Cat allergy is one of the commonest allergic sensitizations and is strongly associated with asthma. Children sensitized to cats are more likely to develop severe asthma than those sensitized to other allergens.
Current treatment options for allergy to cats are largely symptomatic. However, allergen-specific immunotherapy (SIT) is available for desensitization to cats and has been demonstrated to be clinically effective in both allergic rhinitis and asthma. SIT has duration of action that exceeds the treatment period. Furthermore, SIT has the potential to prevent new allergic sensitizations in children and three years of SIT prevented the development of asthma (odds ratio for no-asthma was 4.6 95% CI (1.5-13.7)) over a period of at least 7 years after withdrawal of therapy. However, SIT employing native proteins is associated with a high frequency of treatment-related reactions which can be severe and occasionally, life-threatening. Thus, reducing the allergenicity of immunotherapy approaches represents an important unmet need in the treatment of allergy.
In an attempt to reduce allergenicity, improve safety and reduce treatment times, T cell epitopes from the major cat allergen Fel d 1 with a reduced capacity (versus whole allergen) to cross link IgE on effector cells were identified and used to treat cat allergic subjects. Improvements in clinical and surrogate outcomes were observed in most, but not all, studies. A high frequency of adverse reactions to these early vaccines halted clinical development. More recently, a prototype vaccine, from our own group, consisting of multiple short synthetic peptides was evaluated in clinical studies. The prototype reduced sensitivity to allergen measured through several surrogate clinical outcomes. Most recently the prototype has been refined, by Circassia Ltd., through a comprehensive study of the T cell epitopes of Fel d 1 and evaluated in a series of Phase II clinical trials, only the first of which has been published. Unpublished data from more recent trials is presented in this application and shows that peptide immunotherapy is safe and clinically efficacious, maintaining clinical improvement in a blinded study even after 9 months off treatment, following a short 4 injection regimen.
The recent availability of MHC class II tetramer reagents containing major epitopes of Fel d 1now presents the possibility of studying the immunological effects of an epitope-specific, clinically efficacious intervention directly, ex vivo, allowing us to validate both the T cell epitopes in the vaccine and the tetramer reagents. This unique combination of epitope-specific intervention and epitope-specific tracking of T cells offers the opportunity to investigate the immunological mechanisms of tolerance in a way not previously possible.
We have previously published a series of mechanistic studies of peptide immunotherapy that have partially elucidated mechanisms of action. In this application we provide further unpublished evidence to support our earlier observations of a role for IL-10 in tolerance to allergen and we offer preliminary and tantalizing data that suggests that peptide immunotherapy may down-regulate chemokine receptors involved in targeting of effector T cells to the lung. The current RFA therefore provides a timely opportunity to take this clinical research program to the next level.
In the early 1990’s the ImmuLogic Corporation developed peptide vaccines for cat allergy and ragweed allergy. Clinical trials demonstrated efficacy but this was associated with an unacceptable adverse events profile and clinical development was halted. At the time we were interested in the mechanisms underlying a particularly common adverse event in these trials, which was that of late-onset symptoms of asthma. We went on to show that isolated late asthmatic reactions were the result of activation of allergen-specific T cells. Of interest, administration of peptides was associated with a period of non-responsiveness akin to the induction of specific immunological tolerance. We went on to show that peptides could be used to induce allergen-specific tolerance in the absence of isolated late asthmatic reactions. We established a university spin-out company to develop peptide vaccines (Circassia Ltd., established 1998) and over the last 12 years we Dr. Larché working with Circassia) have developed a portfolio of vaccines for many common allergies including cat, house dust mite, ragweed, grass, birch, mold. The vaccines have been developed in the Larché Laboratory and clinical development has been undertaken by Circassia and its Canadian Joint Venture (with McMaster University, Adiga Life Sciences Inc.). Together, Larché, Circassia and Adiga have created major innovations in the peptide immunotherapy approach which has led to impressive clinical trial results in recent Phase II efficacy trials. We have performed exhaustive MHC restriction epitope mapping of major allergens using a combination of bioinformatics and physical peptide-MHC binding assays. We showed that short synthetic peptides are not associated with IgE-mediated adverse events common with standard immunotherapy. We have shown that administration of low dose peptide immunotherapy is not associated with late asthmatic reactions. We identified the intradermal delivery route as being a key to clinical success. Our most recent clinical trial demonstrated a significant reduction in Total Rhinitis Symptoms Scores (TRSS) following 4 days of allergen exposure in an environmental chamber. The clinical improvement was maintained at a one year follow-up despite the initial treatment being only 4 injections of vaccine and no further treatment for 9 months prior to the outcome measurements (see Figure 2 below).
By comparison with other published or publically available data collected in similar studies, the clinical improvement observed appears larger than other current rhinitis therapies (subcutaneous/sublingual immunotherapy, anti-histamines, inhaled corticosteroids). A further innovation of this approach is the ability to conduct truly blinded clinical trials, since the adverse event profile of our vaccines are indistinguishable from placebo. This has never been possible in trials of subcutaneous or sublingual immunotherapy, as the majority of subjects know when they are receiving active treatment due to local adverse events.
We now wish to capitalize on the unique opportunity offered by this most specific form of allergen immunotherapy, by using MHC class II allergen tetramer reagents to isolate and characterize ex vivo Fel d 1 epitope-specific T cells before and after a Fel d 1 peptide vaccine intervention. The results of this study will synergize with another similar study already underway in which purified, ex vivo Fel d 1-specific T cells are being subjected to gene expression array analysis before, immediately after treatment and one year after treatment with the vaccine (funded by our industrial partner, Adiga Life Sciences). The combined analysis of change in allergen-specific (tetramer+) memory & regulatory T cell frequency, chemokine receptor profiles and cytokine production profiles proposed in this study, with the change in gene expression profiles after therapy (in the related, but separate ongoing Adiga study), will provide the most comprehensive analysis of an antigen/allergen-specific intervention ever undertaken. These studies will lead to an advanced understanding of the mechanisms of immunological tolerance and to the validation of peptide vaccines as modifiers of the immune response, and allergen-specific tetramer reagents as the optimal way to investigate allergen-specific Tcell responses.
For the experiments in Aim 1 we will use frozen PBMC samples that were obtained from a randomized, double- blind, placebo-controlled Phase IIa clinical study of peptide immunotherapy (PIT) in allergic asthmatic subjects of stratified disease severity
Group 1: inhaled short-acting bronchodilator only
Group 2: inhaled corticosteroids only
Group 3: inhaled long-acting bronchodilator and corticosteroids; all groups (n=16; 8 active/8placebo)].
The primary outcome measure of the study was safety not efficacy. The study demonstrated that the vaccine was safe and well tolerated and that there was no deterioration in lung function after each of eight 3nmol vaccine doses. The clinical efficacy of an identical treatment regimen was demonstrated in a related Phase IIb study that measured the difference in total rhinitis symptom score (TRSS) in a population of cat allergic subjects with rhinitis +/- mild asthma after PIT (Figure 1).
Furthermore, a second, more recent, Phase IIb study also confirmed the clinical efficacy of this peptide vaccine, even when outcomes were measured 9 months after the cessation of therapy (Figure 2).
In light of these results, we feel that it is reasonable to assume that the intervention will have achieved a biological response in the safety study from which the proposed samples are to be taken. Before and after this study, PBMC were isolated from 50ml of peripheral blood and cryopreserved (using the Immune Tolerance Network cryopreservation SOP) within 4 hours of venipuncture. We consistently achieve viabilities of >90% when thawing samples from other clinical studies frozen using the same SOP. This frozen archive gives us the unique opportunity to interrogate T cell responses to an allergen-specific intervention targeting T cells. In order to make the best use of our samples, we have spent the last 2 years evaluating flow cytometry-based technologies for characterizing allergen-specific T cells from peripheral blood. We have evaluated several methods;
Due to the low frequency of allergen-specific T cells in peripheralblood, none of these techniques allow detailed characterization (e.g. surface phenotype, intracellular cytokine expression) without expansion/enrichment of the allergen-specific cells in some way. Most frequently this has been achieved by culture of PBMC/T cells with allergen for periods of up to 45 days. The shortcomings of this approach include the fact that (1) it is not possible to fully assess the extent to which in vitro culture affects phenotype and function and (2) when comparing paired PBMC samples, such as those obtained before and after PIT, it is not possible to control several factors (such as viability of cells frozen on different days months apart) that will influence the rate of expansion of the allergen-specific cells. As a result, in vitro expansion for the determination of frequency and phenotype of allergen-specific T cells, particularly in paired samples, is unsatisfactory. This conclusion is based on our own extensive analysis of allergen (Fel d 1)-specific T cells, using 6 MHC class II tetramers in PBMC samples from cat allergic subjects including paired frozen samples from a Phase IIb clinical trial of PIT alluded to above (see Figure 1). We provide some of these results as preliminary data in this application. Our preliminary analysis of the time course of tetramer+ T cell expansion suggests that the frequency of these cells changes inconsistently over time with the highest frequency of tetramer+ cells can be seen anywhere from 9-14 days and, most importantly, that maximal frequency is achieved at different days in different samples (Figure 3).
In extreme cases we have observed differential expansion in tetramer+ cells of 50-100 fold in a 12 day culture (Figure 4):
Is this a real effect of PIT, or atechnical artifact? This and other important questions can only be addressed by direct ex vivo analysis of cells. To do this requires larger volumes of blood and standardized enrichment techniques. In our experience, it is not practical to enrich tetramer+ cells from PBMC by FACS (sorting from tens of millions of PBMC is a lengthy process even on the most advanced machines). As a result we have developed protocols at both the Benaroya and McMaster sites that efficiently enrich tetramer+ T cells using sequential magnetic separation. Co-Investigator Dr. Kwok recently published an analysis of peanut (Ara h 1)-specific T cells isolated from peripheral blood samples using multi-step magnetic enrichment. Enrichment allowed analysis of cell frequency and numerous phenotypic markers with confidence, ex vivo. In parallel, a similar standardized protocol for enrichment of tetramer+ T cells has been developed at McMaster using sequential, automated (RoboSep, StemCell Technologies) magnetic separation (as opposed to the Miltenyi system used by Dr. Kwok). PBMC are isolated by density gradient centrifugation followed by negative selection of CD4+ T cells. The latter are stained for 100min at room temperature with Fel d 1 tetramer(s) and then washed. Tetramer-stained cells are then subjected to positive
selection using magnetic nanobeads conjugated with anti-phycoerythrin (PE) antibodies. Using this technique we are able to obtain substantially enriched populations of tetramer+ cells (up to 14% tetramer+; Figure 5) allowing detailed frequency and phenotype analysis on credible numbers of flow cytometry events (Figure 6), not just a handful of dots on a plot.
These results are achieved ex vivo, without altering frequency or phenotype. We have elected to focus our analysis of tetramer+ T cells on