Clinical Trial Details
— Status: Recruiting
Administrative data
NCT number |
NCT06234761 |
Other study ID # |
ONZ-2023-0280 |
Secondary ID |
|
Status |
Recruiting |
Phase |
|
First received |
|
Last updated |
|
Start date |
January 2, 2024 |
Est. completion date |
December 2028 |
Study information
Verified date |
May 2024 |
Source |
University Hospital, Ghent |
Contact |
Philippe Gevaert, PHD |
Phone |
003293322332 |
Email |
philippe.gevaert[@]ugent.be |
Is FDA regulated |
No |
Health authority |
|
Study type |
Observational
|
Clinical Trial Summary
Background / rationale: Type 2 inflammation is driving several chronic diseases in the
airway. On one hand allergic rhinitis (AR) and allergic asthma (AA) are driven by allergen
expose, while on the other hand eosinophilic Type 2 inflammation with late onset eosinophilic
asthma (LOA) and chronic rhinosinusitis with nasal polyps (CRSwNP) are of non-allergic
ethiology. For late onset type2 asthma, many risk factors have been defined, but clear
insights into disease ethiology are currently lacking. Given the quintessential role of IgE
in disease ethiology of both diseases, understanding the molecular immunological mechanisms
underlying mucosal IgE responses is essential to understand disease ethiology.
Hypothesis: Distinct mechanisms drive local IgE production in AA and LOA Overall objectives:
Elucidate the potential drivers of and immunological pathways leading to local IgE production
in AA and LOA, and understand how dupilumab acts on these mechanisms.
Methods: A unique combination of state-of-the-art methods will be applied, including
single-cell RNA sequencing and receptor profiling, proteomics, determination of the microbial
composition, recombinant antibody screening and disease modelling in cell cultures.
Expected results: The investigators expect for the first time to discern the drivers of local
IgE production in LOA and uncover the immunological pathways leading to local IgE production
in AA and LOA. Moreover, the investigators will obtain insights into the role of Dupilumab in
modulation mechanisms.
Impact: If successful, these insights will answer a long standing, unresolved question in
type 2 disease and might aid in the development of novel directed therapeutics for AA and
LOA.
Description:
Work Package 1 Rationale: High levels of IgE can be detected locally in the nasal cavity and
nasal polyps from patients with CRSwNP. Given the high levels of polyreactive IgE generated
in NP, it is intriguing to speculate how NP IgE responses differ from allergic IgE B cell
responses. For this, a high-resolution analysis of the mechanisms leading to IgE producing B
cells in both settings is needed but has to date not been performed.
Milestone 1.1: To fully characterize the IgE B cell niche in CRSwNP and allergic rhinitis,
the investigators will collect multiple samples from 25 CRSwNP, 25 CRSsNP and 25 allergic
rhinitis patients with HDM allergy. In addition, tonsil biopsies as a proxy for systemic
immune responses will be collected. From 5 HDM allergic patients with high IgE levels nasal
tissue (inferior concha) and tonsil biopsies isolated and subject these tissues to thorough
characterization of the IgE B cell niche.
Milestone 1.2: In this milestone, the investigators will set out to obtain a high detail map
of IgE B cell responses and how they are governed in the type2 niche in CRSwNP and allergic
rhinitis. To this end, a high-resolution single cell map of the nasal polyp , inferior concha
and tonsil tissue will be generated via the 10x genomics 5'VDJ seq protocol. By creating a
high-resolution map of the B cell subsets and their immune receptor profiles, the
investigators aim to understand how chronic type2 IgE B cell responses are governed and
driven in non-allergic and allergic type2 disease. For this analysis DALI (Digital
Addressable lighting Interface) bioinformatic software package will be used. The
investigators will here specifically aim to trace the origins of IgE+ plasmablast or plasma
cell clones through germinal center responses, or directly from naïve B cells in
extrafollicular responses. In addition, NicheNet analysis will allow for the detection of
regulators of IgE B cell responses, such as other immune cells or structural cells. This
software tool analyzes potential sender-receiver pairs for cytokines, chemokines, and
cellular ligands, based on differential gene expression and curated receptor-ligand lists.
Work package 2: While high levels of polyreactive IgE can be detected in nasal secretions and
tissues of patients with CRSwNP, it is unclear whether class switch recombination to IgE is
driven by antigen or by damage associated factors.
Milestone 2.1: To date, the characteristics of IgE in NP remain enigmatic, yet these IgE's
are key to understand their role in disease biology. To elucidate the characteristics of NP
IgE, the investigators will generate 20 monoclonal IgE antibodies from each of 5 patients
with CRSwNP and similarly 20 monoclonal IgE antibodies from each of 5 patients with allergic
rhinitis will be generated. By this, the full-length heavy and light chain sequences of IgE+
clones will be retrieved, and have these produced as recombinant monoclonal antibodies. In
addition, NP IgE antibodies possess polyreactivity will be assessed, rendering them able to
bind to several antigens, a characteristic which has been attributed to so-called natural
IgE.
Milestone 2.2: By performing an unbiased analysis of the nasal cavity proteome, the
investigators aim to gain novel insights into the local factors that regulate IgE B cell
responses. By taking this holistic view in combination with state-of-the-art single cell
analysis methods, the team will for the first time be able to unravel how interplay with a
dysregulated microbiome or extensive tissue damage plays a role in driving IgE B cell
responses. The analysis of the nasal cavity proteome by performing unbiased shotgun MS/MS
mass spectrometry on nasal secretion samples from the same patients. This analysis of the
nasal cavity proteome will allow us to get an unbiased view of the factors that might drive
local IgE responses.
Milestone 2.3: To mechanistically understand how IgE class switch recombination is induced in
nasal polyps a set up an epithelial cell - PBMC (Peripheral Blood Mononuclear Cells)
cocultures and induce IgE class switch recombination in these cultures. First epithelial
cells from patients from CRSwNP or allergic rhinitis will be expanded and put in
air-liquid-interface (ALI) coculture with PBMC from the respective donors. First, the team
will start by mimicking the effect of epithelial damage on IgE class switch recombination by
adding IL-33 (a typical alarmin) to the PBMC-epithelial cell cocultures. Next, these cultures
will incubated with Staphylococcus aureus (a strain of bacteria often colonizing the nasal
cavity of CRSwNP patients) or Candida albicans (a fungus often colonizing the nasal cavity of
CRSwNP patients). Lastly, the investigators will test different combinations of the latter
factors. In these cultures, the induction of germinal center B cell responses (IgG1+ and IgE+
CD20+BCL6+), plasma blasts (IgG1+ and IgE+CD20+CD138+) and plasma cells (IgG1 and
IgE+CD20-CD138+) by multicolor flow cytometry will be assessed. These experiments will
elucidate how instruction by the epithelium of nasal polyp or allergic rhinitis patients upon
relevant triggers affect IgE class switching in B cells.
Work package 3: Assessing the effect of Dupilumab on modulation of local IgE B cell responses
Rationale: As Dupilumab targets the key upstream regulators of IgE class switching, the
investigators will address whether any differences exist in the mechanisms of IgE selection
upon Dupilumab treatment.
Milestone 3.1: Assessing local modulation of IgE B cell responses in CRSwNP by Dupilumab To
test whether Dupilumab influences regulation of local IgE, samples from 5 Dupilumab treated
CRSwNP patients will be collected. Here, total IgE levels in nasal secretions, to see whether
in these patients Dupilumab treatment influenced local IgE production will be analyzed. For
elucidating the molecular immunological mechanisms, the subject nasal polyp tissue of
patients on Dupilumab treatment will analyzed for single cell VDJ seq and compared to the
data obtained in work package 2, and see whether there are any changes in the clonal IgE
responses upon IL-4/IL-13 blocking. In a similar assay as described in work package 2, the
investigators will set up epithelial cell-PBMC cocultures as explained in work package 2 and
block IL-4/-13 signaling by adding dupilumab in the culture medium. The investigators will
here assess the effect on IgE B cell responses by flow cytometry.