Genetic and Functional Characterization of the IgE Repertoire and Compartmental Regulation of IgE Activity in the Pathogenesis of Asthma

Asthma has reached epidemic proportions in the modern world. In the UK alone, 1 in 12 adults and 1 in 5 children suffer, with 3 deaths daily. Many children have to carry around inhalers in case they have an attack at school or at play. Although some medicines do alleviate symptoms they do not cure the disease and the choice is limited. The most effective new treatment for uncontrolled asthmatics is a synthetic antibody (omalizumab) against the key molecule immunoglobulin E (or IgE), itself a type of antibody that primes immune cells in the airways to release mediators that cause asthma in response to inhaled allergens such as grass.

About 10% of asthma sufferers have to cope with severe disease: their care accounts for the majority of NHS asthma costs, ca. £1 billion/year, but this does not prevent the misery of individual sufferers and their families and indirect costs to society.

A fifth of the most severe asthmatics (so-called "non-allergic" or "non-atopic" asthmatics) are excluded from omalizumab treatment because of the assumption that both allergens and the specific IgE antibodies that recognises them are inextricably linked to the disease, so that, if routine tests for allergen sensitivity are negative, these patients will not benefit. We have shown, however, that IgE is elevated in the blood and airways of "non-allergic" asthmatics and hypothesise that IgE is also involved in their disease. This could be because the IgEs in these patients are not among the ones directed against the few environmental allergens commonly tested, or because of the existence of allergen-independent mechanisms of IgE activity. We already have preliminary evidence for both scenarios, and are planning to explore this further in this project.

Every person makes millions of antibodies (their "antibody repertoire") and has the capacity to make many more throughout life. While most antibodies are protective, the system sometimes goes awry when otherwise harmless substances provoke IgE responses, which activate immune cells in the airways. To analyse this process, massive parallel sequencing of the variable parts of the expressed antibody genes is necessary to reveal the relevant differences between individuals with and without disease and the distribution of the antibodies through different organs of the body in sufferers with asthma.

We propose to use novel "next generation sequencing" techniques to compare the antibody repertoires in the nose, lung and blood of asthma patients and healthy controls. This will be accomplished as follows:

(1) We will ask allergic and non-allergic asthmatics and healthy controls to provide samples of blood and snippets or "biopsies" from the linings of their upper (nose) and lower (lung) airways so that we can isolate the relevant IgE producing cells.
(2) We will compile the IgE repertoire by advanced bioinformatics analysis, comparing the blood and upper and lower airways within individuals. We will be able to track the development and migration of IgE producing cells within individuals and re-create the IgE antibodies made in these compartments in the laboratory.
(3) We will study allergen-dependent and independent mechanisms by which these IgE antibodies activate immune cells that cause asthma.

The knowledge gained in these studies will help refine and maybe even redefine the diagnosis and treatment of asthma. If, for example, we do show IgE to have a role in non-atopic asthma, this could pave the way for therapies, such as omalizumab, to become available to and benefit "non-atopic" asthma patients. There are more than 100,000 of these patients in the UK alone. Being allowed access omalizumab, which has revolutionised the lives of patients with severe atopic asthma, would be a major improvement for them. It is also likely that our results may reveal potential new targets for therapy.

Our project aims to elucidate allergen-dependent and allergen-independent IgE pro-inflammatory mechanisms in atopic and non-atopic asthma.

We will characterise the expressed Ig gene repertoire and functions of IgE antibodies in the nose, lung and peripheral blood from 10 non-atopic and atopic asthmatics and healthy controls, distinguished by skin prick tests. Peripheral blood (50 ml) and respiratory mucosal tissues (three nasal and 10 bronchial biopsies) will be obtained from all individuals. Ig repertoires will be captured by cell-direct emulsion RT-PCR and 5'-RACE PCR. The samples will be subjected to deep-sequenceing on the Pacbio RSII system, which delivers the required long-reads with sufficiently high accuracy. Several bioinformatics pipeline and mathematical models (e.g. Hill's diversity, BASELINe, PHYLIP) will be applied to investigate the "natural history" of IgE-expressing B cells.

Paired Ig heavy- and light-chain sequences identified by sequencing and phylogeny analysis will be expressed as recombinant IgE proteins using our in-house rapid expression cloning system (40 IgEs in total). The proteins will be functionally characterized for antigen-specificity and affinity and pro-inflammatory activities using assays such as ELISA, ImmunoCAP ISAC, carbohydrate microarray, SPR, mast cell and basophil activation tests and facilitated antigen binding. We will measure de novo synthesis of total and allergen-specific IgE from cultured mucosal biopsies.

In our recent study of Ig repertoires in allergic rhinitis, we found significant differences between groups of seven allergic patients and three healthy controls. Therefore, we expect to be able to detect significant differences in this project with 10 subjects/group. Appropriate statistical methodology (ANOVA, parametric, non-parametric) will be used, depending on the nature of data set, and post-hoc adjustments (e.g. Bonferroni correction) will be applied.

The results will have broad impact, by revolutionising the diagnosis and treatment of asthma patients, advancing experimental research in the field of allergology and clinical immunology and reviving the efforts of the pharmaceutical companies to develop new asthma drugs.

We will fortify the now very strong indications that it is not just IgEs with specificity for a few common allergens that cause asthma, but also IgEs with specificity for unconventional allergens, and IgEs that act by previously unrecognized mechanisms (Hypothesis 1). This will fundamentally alter the approach to the diagnosis and treatment of asthma, and lead to more personalised treatment, matching the etiology in each individual. As an example, our study of non-atopic asthma will extend access to the non-anaphylactogenic anti-IgEs, omalizumab, to the fifth of severe non-atopic asthmatics. Thus, the impact of this research will also extend to NHS policy markers and NICE by informing decision-making.

We will also discover the precise anatomical sites ("compartments") where the IgE-expressing cell originate in relation to those where they exert their effects in the target organ, the bronchial mucosa in asthma (Hypothesis 2). This will stimulate the development of site-directed interventional medicines and procedures. If these two sites differ, our analytical methods (deep sequencing of the expressed genes) will reveal the direction of IgE trafficking between these compartments. They will also identify the pathogenic IgEs through functional analyses of re-created (recombinant) IgEs, deduced from the massive repertoire. The nature of the IgEs will allow the clinician to specify the optimal treatment, which will directly benefit the individual patients, their families and their carers.

We will localize each of the molecular steps in IgE production, from class switch recombination to clonal selection, and affinity maturation and IgE memory (Hypothesis 3). This will increase the variety of potential targets for intervention in asthma, aiding the pharmaceutical industry in their efforts to develop new drugs for asthma, and ultimately benefiting asthma patients.
The novel methods developed for this project including cell-direct emulsion RT-PCR and 5'-RACE PCR and subsequent deep-sequencing of matched immunoglobulin heavy- and light-chain pairs using the PacBio RS II platform has the potential to impact experimental methods and practices for research colleagues working on asthma and allergy, but also researchers in other fields that involve B cells.

Summing up, this programme may result in a new understanding of the role of IgE in asthma and IgE-associated diseases in general, throw new light on the definition of asthma endotypes, and facilitate the prediction of response to IgE-directed asthma therapies, helping clinicians, the pharmaceutical industry and above all the asthma sufferers and their families and carers.