks suffered by those with severe eosinophilic asthma. However, it 
remains unclear whether this clinical efficacy relates purely to the 
removal of the eosinophil, or additionally to the impact of this on other 
parts of the immune system. 
The BEUTI study will examine if benralizumab leads to a reduction in T2 
mediators and/or activation in airway cells in patients with severe 
eosinophilic asthma. The aim is to take samples of cells from the 
airways during a bronchoscopy (a camera test looking into the lungs) 
before starting benralizumab and after 12 weeks of treatment. These 
investigations will allow us to better understand how benralizumab 
affects the cells within the airways and the pathways involved. 
 
Primary objective 
 
To determine if benralizumab leads to a reduction in T2 mediators 
and/or activation in airway cells in patients with severe eosinophilic 
asthma. 
Secondary objective (s) 
 
1.Determine any change in T2-related signalling pathways and cell 
populations in bronchial biopsies (including those relating to IL-4, IL-5, 
IL-13, TSLP, IL-25 or IL-33) in patients with severe eosinophilic asthma 
before and after 12 weeks of benralizumab using spatial 
transcriptomics. 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 8 of 31 
 
2. Determine any in vitro changes in bronchial epithelial antiviral 
responses to rhinovirus-16 and SARS-CoV-2 before and after 12 weeks 
of benralizumab. 
3. Determine any transcriptomic changes in genes relating to airway 
remodelling before and after 12 weeks of benralizumab. 
4. Determine the effect of benralizumab on small airway dysfunction 
using impulse oscillometry and MRI to measure the change in 
homogeneity of airways ventilation 
Number of Subjects/Patients 
 
12  
Study Type 
 
Observational. Prospective open label phase IV study (single centre) 
Endpoints 
 
1. Change in the number / proportion of T2 related cellular populations 
in BAL including ILC2, Th2, alveolar macrophages, eosinophils and 
basophils pre- and post benralizumab 
2. Change in cellular populations and gene expression of T2-related 
signalling pathways in bronchial biopsies pre- and post benralizumab 
3. Change in type 1 & 3 interferon production and viral replication 
following infection of cultured BECs and alveolar macrophages with RV-
16 and examine the response of cells to SARS-CoV-2 collected pre- and 
post- benralizumab. 
4. Change in cellular populations and gene expression of remodelling-
related pathways in bronchial biopsies pre- and post benralizumab 
5. Change in airway oscillometry measures and MRI measures of 
ventilation pre- and post benralizumab 
Main Inclusion Criteria 
 
1. Patients aged 18 and over with a diagnosis of severe eosinophilic 
asthma for at least the last 6 months 
2. Eligible for benralizumab based on NICE criteria. 
3. Poorly-controlled (ACQ-6 >1.5) 
4. FeNO ≥50ppb at screening despite high dose inhaled corticosteroids 
(at least 1000mcg BDP equivalent) +/- maintenance prednisolone. 
5. Adult-onset (18+) asthma in a minimum of 50% of the study subjects 
Statistical Methodology and Analysis 
 
The primary comparisons are pre- and post-benralizumab therapy.  
Descriptive statistics will be used for demographic and clinical 
characteristics of the study subjects.   
Clinical outcome measures will be compared using either parametric 
paired t-tests or non-parametric U-tests for continuous variables, and 
fisher tests for the analysis of proportions.    
All experimental analyses will be paired, considering patient-specific 
changes. All analyses involving the measurement and comparison of 
multiple variables (e.g. RNA-seq) will include a multiple test correction, 
such as false discovery rate / Bonferroni. 
 
Human Tissue Samples (if applicable) 
 
All human tissue samples will be processed and analysed by the main 
team and collaborators of co-investigator Dr Rocio Martinez-Nunez at 
King’s College London. 
These samples will include blood, cells and tissues from the airways. 
Data collected/storage (if applicable) 
 
We will be using a combination of paper and electronic records.  
Pseudonymised data will be collected by the direct care team and KCL 
research team. All source documentation will be filed both physically 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 9 of 31 
 
and electronically in a secure location within GSTT and KCL sites and on 
password protected computers managed by GSTFT and KCL. The paper 
records will be filed in the study-specific patient folder and will also be 
scanned and filed electronically. 
 
 
 
 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 10 of 31 
 
5 Introduction 
Over 5 million people in the UK have asthma of varying severity. Despite maximum treatment (with 
high dose inhaled corticosteroids plus a second controller and/or systemic corticosteroids), ~5-10% of 
the patients remain symptomatic. These severe asthma patients have a high blood eosinophil count, 
frequent exacerbations and poor prognosis. Severe asthma remains an unmet need with significant 
impact on patients, society and healthcare. 
 
Eosinophils are a type of white blood cell that are linked to allergy and inflammation and are raised in 
people with severe asthma. Increased levels of eosinophils can cause inflammation in the lungs, 
increasing the risk of asthma attacks. The standard treatment for asthma involves taking inhaled 
glucocorticosteroid medication which primarily work by suppressing eosinophilic inflammation in the 
lungs. With this treatment, the majority of people with asthma have no or very few asthma symptoms 
but those with severe asthma have ongoing eosinophilic inflammation despite this treatment and 
remain highly symptomatic and continue to experience frequent asthma attacks.  
 
Commonly in severe asthma, there is bronchial epithelial damage (the junctions holding cells together 
on the airway lining is damaged), there is an increase in cells that make mucus, an increase in the 
thickness of the airway wall and an increase in immune cells. Granules produced by eosinophils (a type 
of white blood cell), can add to this damage and in particular increase the amount of mucus in the 
airways, which can block airways. Some studies have shown that increases in eosinophils in the 
airways can affect the immune responses to viruses and bacteria. 
 
Benralizumab is a relatively new treatment that is approved by NICE for patients with severe asthma 
and ongoing eosinophilic inflammation that remains poorly controlled despite high dose inhaled 
corticosteroid medication.   Benralizumab targets a receptor on the surface of eosinophils called IL-5R 
leading to the rapid death of these cells and consequently a reduction in airways inflammation.  In 
clinical trials, benralizumab has been shown to reduce both symptoms and the number of asthma 
attacks suffered by those with severe eosinophilic asthma. However, it remains unclear whether this 
clinical efficacy relates purely to the removal of the eosinophil, or additionally to the impact of this on 
other parts of the immune system.  
 
The clinical effectiveness of benralizumab evident across phase 3 and real world studies has reaffirmed 
the central role that the eosinophil plays in disease control and exacerbation pathogenesis in patients 
with severe asthma. However, whilst it is generally believed that this fundamentally reflects the 
removal of eosinophils and their toxic granules and mediators from the airway, the full impact of 
benralizumab on T2 immunity may be much broader. Firstly, benralizumab-treated patients stop 
exacerbating when they encounter respiratory viruses such as rhinovirus, suggesting that the deficient 
anti-viral immune responses present in many uncontrolled T2-high asthmatics are restored upon 
benralizumab. Secondly, our centres’ finding that patients continue to remain well-controlled despite 
significantly reducing their inhaled corticosteroid (ICS) use following initiation of benralizumab 
suggests that the other elements of T2 inflammation (including IL-13 driven pathways) are either 
unimportant or suppressed (d’Ancona et al Allergy 2021). Thirdly our observation that benralizumab 
is very effective in FeNO-high patients (indicating increased IL-13-driven activation) and that 
treatment significantly reduces FeNO levels suggests that benralizumab directly or indirectly 
suppresses IL-13 pathways (Hearn et al JACI-IP 2021). Taken together, there is an increasing body of 
data supporting the notion that benralizumab has far-reaching anti-T2 effects that underpin its 
excellent efficacy in clinical practise. Through the use of novel techniques including spatial 
transcriptomics of bronchial biopsies this proposal aims to define and describe these effects. 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 11 of 31 
 
 
The BEUTI study will examine how benralizumab affects the structure and function of the airway cells 
in people with severe eosinophilic asthma and how the immune function of these cells changes with 
treatment. The aim is to take samples of cells from the airways during a bronchoscopy (a camera test 
looking into the lungs) before starting benralizumab and after 12 weeks of treatment. These 
investigations will allow us to better understand how benralizumab affects the cells within the airways. 
 
All potential participants with severe eosinophilic asthma (according to standard NICE criteria for 
biologic eligibility under the NHS) who have already been approved for this treatment in the severe 
asthma multidisciplinary meeting at Guy’s & St Thomas’ NHS Trust will be approached for this study. 
Agreement to take part in this study, will not significantly delay treatment initiation for these patients 
by more than a few weeks. Potential participants will be provided with further written information 
about the study in the form of the patient information sheet (PIS). They will be given ample time to 
read this and there will be a contact telephone number in the PIS should they wish to contact the 
research team for further information. 
 
Hypothesis: 
The depletion of eosinophils by benralizumab leads to additional inhibitory effects on T2 inflammation 
which collectively underpins the improvements in multiple clinical domains and reduction in T2 
biomarkers observed in real world cohort studies. 
 
The important research questions we will aim to answer: 
i) 
What is the effect of benralizumab on T2 signalling. Can benralizumab modulate T2-signalling 
and/or gene expression in addition to triggering eosinophil and basophil depletion? 
ii) 
What additional cell types (e.g. ILC2, alveolar macrophages, mast cells, bronchial epithelial 
cells, airway smooth muscle) are regulated by benralizumab? 
iii) 
Does the marked reduction in exacerbations observed with benralizumab partly reflect 
restoration of the deficient anti-viral immune responses seen in patients with severe asthma? 
6 Trial objectives and purpose 
 
Primary Objective: 
To determine if benralizumab leads to a reduction in T2 mediators and/or activation in airway cells in 
patients with severe eosinophilic asthma. 
The effect of benralizumab on the airway inflammatory (BAL) cell profile of study subjects will be 
assessed using CyTOF.  The CyTOF panel markers are: CD3, CD4, CD8, CD11c, CD14, CD16, CD19, CD20, 
CD25, CD27, CD28, CD38, CD45RA, CD45RO, CD56, CD57, CD66b, CD123, CD127, CD161, CD294, CCR4, 
CC46, CCR7, CXCR3, CXCR5, HLA-DR, IgD, TCRγδ, IL-33, IL-5RA/CD125, IL-6, IL-4, IL-5, IL-13. 
These allow the identification of: CD4+ cells (Naïve, memory, and terminal effector  CD4+ T cell, Th1, 
Th2, Th17, T regs), CD3+ CD4- (MAIT/NKT, γδ T Cell), CD3- CD8+ (Naïve and memory CD8+ T cell), CD3- 
Lymphocytes (NK cells - Early, late), B cells (naïve, memory, plasmablasts), monocytes, dendritic cells 
(myeloid, plasmacytoid), granulocytes (eosinophils, neutrophils, basophils), and ILCs. 
 
Secondary Objectives 
1.     Determine any change in T2-related signalling pathways and cell populations in bronchial biopsies 
(including those relating to IL-4, IL-5, IL-13, TSLP, IL-25 or IL-33) in patients with severe eosinophilic 
asthma before and after 12 weeks of benralizumab using spatial transcriptomics 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 12 of 31 
 
There has been recent progress in high-throughput techniques for single-cell genomic and 
transcriptomic analyses which has specifically led to spatially resolved transcriptomics, or spatial 
transcriptomics which consists of integrating the information gathered from the study of individual 
cells within a tissue as well as their cellular localisation within the same tissue (histology) together 
with the throughput of RNA-sequencing. This has opened up new possibilities to classify these into 
cell types, and characterize variations in their molecular profiles as a function of therapy in the case 
of our study but also in the context of genetics, the microbiome environment, cell–cell interactions, 
developmental processes, aging, and asthma disease. 
 
Single cell sequencing methods allow for establishing the profile of individual cells leading to the 
discovery of novel cell populations and activation status. spatial transcriptomics has not been applied 
in patients with asthma as it has only recently been developed but the technology has been applied 
to post-mortem lung tissues from those who have died as a result of COVID-19 to better understand 
if smoking status or other lung diseases like COPD played a role in COVID severity. This method has 
never been used to study asthma or in understanding the full mechanism of action of benralizumab. 
For example, single cell RNA-seq has shown that IL-13 promotes remodelling in distinct populations 
of airway epithelial cells (Jackson et al. Cell Rep 2020). Spatial transcriptomics will allow identification 
of cellular populations that are modulated by benralizumab treatment - similar to single cell RNA-
sequencing - the potential to reveal novel target populations of benralizumab treatment (including 
their spatial position), and the interactions between them in their tissue of origin. This is important 
because different milieu, provided by different surrounding cells, may differentially modulate one 
same cell type. It will also allow for analysing traditionally difficult to study cells such as mast cells and 
eosinophils present in the tissue, in their location of origin with their in situ interactions. We will 
validate our observations employing immunofluorescence to confirm the expression of novel 
markers/mediators uncovered. Our KCL Genomics Core has the technology to implement these 
experiments (Visium 10x Genomics). Polyadenylated RNA from tissue sections is captured in situ on 
arrays of spatially barcoded DNA probes, providing information of not only gene expression levels but 
also tissue localization. Two sequential cryosections from bronchial biopsies from each patient will be 
performed pre- and post-benralizumab treatment. 
 
2.     Determine any in vitro changes in bronchial epithelial antiviral responses to rhinovirus-16 and 
SARS-CoV-2 before and after 12 weeks of benralizumab. 
Isolated primary bronchial epithelial cells from bronchial brushings will be cultured for a maximum of 
4 passages and infected with rhinovirus 16 and SARS-CoV-2. We will measure transcriptional mRNA 
changes employing RNA-seq on uninfected vs infected bronchial epithelial cells, before and after 12 
weeks of Benralizumab. Unstimulated cells will inform about baseline differences triggered by 
Benralizumab; rhinovirus- and SARS-CoV-2-infected cells will inform about changes triggered by 
Benralizumab that modulate the anti-viral immune response. We will employ these two viruses as 
they are both positive strand RNA viruses but elicit very different anti-viral responses. Moreover, 
rhinovirus is the major cause of asthma exacerbations while SARS-CoV-2 does not trigger as many, 
with underlying mechanisms still poorly understood. We will also assess bronchial epithelial integrity 
by assessing the transepithelial resistance of air-liquid interphase cultures or barrier permeability 
assays. In addition, we will be able to investigate the effects of different variants of SARS-CoV-2 that 
may arise, in addition to the RNA-seq cell data with SARS-CoV-2 omicron. We will aim to employ the 
most relevant variant of omicron for our RNA-seq experiments. 
 
The Co-Investigator on this proposal Dr Martinez-Nunez led the KCL diagnostics response for COVID-
19, including the diagnostics lab, and has already performed BEC infection with SARS-CoV-2 in our 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 13 of 31 
 
institutions’ Cat-3 lab. SARS-CoV-2 infections are routinely done in our School. Her lab also has 
expertise in rhinovirus infections and RNA-sequencing. 
 
The RNA-seq datasets will be analysed to determine genes modulated by benralizumab, as well as 
enrichment of biological pathways employing Ingenuity Pathway Analysis (Qiagen) for which the 
College holds a licence. This will enable exploring other pathways modulated by Benralizumab in 
bronchial epithelial cells, such as those related to epithelial integrity (e.g. occludins, integrins), 
immune profile (alarmins) both at baseline and upon infection.  
 
3.     Determine any transcriptomic changes in genes relating to airway remodelling before and after 
12 weeks of benralizumab 
We will integrate the spatial-associated RNA-seq datasets from bronchial biopsies (spatial 
transcriptomics, secondary objective 1) with bulk RNA-seq in BECs (secondary objective 2) and 
investigate changes in gene expression that relate to airways remodelling (e.g. genes encoding for 
structural proteins, integrins, extracellular matrix proteins etc.). We will focus on analysing non-
stimulated BECs from secondary objective 2, pre- and post- benralizumab, which will show the 
changes in epithelium responses upon benralizumab treatment. 
 
We will further analyse extracellular matrix (ECM) deposition and degradation in biopsies using 
antibodies directed against ECM proteins as well as chemical trichrome staining (Masson’s) to visualise 
connective tissues (collagen), cells and smooth muscle. We will also establish transepithelial 
integrity/permeability assays employing different methods (e.g. dextran permeability through the 
epithelial barrier).  
 
Where feasible, we will dissect larger bronchial biopsies and attempt to isolate other structural cells 
(smooth muscle and fibroblasts) for functional assays associated with inflammation and 
bronchoconstriction and examine: cell contractility, proliferation, migration, protein expression and 
cytokine production. 
 
4.     Determine the effect of benralizumab on small airway dysfunction using impulse oscillometry and 
MRI to measure the change in homogeneity of airways ventilation 
Impulse oscillometry is a far more accurate measure of small airways airflow obstruction than 
standard spirometry, whilst ventilation assessed by inhaled gas MRI has been recently shown to be 
sensitive to anti-T2 therapies and can be used to objectively evaluate the functional consequence of 
clearing eosinophils from the airway lumen (Svenningsen et al. CHEST Oct 2020). 
7 Study design & Flowchart 
7.1 Study Design 
This is a prospective open label phase IV observational study 
 
Primary Endpoint: 
Change in the number / proportion of T2 related cellular populations in BAL including ILC2, Th2, 
alveolar macrophages, eosinophils and basophils pre- and post benralizumab (12 weeks). 
 
Secondary Endpoints: 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 14 of 31 
 
1. 
Change in cellular populations and gene expression of T2-related signalling pathways in 
bronchial biopsies pre- and post benralizumab 
2. 
Change in type 1 & 3 interferon production and viral replication following infection of cultured 
BECs and alveolar macrophages with RV-16 and examine the response of cells to SARS-CoV-2 collected 
pre- and post- benralizumab. 
3. 
Change in cellular populations and gene expression of remodelling-related pathways in 
bronchial biopsies pre- and post benralizumab 
4. 
Change in airway oscillometry measures and MRI measures of ventilation pre- and post 
benralizumab. 
Each recruited participant will be in the study for 3 months and will attend for 8 visits including 
screening and informed consent. Visits will include blood, breathing tests and bronchoscopy. 
Participants will undergo bronchoscopy twice in a 12 week period (pre- and 12 weeks post 
benralizumab treatment). Processing of biological samples and data from clinical measures will be 
mined throughout the study period of 24 months and also when patients revert to ongoing NHS clinical 
care that is standard for those on benralizumab treatment for their asthma. 
Gantt Chart: The total study duration is 24 months.  
Months 
3 
6 
9 
12 
15 
18 
21 
24 
Ethics application and approval 
 
 
 
 
 
 
 
 
Patient recruitment, bronchoscopies and 
sample collection and laboratory procedures 
 
 
 
 
 
 
 
 
BEC and mac culture for anti-viral immunity 
 
 
 
 
 
 
 
 
BEC and Br biopsy Spatial transcriptomics 
 
 
 
 
 
 
 
 
Protein analysis  
 
 
 
 
 
 
 
 
Data analysis 
 
 
 
 
 
 
 
 
Manuscript(s) preparation 
 
 
 
 
 
 
 
 
 
 
Treatment Regimen: 
Benralizumab will be commenced according to UK NICE severe asthma guidance with 30mg sc dosing 
every 4 weeks for first 3 doses, followed by every 8 weeks. 
 
Design and patient flow:  
Visit 1: Consent, Enrollment and schedule of visit dates 
Visit 2: Day 1 Covid-19 test 
Visit 3: Day 2 Bronchoscopy 
Visit 4: Day 10 Subject receives benralizumab dose 1 (30mg subcutaneous injection) 
Visit 5: Day 38 Subject receives benralizumab dose 2 (30mg subcutaneous injection) 
Visit 6: Day 66 Subject receives benralizumab dose 3 (30mg subcutaneous injection) 
Visit 7: Day 83 Covid-19 test 
Visit 8: Day 84 Bronchoscopy 
 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 15 of 31 
 
At visit 1, all eligible participants will give informed consent. They will then have baseline 
characteristics recorded, including information regarding their asthma history and symptoms, asthma 
control score and quality of life questionaire (ACQ-6/mAQLQ) to assess lung function, general medical 
history and all current medications. Breathing tests will be performed (spirometry, fractional exhaled 
nitric oxide (FeNO)). A blood sample will be taken and participants will also be asked for a urine sample 
(pregnancy test for female participants only).  
At this visit, the bronchoscopy will be discussed in more detail, and a separate bronchoscopy consent 
form signed. A convenient date will be arranged for both the bronchoscopy and Covid-19 test.  
 
Visit 2: This visit is one day before the bronchoscopy and participants will have a COVID-19 test and 
consent for bronchoscopy. 
 
Visit 3 and visit 8: Participants will undergo a bronchoscopy, breathing tests, have a blood test, and 
then the bronchoscopy with samples taken of airway cells.  
 
Visit 4/5/6: Participants will receive a dose of benralizumab at monthly intervals during the study and 
will subsequently continue to receive benralizumab every 8 weeks as per standard NHS guidelines. 
Patients will be followed up in the NHS severe asthma clinic as their clinical condition requires. 
Information regarding their clinical condition will be collected from their clinical notes over the 
duration of the study. 
 
Visit 7: This visit is one day before the 2nd bronchoscopy and participants will have a COVID-19 test 
and consent for bronchoscopy. 
 
At all visits unless otherwise stated, the following information and samples will be collected: 
Information regarding their asthma symptoms and adverse events 
Asthma control score and quality of life questionaire (ACQ-6/mAQLQ) 
Breathing tests will be performed (spirometry, fractional exhaled nitric oxide (FeNO)). 
A blood and urine sample (Visit 1 and 4 only) will be taken. 
 
7.2 Flowchart 
 
Screening 
Visit  
Study visits 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
Day 
 
1 
 
COVID 
PCR 
2 
 
Bronch 1 
10 
 
benra 
dose 1 
38 
 
benra 
dose 2 
66 
 
benra 
dose 3 
93 
 
COVID 
PCR 
94 
 
Bronch 2 
Full medical history, 
confirmation of SEA, 
medication review 
X 
 
 
 
 
 
 
 
Physical examination 
X 
X 
X 
X 
X 
X 
X 
X 
Current/recent 
medications, side effects  
X 
 
X 
X 
X 
X 
X 
X 
X 
Nasal and/or throat swab 
for COVID-19 testing (PCR) 
 
X 
 
 
 
 
 
X 
 
Benralizumab 
 
 
 
X 
X 
X 
 
 
Bronchoscopy 
 
 
X 
 
 
 
 
X 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 16 of 31 
 
 
Screening 
Visit  
Study visits 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
Day 
 
1 
 
COVID 
PCR 
2 
 
Bronch 1 
10 
 
benra 
dose 1 
38 
 
benra 
dose 2 
66 
 
benra 
dose 3 
93 
 
COVID 
PCR 
94 
 
Bronch 2 
FeNO 
X 
 
X 
X 
X 
X 
 
X 
Spirometry 
X 
 
X 
X 
X 
X 
 
X 
Oscillometry 
X 
 
X 
X 
X 
X 
 
X 
ACQ-6 
X 
 
X 
X 
X 
X 
X 
X 
mAQLQ 
X 
 
X 
X 
X 
X 
X 
X 
Blood 
X 
 
X 
X 
X 
X 
X 
X 
Urine pregnancy test 
(females only) 
X 
 
 
X 
 
 
 
 
Collection of adverse 
events (AE) 
 
X 
X 
X 
X 
X 
X 
X 
 
8 Subject selection 
 
Population: 
Adult patients with poorly-controlled severe eosinophilic asthma referred to Guy's Severe Asthma 
Centre, Guy's & St Thomas' NHS Trust, who meet NICE criteria to commence biologic therapy with 
benralizumab. Following review of eligibility criteria potential participants will be provided with 
further written information about the study in the form of the Patient Information Sheet (PIS). They 
will be given ample time to read this and there will be a contact telephone number in the PIS should 
they wish to contact the research team for further information. 
 
This is a single centre study. 
 
Each recruited participant will be in the study for 3 months and will attend for 8 visits including 
screening and informed consent. Visits will include blood, breathing tests and bronchoscopy. 
Participants will undergo bronchoscopy twice in a 12 week period (pre- and 12 weeks post 
benralizumab treatment). Processing of biological samples and data from clinical measures will be 
mined throughout the study period of 24 months and also when patients revert to ongoing NHS clinical 
care that is standard for those on benralizumab treatment for their asthma. 
 
Retention: 
The PIS will explain that the study involves 2 bronchoscopies and it is hoped that patients do not drop 
out of the study prior to the second bronchoscopy.  We will work with our Patient Research 
Ambassadors to support retention.  If patients decide to drop out they will continue to receive 
benralizumab as per the plan from the multidisciplinary team (MDT) and patients will be replaced until 
the desired paired sample numbers are reached.  The clinical investigators are experienced in 
recruiting patients as well as performing bronchoscopies on patients with severe asthma and a 
standardised sedation and periprocedural protocol will be used to maintain consistency in sampling 
and patient experience.  
Patients can withdraw from the study at any time.  This will not affect their care or treatment within 
Guy’s Severe Asthma Service and in particular will not affect ongoing delivery of benralizumab. 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 17 of 31 
 
 
Sample Size Justification: 
Based on the observed 85% responder rate to benralizumab in our centre (Kavanagh et al. CHEST 
2021), statistically significant falls in FeNO observed in FeNO-high patients with benralizumab, and 
successful published transcriptomics studies in asthma of a similar number, we believe 12 subjects 
should yield at least 10 clinical responders to benralizumab. Twelve is also an achievable amount from 
a practical perspective to allow timely completion of this study in under 18 months from first subject 
in (FSI). 
8.1 Subject inclusion criteria 
1. 
Patients aged 18 and over with a diagnosis of severe eosinophilic asthma for at least the last 
6 months 
2. 
Eligible for benralizumab based on NICE criteria 
3. 
Poorly-controlled (ACQ-6 >1.5) 
4. 
FeNO ≥50ppb at screening despite high dose inhaled corticosteroids (at least 1000mcg BDP 
equivalent) +/- maintenance prednisolone 
5. 
Adult-onset (18+) asthma in a minimum of 50% of the study subjects 
8.2 Subject exclusion criteria  
1. 
Other severe eosinophilic lung disease including EGPA, chronic eosinophilic pneumonia and 
ABPA 
2. 
Maintenance daily oral corticosteroids (prednisolone) 
3. 
Severe bronchiectasis on CT causing daily sputum production 
4. 
Inability to give written informed consent 
5. 
Current smoking or >20 pack year smoking history 
6. 
Resting oxygen saturations <94% on air 
7. 
Any severe cardiac or other non-asthma related co-morbidity that would make bronchoscopy 
and/or sedation high-risk 
8. 
Symptoms suggestive of a respiratory viral / bacterial infection within the last 3 weeks 
9. 
Acute exacerbations of asthma requiring high dose prednisolone within the last 3 weeks 
10. 
A change in dose of maintenance inhaled and/or oral corticosteroid dose within the last 3 
weeks 
11. 
Positive strongyloides serology following screening 
12. 
Pregnancy or lactation 
13. 
Hypersensitivity to benralizumab or any of the excipients 
9 Study procedures 
9.1 Subject recruitment 
Members of the patient's existing direct care team will have access to patient records for discussion 
at the Guy's and St Thomas' hospital NHS Trust (GSTT) severe asthma clinic meetings. Potential 
participants will be identified at site by the direct care team using clinic lists. Adult patients with 
poorly-controlled severe eosinophilic asthma referred to Guy's Severe Asthma Centre clinic at Guy's 
& St Thomas' NHS Trust, who meet NICE criteria to commence biologic therapy with benralizumab will 
be approached by the direct care team.  
 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 18 of 31 
 
Following review of eligibility criteria potential participants will be provided with further written 
information about the study in the form of the Patient Information Sheet (PIS). They will be sent the 
PIS at least a week before their clinic visit and given as much time as they need read the PIS and to ask 
questions. The PIS and the Informed Consent Form (ICF) will be explained in clear, simple language. 
They will be provided with a contact telephone number should they wish to contact the research team 
for further information. The patients will have as much time as they require to decide whether to take 
part and will be given the opportunity to go home and discuss with family/friends if they want to. 
 
The bronchoscopy procedure will also be discussed in detail prior to obtaining consent. 
The asthma direct care team at GSTFT are integrated with the research team and these activities will 
be completed by both teams. 
Informed Consent: 
The process for obtaining participant informed consent will be in accordance with the REC guidance, 
and GCP and other regulatory requirements that might be introduced.  
The chief investigator and/or the direct care team will conduct the informed consent process by 
explaining to the participant the nature of the study; its purpose; the procedures involved; the 
expected duration; the potential risks and benefits involved; and the fact that participation is 
completely voluntary and consent may be withdrawn at any time with no consequences for their 
subsequent medical care. 
Participants will also be asked to review and sign a standard bronchoscopy consent form currently 
used by the respiratory department as per trust policy 
 
The CI/PI/delegate and the participant or other legally authorised representative shall both sign and 
date the Informed Consent Form before the person can participate in the study. 
The participant will keep a copy of the PIS and a signed and dated Consent Form. The original will be 
retained in the Trial Master File. A second copy, along with the PIS, will be filed in the participant’s 
medical notes and a signed and dated note made in the notes of when the PIS was provided and that 
informed consent was obtained for the study.  
The decision regarding participation in the study is entirely voluntary. The investigator or their 
nominee shall emphasize to the participant that consent regarding study participation may be 
withdrawn at any time without penalty or affecting the quality or quantity of their future medical care, 
or loss of benefits to which the participant is otherwise entitled. 
 
Participation in the study is voluntary and recruited patients will not be paid. 
 
Participants will also be made aware of and consent to the fact that their GP will be informed of their 
participation. Each participant will authorise in writing that portions of their medical records and 
source data related to the project may be reviewed by a monitor, an auditor or a regulatory inspector, 
in accordance with applicable regulatory requirements. They will be made aware that Data protection 
will be handled in compliance with UK law.  
 
9.2 Screening Procedures 
Members of the patient's existing direct care team will have access to patient records for discussion 
at the Guy's and St Thomas' hospital NHS Trust (GSTT) severe asthma clinic meetings.  
 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 19 of 31 
 
Where the participant has been approved to receive benralizumab for clinical reasons and they meet 
the inclusion criteria for the study, the identified participant will then be approached by a member of 
the GSTT asthma direct care team who will discuss the study in detail with their patient. The patient 
will be given a copy of the participant information sheet (PIS) or one will be sent to them by post or 
email to consider before their next scheduled clinic visit. The patient will be given adequate time to 
decide whether or not they wish to take part in the study and be encouraged to ask any questions. 
 
9.3 Schedule of assessments for each visit  
See also section 7.2 
Design and patient flow:  
Visit 1: Consent, Enrollment and schedule of visit dates 
Visit 2: Day 1 Covid-19 test 
Visit 3: Day 2 Bronchoscopy 
Visit 4: Day 10 Subject receives benralizumab dose 1 (30mg subcutaneous injection) 
Visit 5: Day 38 Subject receives benralizumab dose 2 (30mg subcutaneous injection) 
Visit 6: Day 66 Subject receives benralizumab dose 3 (30mg subcutaneous injection) 
Visit 7: Day 83 Covid-19 test 
Visit 8: Day 84 Bronchoscopy 
 
9.4 Follow up Procedures 
The study participants will be referred back to the care of GSTT asthma clinic staff for continued 
standard care. There will be no study specific follow up. 
9.5 End of Study Definition  
The study is planned over a 2 year period. Last visit of the last subject will take place by 18 months of 
the study start date i.e. all 12 participants would have had 2 bronchoscopies by 18 months. After this 
point we have planned 6 months for processing of clinical samples, molecular work, data analysis and 
interpretation. 
10 Laboratories  
10.1 Central/Local Laboratories  
All the samples collected in this study will be processed in the laboratories of Prof. David Jackson and 
Dr Rocio Martinez Nunez within King’s College London at Guy’s hospital campus.  
10.2 Sample Collection/Labelling/Logging  
Samples will be collected by the study direct care team. Samples will be immediately stored on ice or 
room temperature and labelled with patient code, visit number and the time of collection recorded 
on study worksheets, Each sample type will have specific SOPs and guidance on information that 
must be recorded at the appropriate environmental conditions to maintain sample integrity. The KCL 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 20 of 31 
 
research team or delegated personnel who are appropriately trained will be contacted to inform 
them that the sample is ready for collection or that the sample is being delivered to the laboratory. 
10.3 Sample Analysis Procedures  
The aim is to study the influence of benralizumab on structural and inflammatory airway cells in 
patients with severe eosinophilic asthma. 
Our primary outcome measure is: 
The change in the number / proportion and/or activation status of Type-2 related cellular 
populations in samples derived from bronchoscopy including bronchial biopsies, bronchoalveolar 
lavage (BAL) and bronchial brushings. These cells include ILC2, Th2, alveolar macrophages, epithelial 
cells, eosinophils and basophils pre- and post- 12 weeks of benralizumab using molecular 
techniques. 
Secondary Endpoints:  
To investigate the following before and after completing treatment with 12 weeks of benralizumab. 
 
1. Changes in epithelial barrier integrity using cell biology methods such as transepithelial resistance 
or barrier permeability. 
2. Changes in epithelial antiviral responses in untreated vs respiratory virus-infected cells including 
rhinovirus-16 and SARS-CoV-2 using protein and nucleic acid analyses. 
3. Changes in the antiviral responses of alveolar macrophages to rhinovirus-16 or other respiratory 
virus infection using protein and nucleic acid analyses. 
4. Changes in epithelial responses to cytokines/chemokines and/or exposure to other cell types (co-
cultures). 
5. Changes in airway remodelling by histological examination. 
6. Changes in the profile and function of isolated structural cells (such as airway smooth muscle and 
epithelial cells but not restricted to) dissected from larger biopsies by molecular and cellular 
methods. 
7. To evaluate the impact on peripheral airways by obtaining bronchoalveolar lavage (BAL) and 
performing nucleic acid and protein analyses 
8. Peripheral airway dysfunction using tests such as impulse oscillometry but not restricted to. 
9. Change in FeNO levels. 
Study procedures- Clinical Assessment 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 21 of 31 
 
The CRF will be completed and include clinical details about asthma and other relevant medical 
conditions and medications.  
Spirometry  
Spirometry will be conducted in hospital using a spirometer conforming to ATS/ERS standards as 
specified by the manufacturer’s instructions.  FEV1 (L), FVC (L), FEV1/FVC ratio, FEF25-75 (% of 
predicted value) and PEF (L/min) will be recorded. FEV1, FVC and PEF will be documented as both 
absolute values and as a percentage of the predicted value. 
Oscillometry 
Oscillometry is a non-invasive measure of pulmonary resistance and reactance. By measuring 
oscillating pressures/flow signals of moving air within the lungs, when generated at different 
frequencies, it provides measures of airway mechanics from different lung regions.  Generally lower 
frequencies, in particular frequency dependent changes in resistance between 5 and 20 Hz and 
capacitive reactance at 5 Hz are thought to reflect changes arising in the more distal airways. 
Oscillometry will be performed pre- and post-bronchodilator.  
Fractional nitric oxide (FeNO) 
FeNO is a surrogate marker of T2-airway inflammation. FeNO will be measured using a NIOX VERO 
device, as specified by the manufacturer’s instructions. This includes collection by controlled 
exhalation at the recommended controlled expiratory flow rate of 50ml/s for greater than 6 
seconds. 
Blood sampling 
Blood will be collected for routine full blood count (including blood eosinophil and basophils), 
biochemistry and coagulation measurements pre-bronchoscopy. In addition plasma/ serum will be 
stored for proteomic/nucleic acid analyses.  
Bronchoscopy 
Bronchoscopy will be conducted according to established guidelines, and will be performed by 
respiratory physicians experienced in bronchoscopy. Participants will receive nebulised salbutamol 
(2.5mg) and ipratropium bromide (500mcg) prior to the procedure. Local anaesthesia will be 
achieved with gel applied to the nasal passages and lignocaine spray to the throat, soft palate and 
pharynx. Intravenous sedation using midazolam (maximum 5 mg) and alfentanyl (maximum 500 
mcg) will be given to achieve conscious sedation. A flexible bronchoscope will then be passed either 
through the nose or mouth into the lungs with additional lignocaine spray being applied to prevent 
coughing. 
i) 
Bronchial brushings will be performed using single-use brushes. Up to a total of 6 brushes 
will be taken. 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 22 of 31 
 
ii) 
Bronchial biopsies will be performed from the lower and middle lobes using single use 
forceps.  Up to 6 evaluable biopsies will be taken.  
iii) 
Bronchoalveolar lavage (BAL): the bronchoscope will be inserted in a sub-segmental bronchi 
and 60ml aliquots of pre-warmed 0.9% saline will be instilled followed by gentle suction to recover 
lining fluid. This will be repeated up to 3 times.  
At the first bronchoscopy (Visit 3) samples will be collected from the right side of the lung and at 
second bronchoscopy (Visit 8) samples will be taken from the left side.  
The bronchoscopy will be rescheduled if the patient has had an asthma exacerbation in the 
preceding 6 weeks.  
Study Procedures- molecular 
Epithelial brushes processing 
Two brushings per patient will be stored at -80 C in RNA stabilising agent for future analysis. Four 
brushings will be processed for basal airway epithelial isolation by culturing in selective medium as 
previously described. Cultured cells, which are mainly basal epithelium, will be processed and 
infected/non infected with respiratory viruses including (but not restricted to) rhinovirus and SARS-
CoV-2 to assess genome-wide changes, both at baseline and in their antiviral response upon 
benralizumab treatment. We will first set up time courses of 8,24 and 48h given that the responses 
to rhinovirus and SARS-CoV-2 in bronchial epithelium differs in its dynamics, and sequence a single 
time point that is appropriate. We will keep RNA samples for all time points assessed for future 
validation purposes, together with supernatants to enable analysis of cellular mediators. 
Cells infected/uninfected with virus will be subjected to RNA-seq after treatment. Top candidates 
based on statistical and biological significance will be validated by qPCR; western blotting/ELISA or 
other protein detection approaches. In the validation experiments we will also employ UV-
inactivated virus as additional control. Cell culture supernatants will be collected to assess virus 
titres and RNA, as well as for future protein expression analyses. Cell lysates will also be stored for 
future protein level assessment using different platforms (proteomics; western blotting; ELISA or 
similar such as Luminex, O-link, etc.). Same passage cells will also be frozen for future analyses and 
exploratory studies detailed above.  
Endobronchial biopsies 
Up to 6 biopsies will be obtained:  
Spatial transcriptomics: the optimal conditions of hybridization and staining will be firstly titrated in 
the lab using currently existing biopsy samples, to determine optimal conditions to process all 
samples. Biopsies will be fixed and both cryopreserved (snap frozen) as well as processed in paraffin 
wax for histological applications such as Visium Spatial Gene Expression protocols.  
Bronchoalveolar lavage processing 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 23 of 31 
 
An aliquot of bronchoalveolar lavage (BAL) will be stored for microbiome analysis. The BAL will then 
be spun to obtain cell pellets. Supernatant will be aliquoted for future analyses including 
microbiome, RNA and protein expression.  
An aliquot of at least 1 million cells will be frozen for future analyses. Cells will be plated to isolate 
alveolar macrophages as previously described. Purified alveolar macrophages will be 
infected/uninfected with respiratory viruses (including but not restricted to rhinovirus and SARS-
CoV-2), processed for RNA and protein measurements and stored at -80C for future analyses. 
Supernatants will also be collected for future analyses. 
Blood samples 
Blood samples will be spun on the same day of extraction. Serum/plasma will be frozen for 
proteomics/nucleic acid analysis. An aliquot of whole blood cells will be stored in an RNA stabilising 
agent and aliquots of whole blood cells and peripheral blood mononuclear cells obtained by gradient 
centrifugation will also be frozen in freezing medium for future analyses. 
10.4 Sample Storage Procedures 
Samples will be appropriately stored in temperature controlled fridge, freezers and liquid nitrogen 
dewars which are remotely monitored 24hr. These temperature records will be filed for laboratory 
audit purposes to ensure and to show that the integrity of the stored samples have not been 
compromised. Inventory logs of all study samples will be maintained and storage positions in the 
freezers and dewar will be clearly recorded. 
The samples will be stored in secure freezers in laboratories within the School of Immunology and 
Microbial Sciences at King's College London and GSTT sites linked to the direct care team. 
Overall custodial arrangements will be with the Chief Investigator and access will only be provided to 
the study team or delegates of the study team (as decided by the chief Investigator). 
The HTA site license number is 12521 for the KCL Guy's campus site. The designated individual is 
Cheryl Gillett. 
10.5 Data Recording/Reporting  
Enrolment into the study will be documented in each participant’s medical notes.  Data collection 
will comprise of a paper case report form (CRF) including participant demographics, asthma 
characteristics (e.g. age of onset, atopic status, T2 biomarker levels, presence of nasal polyposis etc), 
pulmonary function test results and record of bronchoscopy procedures. 
All data will be handled within the conditions of the General Data Protection Regulations (GDPR) 
which came into force in May 2018 with the participant’s informed consent form.  Confidential 
information, both medical records and personal information will only be accessible by members of 
the GSTT direct care team. Recruited participants will be pseudonymised (given a code) and any data 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 24 of 31 
 
records will be referred to by this code. Data will be archived according to current ICH GCP 
guidelines.  Laboratory books containing results of the study will not contain personal information. 
A combination of paper and electronic records will be used. The paper records will be filed in the 
study-specific folder and will also be scanned and filed electronically on KCL and GSTT servers on 
computers that are password protected. 
Molecular data: 
Sequencing datasets: sequencing datasets will be stored in local servers at King’s College London 
(KCL) and in double-password protected hard drives which will be kept in a locked cabinet. 
Integration of the transcriptomics and clinical datasets will be done in local machines at KCL; if more 
computational power is required we will leverage on the cloud resources such as our local high 
Performance Computer (Rosalind) or Cyverse. Patient datasets will be pseudonymised and all data 
handling in the cloud will be done in accordance with GDPR regulations. 
Strict measures will be in place to safeguard the confidentiality of all data collected. All laboratory 
specimens and forms, laboratory reports, study data collection tools and stored samples will be 
identified using unique study numbers alone, to maintain participant confidentiality. No personal 
identifier information will be shared or recorded on any of these forms or samples and their 
associated databases. Personal identifiers (e.g. name and contact details) will only be collected for 
(1) informed consent and (2) logistical purposes. Personal identifiers will appear on consent forms 
and paper enrolment logs only. These will NOT include any sensitive study information. The unique 
study number will link personal identifiers to study information. All electronic data will be stored in a 
password protected database systems on password protected computers, servers and networks. No 
individuals will ever be identified when reporting study findings or making sequencing data publicly 
available at the time of publication. 
10.6 Sample Receipt/Chain of Custody/Accountability  
Firstly, a materials transfer agreement will be in place between the GSTT and KCL site for sample 
transfer and chain of custody processes.  Laboratory SOPs are in place to meet GCLP and HTA 
compliance. Sample receipt and processing worksheets will be in place for each sample type to 
record the patient code number, visit number, date, time and condition of the samples upon arrival 
to the laboratory. Sample labelling will be maintained throughout the processing and storing 
procedure. 
10.7 Sample Transfer to sites outside the Organisation 
Where study samples will need to be transferred outside the organisation to fulfil the endpoints of 
the study. These will be appropriately packed and sent with a medical courier with documentation of 
sample code, sample type, volume and safety guidelines and transported at an appropriate 
temperature. 
In the study, biological samples [blood, lung biopsies and lavage fluid and airway cells] will be 
collected from patients in accordance with the patient consent form and patient information sheet 
and shall include all tissue samples or other biological materials and any derivatives, portions, 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 25 of 31 
 
progeny or improvements as well as all patient information and documentation supplied in relation 
to them. Further, the custodian of the Materials will use the Materials for the Study only. For the 
avoidance of doubt Recipient shall only use the Material in accordance with the consent provided by 
the Study Donors (if applicable) and shall always use the Materials with dignity and respect and shall 
always use good laboratory practice in handling Materials 
 
The chief investigator and study co-investigator [Dr Rocio Martinez-Nunez at King’s College London] 
will process, store and dispose of the study samples [blood, lung biopsies and lavage fluid and airway 
cells] in accordance with all applicable legal and regulatory requirements, including the Human 
Tissue Act 2004 and any amendments thereto. While Provider uses reasonable endeavours to 
ensure the quality of the Materials, the Materials are provided ‘as is’ and it makes no representation 
and gives no warranty or undertaking in relation to the Materials, including but not limited to its 
safety, fitness for purpose or use of any kind. 
 
After ethics approval for the study has expired, the study samples [blood, lung biopsies and lavage 
fluid and airway cells] will be disposed of in accordance with the Human Tissue Act 2004, and any 
amendments thereto, or transferred to a licensed tissue bank. 
 
11  Assessment of Safety  
An Adverse event (AE) is the development of an undesirable medical condition or the deterioration 
of a pre-existing medical condition following or during exposure to a pharmaceutical product, 
whether or not considered causally related to the product. An undesirable medical condition can be 
symptoms (e.g. nausea, chest pain), signs (e.g. tachycardia, enlarged liver) or the abnormal results of 
an investigation (e.g. laboratory findings, electrocardiogram). In clinical studies, an AE can include an 
undesirable medical condition occurring at any time, including run-in or washout periods, even if no 
study treatment has been administered. The term AE is used to include both serious and non-serious 
AEs. 
An SAE is an AE occurring during any study phase (i.e., run-in, treatment, washout, follow- up), that 
fulfils one or more of the following criteria: 
• 
Results in death 
• 
Is immediately life-threatening 
• 
Requires in-patient hospitalization or prolongation of existing hospitalization 
• 
Results in persistent or significant disability/incapacity or substantial disruption of the ability 
to conduct normal life functions 
• 
Is a congenital anomaly or birth defect 
• 
Is an important medical event that may jeopardize the subject or may require medical 
intervention to prevent one of the outcomes listed above 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 26 of 31 
 
An adverse drug reaction is the development of an undesirable medical condition or the 
deterioration of a pre-existing medical condition following or during exposure to a medicinal 
product, suspected to be causally related to the product. 
Participants will be asked about the occurrence of any AEs at each visit and will be asked to report 
AEs to the study direct care team between visits. AEs will be assessed by the CI/PI for causality, 
intensity, seriousness and expectedness. All AEs will be recorded in the CRF and reported to the 
Sponsor as per their guidelines. Any adverse events that do occur and are considered by the PI to be 
related to the investigations will be expedited to the Sponsor and REC within 7 days. Lists of the AEs 
will be provided to the Sponsor when requested. 
All SAEs will be recorded on a Serious Adverse Event Form and will be reported within 24 hours of 
awareness to the Sponsor.  All SAEs occurring to a research participant that the CI feels was related 
i.e. resulted from administration of any of the research procedures, and unexpected i.e. the type of 
event not listed in the protocol as an expected occurrence will be reported to the appropriate REC. 
11.1 Ethics Reporting 
Reports of related and unexpected SAEs will be submitted to the Main REC within 15 days of the 
chief investigator becoming aware of the event, using the NRES template. The form will be 
completed in typescript and signed by the chief investigator. The main REC will acknowledge receipt 
of safety reports within 30 days. A copy of the SAE notification and acknowledgement receipt will be 
sent to the R&D Directorate.  
11.2 Trial Steering Committee (if applicable) 
Please outline if there will be any data monitoring/steering/safety committees set up for this study. 
Describe the extent of the role of this committee and their involvement within the study. 
11.3 Ethics & Regulatory Approvals 
State the name and address of the REC to which the study protocol and other documentation will 
be submitted.   
Which REC has not yet been determined. 
12 Compliance and withdrawal 
12.1 1Subject compliance 
The direct care team will administer benralizumab subcutaneously therefore compliance will not be 
an issue. 
12.2 Withdrawal / dropout of subjects 
Treatment Regimen: 
Benralizumab will be commenced according to UK NICE severe asthma guidance with 30mg sc dosing 
every 4 weeks for first 3 doses, followed by every 8 weeks. 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 27 of 31 
 
No change in any background asthma medications (e.g change in ICS type or dose) is permitted during 
the study. Given the importance of measuring the effect of 3 doses of benralizumab following the first 
bronchoscopy, the subject will be excluded if they do not receive the second and third benralizumab 
dose at the correct time intervals (+/- 7 days for each dose) following the first benralizumab injection. 
12.3 Protocol Compliance  
Any protocol deviation will be recorded and discussed with the CI to identify if a serious breach has 
occurred. 
 
13 Data 
13.1 Data to be collected 
The study is planned over a 2 year period. 
Last visit of the last subject will take place by 18 months of the study start date i.e. all 12 participants 
would have had 2 bronchoscopies by 18 months. 
After this point we have planned 6 months for processing of clinical samples, molecular work, data 
analysis and interpretation. 
The laboratory measures gathered from blood and bronchoscopy samples will be analysed and 
correlated to clinical physiological parameters. 
Main comparisons will be measures and pre- and post benralizumab treatment. 
 
Epithelial brushes processing 
Two brushings per patient will be stored at -80 C in RNA stabilising agent for future analysis. Four 
brushings will be processed for basal airway epithelial isolation by culturing in selective medium as 
previously described. Cultured cells, which are mainly basal epithelium, will be processed and 
infected/non infected with respiratory viruses including (but not restricted to) rhinovirus and SARS-
CoV-2 to assess genome-wide changes, both at baseline and in their antiviral response upon 
benralizumab treatment. We will first set up time courses of 8,24 and 48h given that the responses to 
rhinovirus and SARS-CoV-2 in bronchial epithelium differs in its dynamics, and sequence a single time 
point that is appropriate. We will keep RNA samples for all time points assessed for future validation 
purposes, together with supernatants to enable analysis of cellular mediators. 
Cells infected/uninfected with virus will be subjected to RNA-seq after treatment. Top candidates 
based on statistical and biological significance will be validated by qPCR; western blotting/ELISA or 
other protein detection approaches. In the validation experiments we will also employ UV-inactivated 
virus as additional control. Cell culture supernatants will be collected to assess virus titres and RNA, 
as well as for future protein expression analyses. Cell lysates will also be stored for future protein level 
assessment using different platforms (proteomics; western blotting; ELISA or similar such as Luminex, 
O-link, etc.). Same passage cells will also be frozen for future analyses and exploratory studies detailed 
above.  
Endobronchial biopsies 
Up to 6 biopsies will be obtained:  
Spatial transcriptomics: the optimal conditions of hybridization and staining will be firstly titrated in 
the lab using currently existing biopsy samples, to determine optimal conditions to process all 
samples. Biopsies will be fixed and both cryopreserved (snap frozen) as well as processed in paraffin 
wax for histological applications such as Visium Spatial Gene Expression protocols.  

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 28 of 31 
 
Bronchoalveolar lavage processing 
An aliquot of bronchoalveolar lavage (BAL) will be stored for microbiome analysis. The BAL will then 
be spun to obtain cell pellets. Supernatant will be aliquoted for future analyses including microbiome, 
RNA and protein expression.  
An aliquot of at least 1 million cells will be frozen for future analyses. Cells will be plated to isolate 
alveolar macrophages as previously described. Purified alveolar macrophages will be 
infected/uninfected with respiratory viruses (including but not restricted to rhinovirus and SARS-CoV-
2), processed for RNA and protein measurements and stored at -80C for future analyses. Supernatants 
will also be collected for future analyses. 
Blood samples 
Blood samples will be spun on the same day of extraction. Serum/plasma will be frozen for 
proteomics/nucleic acid analysis. An aliquot of whole blood cells will be stored in an RNA stabilising 
agent and aliquots of whole blood cells and peripheral blood mononuclear cells obtained by gradient 
centrifugation will also be frozen in freezing medium for future analyses. 
13.2 Data handling and record keeping 
The direct care team and KCL research team are responsible for data collection, recording and quality. 
All data will be handled within the conditions of the General Data Protection Regulations (GDPR) which 
came into force in May 2018 and the participant’s informed consent form.  Confidential information, 
both medical records and personal information will only be accessible by members of the clinical 
research team and any data removed from the site will be anonymous and de-identified. Data will be 
archived according to current ICH GCP guidelines.  Laboratory books containing results of the study 
will not contain personal information. 
Data collection forms:  
Enrolment into the study will be documented in each participant’s medical notes.  Data collection will 
comprise of a paper case report form (CRF) including participant demographics, asthma characteristics 
(e.g. age of onset, atopic status, T2 biomarker levels, presence of nasal polyposis etc), pulmonary 
function test results and record of bronchoscopy procedures. 
Strict measures will be in place to safeguard the confidentiality of all data collected. All laboratory 
specimens and forms, laboratory reports, study data collection tools and stored samples will be 
identified using unique study numbers alone, to maintain participant confidentiality. No personal 
identifier information will be recorded on any of these forms or samples and their associated 
databases. Personal identifiers (e.g. name and contact details) will only be collected for (1) informed 
consent and (2) logistical purposes. Personal identifiers will appear on consent forms and paper 
enrolment logs only. These will NOT include any sensitive study information. The unique study number 
will link personal identifiers to study information. All electronic data will be stored in a password 
protected database systems on password protected computers, servers and networks. No individuals 
will ever be identified when reporting study findings or making sequencing data publicly available at 
the time of publication. 
Specified retention period: 15 years after the end of the study 
14 Statistical considerations 
Statistician 
Name: Dr Rocio T Martinez-Nunez 
Address: King's Centre for Lung Health, Faculty of Life Sciences & Medicine, School of Immunology 
and Microbial Sciences, 5th Floor Tower Wing, Guy’s Campus, King’s College London, London SE1 
9RT 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 29 of 31 
 
Email: rocio.martinez_nunez@kcl.ac.uk 
 
14.1 Sample size calculation (some pilot/feasibility studies may not 
require a formal sample size calculation) 
Sample Size Justification: 
Based on the observed 85% responder rate to benralizumab in our centre (Kavanagh et al. CHEST 
2021), statistically significant falls in FeNO observed in FeNO-high patients with benralizumab, and 
successful published transcriptomics studies in asthma of a similar number, we believe 12 subjects 
should yield at least 10 clinical responders to benralizumab. Twelve is also an achievable amount from 
a practical perspective to allow timely completion of this study in under 18 months from first subject 
in (FSI). 
14.2 Statistical analysis 
Descriptive statistics will be used for demographic and clinical characteristics of the study subjects.  
The primary comparisons are pre- and post-benralizumab therapy.  Clinical outcome measures 
(asthma control scores, exacerbation frequency, medication use, lung function obtained at baseline 
and following benralizumab treatment will be evaluated using standard statistical tests. These 
statistical tests are either parametric paired t-tests or non-parametric U-tests for continuous variables, 
and fisher tests for the analysis of proportions. We will also leverage on local statisticians when 
needed. 
Statistical comparisons in RNA-seq will be undertaken using currently available pipelines such as 
DESeq2. DESeq2 has in-built modelling based on binomial distributions of RNA-seq datasets and is well 
established and validated. Likewise we will follow the most up to date pipelines for analysis of spatial 
transcriptomics as per 10xgenomics, which include combining spatial information with single cell RNA-
seq-type analyses. 
   
Employing spatial transcriptomics, we will compare the effect of benralizumab on cell-specific gene 
expression changes, cell numbers and position within bronchial biopsies, as well as cell-to-cell 
interactions.  
Employing RNA-seq on BECs, we will compare (1) unstimulated BECs pre- and post- benralizumab, and 
(2) SARS-CoV-2/RV-16 infected BECs pre- and post- benralizumab, determining the effect of 
benralizumab on the antiviral response of BECs to each one of these viruses separately.  
All analyses will be paired, considering patient-specific changes.  
All analyses involving the measurement and comparison of multiple variables (e.g. RNA-seq) will 
include a multiple test correction, such as false discovery rate / Bonferroni. 
 
 
15 Ethical considerations 
Study will go through R&D and REC approval. 
Patients will give informed consent 
16 Financing and Insurance 
The study is being funded by Astra Zeneca. 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 30 of 31 
 
The Chief Investigator is employed by KCL so additional indemnity cover is provided by the College As 
the study is co-sponsored by GSTFT, clinical negligence is provided via the NHS Indemnity Scheme.  
17 Reporting and dissemination 
Any pilot data arising from this study, and which is statistically robust will be presented at conferences. 
At the end of the study period, following data analysis completion, a manuscript will be prepared for 
peer review and publication.  
 
 
Tables, Figures, References 
Appendices 
Including (where relevant): 
Patient information sheet 
Patient consent form 
Data collection forms and validation information 
Ethics form 
Summary of product characteristics 
Investigators brochure 
 
 
Useful reading/websites 
 
Integrated Research Application System (IRAS) 
https://www.myresearchproject.org.uk/  
 
Health Research Authority (HRA) 
www.hra.nhs.uk  
 
HRA Guidance for Patient Information Sheet and Informed Consent  
http://www.hra.nhs.uk/research-community/before-you-apply/participant-information-sheets-and-
informed-consent/  
 
CONSORT statement  
  
 
ICH Harmonised Tripartite Guidelines for Good Clinical Practice (1996) 
http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E6/E6_R1_Guidel
ine.pdf  
 
Martin Bland et al, Statistical guide for research grant applications 
http://www-users.york.ac.uk/~mb55/guide/guide.htm  
Includes detailed information and definitions of many aspects required for a research protocol.  
 
Declaration of Helsinki  
(http://www.wma.net/en/30publications/10policies/b3/index.html) 
 
 

  
 
GSTFT – Observational Research Protocol 
BEUTI STUDY Jackson KCL 
IRAS: 310865 
 
Protocol Version 1.0  15/12/2022                                                                                      Page 31 of 31 
 
Appendix 1 – Information with regards to Safety Reporting in Non-CTIMP 
Research 
 
Who 
When 
How 
To Whom 
SAE 
Chief 
Investigator 
-Report to Sponsor 
within 24 hours of 
learning of the event 
 
-Report to the MREC 
within 15 days of 
learning of the event 
 
SAE Report form for Non-
CTIMPs, available from NRES 
website. 
Sponsor and MREC 
Urgent Safety 
Measures  
Chief 
Investigator  
Contact the Sponsor and 
MREC Immediately 
 
Within 3 days  
By phone 
 
 
 
 
Substantial amendment 
form giving notice in writing 
setting out the reasons for 
the urgent safety measures 
and the plan for future 
action. 
Main REC and 
Sponsor  
 
 
 
Main REC with a 
copy also sent to 
the sponsor. The 
MREC will 
acknowledge this 
within 30 days of 
receipt.  
Progress 
Reports  
Chief 
Investigator  
Annually ( starting 12 
months after the date of 
favourable opinion) 
Annual Progress Report 
Form (non-CTIMPs) available 
from the NRES website 
Main REC 
Declaration of 
the conclusion 
or early 
termination of 
the study 
Chief 
Investigator  
Within 90 days 
(conclusion) 
 
Within 15 days (early 
termination) 
 
The end of study should 
be defined in the protocol 
End of Study Declaration 
form available from the 
NRES website 
Main REC with a 
copy to be sent to 
the sponsor  
Summary of 
final Report  
Chief 
Investigator 
Within one year of 
conclusion of the 
Research 
No Standard Format 
However, the following 
Information should be 
included:- 
Where the study has met its 
objectives, the main findings 
and arrangements for 
publication or dissemination 
including feedback to 
participants 
Main REC with a 
copy to be sent to 
the sponsor