Patient stratification and the unmet need in asthma.

Asthma is often described as an inflammatory disease of the lungs and in most patients symptomatic treatment with bronchodilators or inhaled corticosteroids is sufficient to control disease. Unfortunately there are a proportion of patients who fail to achieve control despite treatment with the best current treatment. These severe asthma patients have been considered a homogeneous group of patients that represent the unmet therapeutic need in asthma. Many novel therapies have been tested in unselected asthma patients and the effects have often been disappointing, particularly for the highly specific monoclonal antibody-based drugs such as anti-IL-13 and anti-IL-5. More recently, it has become clear that asthma is a syndrome with many different disease drivers. Clinical trials of anti-IL-13 and anti-IL-5 have focused on biomarker-defined patient groups and these trials have driven the clinical progression of these drugs. Work on asthma phenotyping indicates that there is a group of asthma patients where T helper cell type 2 (Th2) cytokines and inflammation predominate and these type 2 high (T2-high) patients can be defined by biomarkers and response to therapies targeting this type of immunity, including anti-IL-5 and anti-IL-13. However, there is still a subset of T2-low patients that do not respond to these new therapies. This T2-low group will represent the new unmet medical need now that the T2-high-targeting therapies have made it to the market. This review will examine the current thinking on patient stratification in asthma and the identification of the T2-high subset. It will also look at the T2-low patients and examine what may be the drivers of disease in these patients.

Development of a mouse model for chronic cat allergen-induced asthma.

BACKGROUND: Allergic asthma is a chronic inflammatory airway disease caused by exposure to airborne allergens. In order to develop novel therapies for allergic asthma, models that are relevant to human disease are needed. METHODS: Female BALB/c mice were presensitised subcutaneously with alum-adsorbed recombinant cat allergen Fel d 1, followed by intranasal challenges with cat dander extract spiked with recombinant Fel d 1 for 7 weeks. For reference, mice were presensitised and challenged with ovalbumin following the same protocol. Airway hyperresponsiveness, serum antibodies, airway inflammation and cell infiltration, and cytokines in lung tissue and bronchoalveolar lavage were measured. RESULTS: Mice presensitised with recombinant Fel d 1 and challenged with cat dander extract or presensitised and challenged with ovalbumin showed airway hyperresponsiveness in response to metacholine. Mice of the cat allergen model showed influx of neutrophils, eosinophils and lymphocytes in bronchoalveolar lavage, combined with increased levels of IL-17a and increased IL-4 mRNA expression in lung tissue. In contrast, mice sensitised and challenged with ovalbumin showed a predominant influx of eosinophils in bronchoalveolar lavage and had an increased expression of IL-5 in lung tissue. Both protocols induced features of lung tissue remodelling and allergen-specific antibody responses. CONCLUSIONS: The presented mouse model for cat allergen-induced asthma exhibits hallmarks of chronic allergic asthma, like airway hyperresponsiveness, a mixed neutrophilic/eosinophilic infiltration in bronchoalveolar lavage, expression of IL-17a and signs of remodelling in lung tissue. The model will provide a relevant platform for the development of novel treatment strategies.

Covalent coupling of vitamin D3 to the major cat allergen Fel d 1 improves the effects of allergen-specific immunotherapy in a mouse model for cat allergy.

BACKGROUND: Allergen-specific immunotherapy (SIT) is currently the only curative treatment for allergy but the treatment needs to be improved. We hypothesize that covalent coupling of immunomodulating vitamin D3 to the major cat allergen Fel d 1 can enhance the beneficial effects of SIT to cat allergy. METHODS: We treated mice sensitized to Fel d 1 with subcutaneous injections of two doses of recombinant Fel d 1 coupled to 1α,25-dihydroxyvitamin D3 (rFel d 1:VD3) and compared to treatment with the same doses of rFel d 1 in a mouse model for cat allergy. Airway hyperresponsiveness (AHR), cytokines and cells in bronchoalveolar lavage (BAL), in vitro activation of splenocytes to rFel d 1, and Fel d 1-specific immunoglobulins were evaluated. RESULTS: Treatment with both doses of rFel d 1:VD3 decreased AHR, cellular influx and Th2 cytokines in BAL compared to untreated mice. High- and low-dose rFel d 1 treatment also decreased AHR and BAL Th2 cytokines, with less decrease for the low-dose treatment. Importantly, the total number of cells and eosinophils in BAL was markedly reduced at both high- and low-dose rFel d 1:VD3 compared to treatment with rFel d 1 alone. Finally, treatment with both rFel d 1 and rFel d 1:VD3 induced Fel d 1-specific serum IgG. CONCLUSION: Our results indicate a beneficial therapeutic effect of rFel d 1:VD3 on airway inflammation, AHR and rFel d 1-specific immune responses and thus suggest that this novel immunomodulatory candidate may improve both the efficacy and safety of SIT.

Crystal structure of the dog lipocalin allergen Can f 2: implications for cross-reactivity to the cat allergen Fel d 4.

The dog lipocalin allergen Can f 2 is an important cause of allergic sensitization in humans worldwide. Here, the first crystal structure of recombinant rCan f 2 at 1.45 A resolution displays a classical lipocalin fold with a conserved Gly-Xaa-Trp motif, in which Trp19 stabilizes the overall topology of the monomeric rCan f 2. Phe38 and Tyr84 localized on the L1 and L5 loops, respectively, control access to the highly hydrophobic calyx. Although the rCan f 2 calyx is nearly identical with the aero-allergens MUP1, Equ c 1 and A2U from mouse, horse and rat, respectively, no IgE cross-reactivity was found using sera from five mono-sensitized subjects. However, clear IgE cross-reactivity was demonstrated between Can f 2 and the cat allergen Fel d 4, although they share less than 22% sequence identity. This suggests a role for these allergens in co-sensitization between cat- and dog-allergic patients.

The major cat allergen, Fel d 1, in diagnosis and therapy.

Sensitization to cat is a common cause of allergic disease all over the world. Symptoms range from mild rhinoconjunctivitis to potentially life-threatening asthmatic exacerbations. In vivo and in vitro diagnostics of cat allergy is currently based on cat dander extract. As allergen extracts from natural sources are heterogeneous in composition, the allergen content may vary. With the introduction of allergens produced by recombinant techniques, a large panel of recombinant allergenic molecules including the major cat allergen, recombinant Fel d 1, has become available for immunological investigations, diagnosis and treatment. Studies have shown that this single allergen, which belongs to the uteroglobin protein family, is at least as good as cat dander extract in identifying cat-allergic patients. The introduction of recombinant Fel d 1-based tests into clinical practice will increase our knowledge of this single allergen molecule as a diagnostic tool and improve the selection for therapy of cat allergy. Several different modes for allergen-specific immunotherapy of cat allergy based on Fel d 1 have been developed. These include Fel d 1 hypoallergens and allergen constructs where Fel d 1 is coupled to immunomodulatory proteins or carriers. The approaches have been evaluated in experimental in vitro and in vivo model systems with promising results. In addition, immunotherapy with Fel d 1 peptides containing T-cell epitopes has been tested in clinical trials. After initial problems with adverse reactions, more recent data show that peptide immunotherapy modulates the immune response to Fel d 1 and reduces early- and late-phase effector reactions in cat-allergic patients.

Cat sensitization identified by recombinant Fel d 1 several years before symptoms--results from the BAMSE cohort.

Exposure to cat is one of the most important causes of allergic disease. The objective of this study was to investigate IgE reactivity to the recombinant major cat allergen, rFel d 1, as an early marker of cat sensitization. Based on questionnaires, 144 children with allergic symptoms due to cat, or where such symptoms were suspected, were selected from the birth cohort BAMSE and allocated into three study groups. Blood samples taken at age 4 and 8 yrs were analysed for IgE to rFel d 1 and cat dander extract (CDE) by quantitative ELISA (cut-off limit 0.037 kU(A)/l) and the ImmunoCAP System (cut-off limit 0.35 kU(A)/l), respectively. At 4 yrs, 25/33 children with certain allergic symptoms to cat had IgE to both rFel d 1 and CDE, while 14/42 of those suspecting symptoms at 4 had IgE to rFel d 1 and 9/42 to CDE. In a group developing symptoms after 4 yrs, 60/69 had IgE to rFel d 1 and 57/69 to CDE at 8, while 33/69 had IgE to rFel d 1 already at 4 and 26/69 to CDE. This was the only one of the three study groups where a significant increase in the IgE levels to rFel d 1 was found from 4 to 8 yrs (p < 0.001), even when only children with IgE to rFel d 1 already at 4 were included (p < 0.001). We show that the single major cat allergen rFel d 1 is at least as good as CDE in the diagnosis of cat allergy in childhood. With a sensitive rFel d 1 assay cat sensitization can be detected several years before symptoms to cat are reported.

Cloning and characterisation of two IgE-binding proteins, homologous to tropomyosin and alpha-tubulin, from the mite Lepidoglyphus destructor.

BACKGROUND: The dust mite Lepidoglyphus destructor is a major source of mite allergy in European rural environments, but it also causes allergy in urban populations around the world. We have previously cloned, sequenced and expressed several allergens from L. destructor (Lep d 2, Lep d 5, Lep d 7 and Lep d 13). The aim of this study was to identify and clone additional allergens from L. destructor, and to evaluate their IgE-binding reactivities. METHODS: PCR and screening with sera from L. destructor-sensitised individuals were used to isolate new clones from a phage display L. destructor cDNA library. The complete coding sequences of the clones were determined and expressed as His(6)-tagged recombinant proteins in Escherichia coli. The recombinant proteins were analysed by SDS-PAGE, immunoblotting and mass spectrometry. RESULTS: Two new clones, showing homology to tropomyosin and alpha-tubulin in several species, were isolated from the phage display L. destructor cDNA library. Due to its homology to group 10 dust mite allergens, the tropomyosin clone was named Lep d 10. The IgE-binding frequencies of the recombinant Lep d 10 and alpha-tubulin were 13% (18/136) and 12% (11/95), respectively, among subjects with IgE reactivity to mites and/or crustaceans. CONCLUSIONS: Two new allergens from L. destructor have been identified and can now be added to the repertoire of recombinant L. destructor allergens. In addition, both these allergens belong to highly conserved protein families and may be important for evaluation of allergenic cross-reactivity.