Severe Asthma Clinical Trial
Official title:
P-CLESA: Phenotyping Circulating and Lung Resident Eosinophils in Severe Asthma
Title: Phenotyping circulating and lung resident eosinophils in severe asthma (3 years).
Background: Asthma is a long-term condition that affects the airways. When a person with
asthma comes into contact with something that irritates their sensitive airways, the lungs
respond with contracting the muscles around the airway tubes, an inflammation process and
mucus production. The airway will become narrower and inflamed making it hard to breathe and
results in symptoms such as wheezing and coughing. The treatment of asthma consists of using
inhalers that work to widen the airway to relief these symptoms. Often severe asthmatics have
difficulty in controlling their disease, despite good medical care and taking asthma
medicines. At the moment there is no cure for asthma. A new medicine called Mepolizumab
(anti-Interleukin(IL)-5 therapy) has now shown to improve the symptoms of asthma particularly
patients with severe asthma in whom the normal medicines prescribed for asthma are not highly
effective in controlling their disease. You have been chosen receive this new medicine as we
believe it will improve the control of your disease. The aim for this study is to understand
the effect of Mepolizumab on a particular type of cell, called an eosinophil, which in
present lungs and blood of all people but is increased in asthma patients.
Rationale: The relationship between subsets of circulating and lung resident eosinophils in
severe asthma and Mepolizumab (anti-IL-5 therapy) efficacy has not been explored.
Objectives: To determine the gene expression and release of inflammatory proteins (mediator
profiles) of eosinophils from the circulation and the lung, specifically blood and tissue
resident, in patients with severe asthma at baseline and on Mepolizumab therapy.
Study 1: Phenotype subsets of circulating eosinophils in patients with severe asthma at one
time-point Recruit: 15 biologic naïve SA and 15 SA currently on Mepo therapy. Blood
eosinophils will be isolated by negative selection. Single-cell RNA-seq 10xGenomics and
bulk-RNA-seq to be used to simultaneously measure gene and cell surface protein expression in
the same cell to understand cellular heterogeneity in asthmatic eosinophils and identify
novel targets and biomarkers for non-responsiveness Study 2: Phenotype subsets of circulating
and lung eosinophils in patients with severe asthma on Mepolizumab therapy over one year.
Treat 30 appropriately characterised severe asthmatics (Eos>300/ul) with Mepolizumab Blood
eosinophils will be isolated by negative selection. Single-cell RNA-seq 10xGenomics and bulk
-RNA -seq to be used to understand cellular heterogeneity in asthmatic eosinophils post Mepo
Therapy. Sampling at baseline, 3 and 12 months post Mepo Therapy.
Bronchoscopy performed on 30 patients, sampling endobronchial lung biopsy at baseline and 1
yr post Mepo Therapy. Single-cell RNA-seq 10xGenomics on lung resident eosinophils at
baseline and 1yr post Mepolizumab therapy. Immunohistochemistry will also be performed to
characterise cellular content and structure.
Background: Severe asthma has been defined as "asthma which requires treatment with high dose
inhaled corticosteroids (ICS) plus a second controller (and/or systemic corticosteroids) to
prevent it from becoming 'uncontrolled' or which remains 'uncontrolled' despite this therapy"
(1), representing a most difficult group of participants to manage. It is a heterogeneous
condition and the use of mathematical approaches led to the definition of clinical subtypes
(phenotypes) from cohorts in Europe, USA and Asia. With the introduction of monoclonal
antibody therapies such as anti-IgE antibody (omalizumab), anti-Interleukin (IL)-5/anti-IL5Rα
antibody (Mepolizumab, reslizumab and benralizumab) and anti-IL4Rα (dupilumab), personalised
medicine has been initiated as these therapies are effective in patients with allergic and/or
in asthma patients with increased numbers of a particular type inflammatory cell called an
eosinophil (eosinophilic asthma), respectively. Introduction of these targeted therapies has
helped a proportion of participants with severe asthma particularly those with eosinophilic
severe asthma, but further progress will come with a better understanding of all the driving
pathways underlying these phenotypes.
The biology and function of the Eosinophil Eosinophils are characterised by the presence of
specific secondary granules, in their cytoplasm, containing toxic cationic proteins(2).
Research studies on eosinophil biology indicate that eosinophils function in many ways
including cytokine production and the development of airway inflammation. Under physiological
conditions, only small numbers of eosinophils are released from the bone marrow. However,
eosinophil production is dramatically increased as a result of so-called Th2 cell responses
associated with allergic diseases such as asthma (2-4). There is also evidence that
eosinophils contribute to homeostatic immune processes (4, 5). This increase in eosinophil
production is driven by a dedicated set of cytokines, namely IL-3, IL-5, and granulocyte
macrophage-CSF (GM-CSF) (2, 4). Amongst these, the Th2-associated cytokine IL-5 is the most
specific cytokine for the eosinophil lineage and is responsible for the expansion of
eosinophils from committed bone marrow progenitors, their release into the blood and their
survival following migration into the tissues(2, 4, 6). At the site of injury, eosinophils
can release their cytotoxic granule proteins as well as preformed cytokines and lipid
mediators, to exacerbation of inflammation and tissue damage, which is particularly
deleterious when Th2 responses are directed against allergens (2, 4).
Recruitment of eosinophils from the circulation requires blood eosinophils to become
activated, leading to their attachment to activated endothelium and movement of white blood
cells from the capillaries to the tissues surrounding them (extravasation(7, 8)). Arrest of
eosinophils in vessels and their extravasation into the airway wall and through the bronchial
tissue and epithelium to the airway lumen are mediated by a set of proteins called
integrins(7, 8). Blood eosinophils from subjects with allergy or asthma, have a greater
degree of adhesion or migration compared to those from normal volunteers (9).
Pharmacodynamic biomarkers for Mepolizumab treatment response Mepolizumablizumab binds with
high specificity and affinity to human IL-5(10), the key T2 cytokine responsible for
regulation of blood and tissue eosinophils(11). Mepolizumab prevents IL-5 from binding to the
IL-5 receptor complex expressed on the eosinophil cell surface and thus inhibits IL-5
signaling, blocking eosinophil survival and proliferation. Although the exact mechanism of
action of IL-5 inhibitors is not fully elucidated, the desired goal is to neutralize the
effect of activated eosinophils in blood and tissues and lung, particularly the lung for
asthma in order to achieve therapeutic effect.
In asthma, an association between eosinophil overexpression and asthma severity was made in
1990(12) and subsequent studies have since repeated the view that blood, or sputum eosinophil
counts can be used to characterize participants with severe eosinophilic asthma (13). In the
SIRIUS trial Mepolizumab at a dose of 100 mg administered subcutaneously was shown to reduce
the requirement for daily oral corticosteroids while at the same time improving asthma
control, lung function, and quality of life and reduced exacerbations(14). In the MENSA trial
participants treated with Mepolizumab reported significantly reduced exacerbations and an
improvement in asthma control questionnaire scores(15). The Mepolizumab investigators
identified blood eosinophils, rather than sputum eosinophils, as a good predictor of the
clinical efficacy of Mepolizumab, providing an accessible biomarker for severe eosinophilic
asthma. Moreover, secondary analysis(16) of the DREAM(17) and MENSA(15) studies identified
clinical efficacy of Mepolizumab in severe asthma participants with baseline eosinophil
counts of ≥150-300 cells/µl and ≥300 cells/µl.
Subsets of eosinophils in asthma In mice subsets of eosinophils have been identified(18): i)
lung resident eosinophils (rEos) that are IL-5-independent cells with a ring-shaped nucleus
and ii) inflammatory eosinophils (iEos), which were defined as IL-5-dependent cells with a
segmented nucleus. In a murine model of asthma, Mesnil and colleagues reported that the
features of lung rEos remained unchanged but rEos were co-localised with iEos in the lung and
that mice lacking rEos had an increased Th2 cell response to inhaled allergens. Studies in
man reported that parenchymal rEos found in non-asthmatic human lungs were phenotypically
distinct from the iEos isolated from the sputa of eosinophilic asthmatic patients. These data
highlight the existence of distinctive circulatory and lung resident subsets of eosinophils
in man.
Eosinophils secrete many molecules involved in asthma pathology including cytotoxic granule
constituents cytokines and chemokines such as, lipid mediators leukotrienes(2, 19). While the
role of IL-5 is a confirmed in eosinophil activation and asthma, the comparative activities
of other cytokines such as GM-CSF and IL-3, also implicated in asthma, are yet to be fully
determined. Eotaxins (CCL11 and CCL24) are considered to be important chemokines involved in
the recruitment of eosinophils into the airways while IL-4 and IL-3 are reported to be
overexpressed in the airways of severe asthmatics(20, 21). In addition to these Th2-type
cytokines, IFNs and TNFα also upregulate eosinophils and prolong their survival(22, 23).
Furthermore, the receptors TSLPR, IL33R and CCR3 present on activated eosinophils were found
to be elevated in severe eosinophilic asthma (24) Thus, an investigation of cytokine
stimulation of subsets of eosinophils, the possible crosstalk between stimulated eosinophils
and the resulting differential mediator release and protein expression will likely provide a
better understanding of the underlying pathophysiological signaling pathways involved in
severe asthma.
Observational Study This will be an observational study of participants diagnosed with severe
asthma being followed up and managed according to agreed UK guidelines for management of
severe asthma. In this group, participants will have been treated with Mepolizumab according
to the blood eosinophil count. These participants will be recruited and phenotyped according
to eosinophilic molecular phenotype by analysis of blood and lung transcriptomics and
proteomics. Participants will be evaluated at 0, 3 and 12 months. Incidental clinical finding
will be forwarded to the clinical team and GP, as warranted.
Study Rationale The relationship between subsets of circulating and lung resident eosinophils
in severe asthma and anti-IL-5 therapy efficacy has not been explored.
Objectives To determine the transcriptomic and mediator profiles of eosinophils from the
circulation and the airway, specifically blood and tissue resident, in participants with
severe asthma at baseline and on Mepolizumablizumab therapy.
Study Model Unbiased study to identify all subsets of circulating and lung resident
eosinophils in biologic naïve participants with severe asthma at baseline and at time-points
post treatment with Mepolizumab (anti-IL-5 Rx). Severe asthma participants (Eos>300/ul) will
be recruited from the Severe Asthma clinic at the Royal Brompton Hospital, and, as much as
feasible, participants will not be on OCS therapy.
Primary Objectives (i) Phenotype subsets of circulating eosinophils from participants with
severe asthma at one time-point (ii) Phenotype subsets of circulating and lung eosinophils
from participants with severe asthma on Mepolizumab therapy over one year.
Study 1: Phenotype subsets of circulating eosinophils in participants with severe asthma at
one time-point
Recruit: 15 biologic naïve SA not on maintenance OCS and 15 SA currently on Mepolizumab
therapy with good clinical response.
Blood samples (90ml) will be taken from these participants. Blood eosinophils will be
isolated from these samples which will then be subtyped (phenotype) by simultaneously
measuring gene and cell surface protein expression in the same cell. Blood eosinophils will
also be cell-cultured in the laboratory and stimulated with a variety of cytokines and
chemokines the release of inflammatory mediators determined. Together, this will increase our
understanding of the cellular heterogeneity in asthmatic eosinophils and identify the
difference between peripheral blood eosinophil populations in untreated and
Mepolizumablizumab treated participants .
Study 2: Phenotype subsets of circulating and lung eosinophils from participants with severe
asthma on Mepolizumab therapy over one year.
Recruit 36 severe asthma participants appropriately characterised severe asthmatics
(Eos>300/ul), not on OCS maintenance therapy, to achieve paired biopsy samples from 30
participants
Blood samples will be taken at baseline, 3 and 12 months post-Mepolizumab Therapy. Blood
eosinophils will then be isolated and phenotyped by simultaneously measuring gene and cell
surface protein expression in the same cell. Blood eosinophils will also be cell-cultured in
the laboratory and stimulated with a variety of cytokines and chemokines the release of
inflammatory mediators determined. Together, this will increase our understanding of cellular
heterogeneity in asthmatic eosinophils post Mepolizumab Therapy.
Bronchoscopy will be performed on 36 participants, sampling endobronchial lung biopsy at
baseline and 1 yr post Mepolizumab Therapy; the second procedure will be performed within 2
weeks of the last Mepolizumab injection. Single-cell RNA-seq 10xGenomics and bulk -RNA -seq
will be performed, using the optimized protocol of the Gronigen group, on lung tissue
eosinophils through enzymatic disassociation of lung tissue (at 4oC) at baseline and 1yr post
Mepolizumab therapy. Immunohistochemistry (IHC) will also be performed to characterise
cellular content and structure. Four biopsies will be reserved for RNA-seq and two for IHC.
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