A phase II study of Bruton's tyrosine kinase inhibition for the prevention of anaphylaxis.

BACKGROUNDIgE-mediated anaphylaxis is a potentially fatal systemic allergic reaction for which there are no currently FDA-approved preventative therapies. Bruton's tyrosine kinase (BTK) is an essential enzyme for IgE-mediated signaling pathways and is an ideal pharmacologic target to prevent allergic reactions. In this open-label trial, we evaluated the safety and efficacy of acalabrutinib, a BTK inhibitor that is FDA approved to treat some B cell malignancies, in preventing clinical reactivity to peanut in adults with peanut allergy.METHODSAfter undergoing graded oral peanut challenge to establish their baseline level of clinical reactivity, 10 patients had a 6-week rest period, then received 4 standard doses of 100 mg acalabrutinib twice daily and underwent repeat food challenge. The primary endpoint was the change in patients' threshold dose of peanut protein to elicit an objective clinical reaction.RESULTSAt baseline, patients tolerated a median of 29 mg of peanut protein before objective clinical reaction. During subsequent food challenge on acalabrutinib, patients' median tolerated dose significantly increased to 4,044 mg (range 444-4,044 mg). 7 patients tolerated the maximum protocol amount (4,044 mg) of peanut protein with no clinical reaction, and the other 3 patients' peanut tolerance increased between 32- and 217-fold. 3 patients experienced a total of 4 adverse events that were considered to be possibly related to acalabrutinib; all events were transient and nonserious.CONCLUSIONAcalabrutinib pretreatment achieved clinically relevant increases in patients' tolerance to their food allergen, thereby supporting the need for larger, placebo-controlled trials.TRIAL REGISTRATIONClinicalTrials.gov NCT05038904FUNDINGAstraZeneca Pharmaceuticals, the Johns Hopkins Institute for Clinical and Translational Research, the Ludwig Family Foundation, and NIH grants AI143965 and AI106043.

Bruton's tyrosine kinase inhibition for the prevention of anaphylaxis: an open-label, phase 2 trial.

IgE-mediated anaphylaxis is a potentially fatal systemic allergic reaction for which there are no known preventative therapies. Bruton's tyrosine kinase (BTK) is an essential enzyme for IgE-mediated signaling pathways, and is an ideal pharmacologic target to prevent allergic reactions. In this open-label trial (NCT05038904), we evaluated the safety and efficacy of acalabrutinib, a BTK inhibitor that is FDA-approved to treat some B cell malignancies, in preventing clinical reactivity to peanut in adults with IgE-mediated peanut allergy. After undergoing a graded oral peanut challenge to establish their baseline level of clinical reactivity, all patients then received four standard doses of 100 mg acalabrutinib twice daily and underwent repeat food challenge. The primary endpoint was the change in patients' threshold dose of peanut protein to elicit an objective clinical reaction. At baseline, patients tolerated a median of 29 mg of peanut protein before objective clinical reaction. During subsequent food challenge on acalabrutinib, patients' median tolerated dose significantly increased to 4,044 mg (range, 444 - 4,044 mg). 7 of 10 patients tolerated the maximum protocol amount (4,044 mg) of peanut protein with no objective clinical reaction, and the other 3 patients' peanut tolerance increased between 32- and 217-fold. Three patients experienced a total of 4 adverse events that were considered by the investigators to be possibly related to acalabrutinib; all events were transient and nonserious. These results demonstrate that acalabrutinib pretreatment can achieve clinically-relevant increases in patients' tolerance to their food allergen, thereby supporting the need for larger, placebo-controlled trials.

The efficacy of omalizumab treatment in chronic spontaneous urticaria is associated with basophil phenotypes.

BACKGROUND: The mechanisms underlying disease pathogenesis in chronic spontaneous urticaria (CSU) and improvement with omalizumab are incompletely understood. OBJECTIVES: This study sought to examine whether the rate of clinical remission is concordant with baseline basophil features or the rate of change of IgE-dependent functions of basophils and/or plasmacytoid dendritic cells during omalizumab therapy. METHODS: Adults (n = 18) with refractory CSU were treated with omalizumab 300 mg monthly for 90 days. Subjects recorded daily urticaria activity scores, and clinical assessments with blood sampling occurred at baseline and on days 1, 3, 6, 10, 20, 30, 60, and 90 following omalizumab. At baseline, subjects were categorized by basophil functional phenotypes, determined by in vitro histamine release (HR) responses to anti-IgE antibody, as CSU-responder (CSU-R) or CSU-non-responder (CSU-NR), as well as basopenic (B) or nonbasopenic (NB). RESULTS: CSU-R/NB subjects demonstrated the most rapid and complete symptom improvement. By day 6, CSU-R/NB and CSU-NR/NB had increased anti-IgE-mediated basophil HR relative to baseline, and these shifts did not correlate with symptom improvement. In contrast, CSU-NR/B basophil HR did not change during therapy. The kinetics of the decrease in surface IgE/FcεRI was similar in all 3 phenotypic groups and independent of the timing of the clinical response. Likewise, plasmacytoid dendritic cells' surface IgE/FcεRI decline and TLR9-induced IFN-α responses did not reflect clinical change. CONCLUSIONS: Changes in basophil IgE-based HR, surface IgE, or FcεRI bear no relationship to the kinetics in the change in clinical symptoms. Baseline basophil count and basophil functional phenotype, as determined by HR, may be predictive of responsiveness to omalizumab.

Targeting the FcεRI Pathway as a Potential Strategy to Prevent Food-Induced Anaphylaxis.

Despite attempts to halt it, the prevalence of food allergy is increasing, and there is an unmet need for strategies to prevent morbidity and mortality from food-induced allergic reactions. There are no known medications that can prevent anaphylaxis, but several novel therapies show promise for the prevention of food-induced anaphylaxis through targeting of the high-affinity IgE receptor (FcϵRI) pathway. This pathway includes multiple candidate targets, including tyrosine kinases and the receptor itself. Small molecule inhibitors of essential kinases have rapid onset of action and transient efficacy, which may be beneficial for short-term use for immunotherapy buildup or desensitizations. Short courses of FDA-approved inhibitors of Bruton's tyrosine kinase can eliminate IgE-mediated basophil activation and reduce food skin test size in allergic adults, and prevent IgE-mediated anaphylaxis in humanized mice. In contrast, biologics may provide longer-lasting protection, albeit with slower onset. Omalizumab is an anti-IgE antibody that sequesters IgE, thereby reducing FcϵRI expression on mast cells and basophils. As a monotherapy, it can increase the clinical threshold dose of food allergen, and when used as an adjunct for food immunotherapy, it decreases severe reactions during buildup phase. Finally, lirentelimab, an anti-Siglec-8 antibody currently in clinical trials, can prevent IgE-mediated anaphylaxis in mice through mast cell inhibition. This review discusses these and other emerging therapies as potential strategies for preventing food-induced anaphylaxis. In contrast to other food allergy treatments which largely focus on individual allergens, blockade of the FcϵRI pathway has the advantage of preventing clinical reactivity from any food.

Dependence of Optimal Histamine Release on Cell Surface IgE Density on Human Basophils: Nature of the Stimulus.

BACKGROUND: The characteristics of the aggregation reaction that follows allergen binding to cell surface IgE on basophils and mast cells depend on a variety of factors that include the density of IgE and the affinity of the allergen for IgE. For simple bivalent stimuli, one prediction is that the location of the optimum for aggregation is not dependent on IgE density, only the affinity for IgE. However, this behavior does not occur for stimulation with an anti-IgE antibody (Ab) during the treatment of patients with omalizumab. METHODS: This study re-examined the stability of the optimum for histamine release, relative to cell surface IgE density, using the simple bivalent penicillin hapten (BPO2) or a bivalent monoclonal anti-IgE Ab. RESULTS: The results validated one prediction for one bivalent hapten, BPO2. Across a range of BPO-specific IgE density of 270-23,500/cell, optimal histamine release remained constant (10 nM BPO2). In contrast, across a range of approximately 6,000-110,000/cell, optimal histamine release shifted 8- to 30-fold for anti-IgE Ab. The distinguishing characteristic between the 2 bivalent stimuli was the difference in their crosslink re-equilibration. Recent modeling of histamine release suggested that the SYK-to-receptor ratio could determine the position of histamine release optimum. The study showed that there were significant shifts in the SYK-to-receptor ratio (from 1: 6 to 5: 1) but the basophil's ability to sense this ratio was restricted to transient crosslinks, as occurred with anti-IgE Ab. CONCLUSIONS: The results suggest that ligand crosslinking dynamics couple with SYK and receptor expression levels to determine qualitative characteristics of the dose response curve for secretion.

Basophil activation testing.

Both the treatment of patients with allergic diseases and the study of allergic disease mechanisms depend on a wide variety of assays that in various ways assess the presence and function of IgE antibody. The study of allergic diseases could benefit from the study of its 2 principle cellular participants, mast cells and basophils, but the basophil is more accessible than mast cells for ex vivo studies. Its functionality is tested by using 2 predominant methodologies: the secretion of mediators of allergic inflammation and the expression of proteins on the plasma membrane after stimulation. Each approach has benefits. There are also many operational details to consider regardless of which general approach is taken, and proper interpretation of the methods requires a good understanding of the reagents used and the receptors expressed on basophils and a detailed understanding of the factors regulating aggregation of cell-surface IgE.

Omalizumab increases the intrinsic sensitivity of human basophils to IgE-mediated stimulation.

BACKGROUND: Treatment of allergic patients with omalizumab results in a paradoxical increase in their basophil histamine release (HR) response ex vivo to cross-linking anti-IgE antibody. It is not known whether this change in response is associated with an increase in intrinsic cellular sensitivity, which would be a paradoxical response. OBJECTIVE: We sought to determine whether the increase in response to anti-IgE antibody is a reflection of an increased cellular sensitivity expressed as molecules of antigen-specific IgE per basophil required to produce 50% of the maximal response. METHODS: Patients were treated with omalizumab or placebo for 12 weeks (NCT01003301 at ClinicalTrials.gov), and the metric of basophil sensitivity was assessed at 4 time points: baseline, 6 to 8 weeks, 12 weeks (after which treatment stopped), and 24 weeks (12 weeks after the end of treatment). RESULTS: As observed previously, treatment with omalizumab resulted in a marked increase in the maximal HR induced by cross-linking anti-IgE antibody. This change was accompanied by a marked shift in intrinsic basophil sensitivity, ranging from 2.5- to 125-fold, with an average of 6-fold at the midpoint of the treatment to 12-fold after 12 weeks. The magnitude of the increase in cellular sensitivity was inversely related to the starting sensitivity or the starting maximum HR. The increased cellular sensitivity also occurred when using leukotriene C4 secretion as a metric of the basophil response. Twelve weeks after the end of treatment, cellular sensitivity was found to shift toward the baseline value, although the return to baseline was not yet complete at this time point. CONCLUSIONS: Treatment with omalizumab results in a markedly increased sensitivity of basophils to IgE-mediated stimulation in terms of the number of IgE molecules required to produce a given response. These results provide a better quantitative sense of the phenotypic change that occurs in basophils during omalizumab treatment, which has both mechanistic and clinical implications.

IgE-dependent signaling as a therapeutic target for allergies.

Atopic diseases are complex, with many immunological participants, but the central element in their expression is IgE antibody. In an atopic individual, the immune system pathologically reacts to environmental substances by producing IgE, and these allergen-specific IgE antibodies confer to IgE receptor-bearing cells responsiveness to the environmental substances. Mast cells and basophils are central to the immediate hypersensitivity reaction that is mediated by IgE. In humans, there are various other immune cells, notably dendritic cells and B cells, which can also bind IgE. For mast cells, basophils and dendritic cells, the receptor that binds IgE is the high-affinity receptor, FcɛRI. For B cells and a few other cell types, the low affinity receptor, FcɛRII, provides the cell with a means to sense the presence of IgE. This overview will focus on events following activation of the high-affinity receptor because FcɛRI generates the classical immediate hypersensitivity reaction.

Suppression of the basophil response to allergen during treatment with omalizumab is dependent on 2 competing factors.

BACKGROUND: A recent study of subjects with peanut allergy treated with omalizumab generated some results that were concordant with a study of subjects with cat allergy treated with omalizumab. However, there were differences that provided additional insight into the nature of the cellular responses in allergic subjects. OBJECTIVE: We sought to determine the cause for failure to suppress the allergen-induced basophil response during treatment with omalizumab. METHODS: Patients with peanut allergy were treated with omalizumab. Clinical, serologic, and cellular indices relevant to the response of the subjects and their peripheral blood basophil values (specific/total IgE ratio, cell-surface FcεRI expression, and histamine release responses to anti-IgE antibody or peanut allergen) were obtained at 3 times. RESULTS: After treatment, approximately 60% of the subjects' basophil responses to peanut allergen did not significantly decrease. In 40% of cases, the in vitro basophil response to peanut allergen increased 2- to 7-fold. The increases were associated with 2 primary factors: a high (>10%) specific/total IgE ratio and an increase in the intrinsic response of the basophil to IgE-mediated stimulation. The extent to which the basophil response to peanut allergen increased was inversely correlated with improvement in the patient's ability to tolerate ingestion of peanut. CONCLUSION: The basophil response during treatment with omalizumab is a consequence of 2 competing factors: suppression of allergen-specific IgE on the cell surface versus increased intrinsic sensitivity to IgE-mediated stimulation. In subjects with peanut allergy, the basophil response appears to mitigate against the ability of omalizumab to improve the patient's tolerance of oral allergen.

Kinetics of mast cell, basophil, and oral food challenge responses in omalizumab-treated adults with peanut allergy.

BACKGROUND: Monoclonal antibodies directed at IgE demonstrate clinical efficacy in subjects with peanut allergy, but previous studies have not addressed the kinetics of the clinical response or the role of mast cells and basophils in the food-induced allergic response. OBJECTIVE: We sought to determine the kinetics of the clinical response to omalizumab and whether clinical improvement is associated with either mast cell or basophil suppression. METHODS: Subjects with peanut allergy were treated with omalizumab for 6 months and assessed for clinical and cellular responses. At baseline, subjects had a double-blind, placebo-controlled oral food challenge (OFC), skin prick test titration (SPTT), and basophil histamine release (BHR) to peanut. BHR was repeated at week 2 and then weekly until it decreased to less than 20% of baseline values. The OFCs and SPTTs were repeated after the BHR reduction (or at week 8 if BHR did not decrease) and again at 6 months. RESULTS: Fourteen subjects enrolled in the study. At the second food challenge, there was a significant increase in the threshold dose of peanut inducing allergic symptoms (80 to 6500 mg, P < .01). Peanut-induced BHR was either completely suppressed (n = 5) or 10-fold more allergen was required to induce maximal BHR (n = 9), and SPTT responses were not significantly changed from baseline. After 6 months of omalizumab, further changes in the OFC threshold dose or BHR were not observed, but a significant suppression in SPTTs was identified. CONCLUSIONS: The clinical response to omalizumab occurs early in treatment when the basophil, but not the mast cell, is suppressed, supporting a role for the basophil in acute food reactions.

Assessing basophil functional measures during monoclonal anti-IgE therapy.

Assessing the impact of therapeutic interventions on the clinical and immunologic responses of allergic subjects is a topic of extensive investigation. Available approaches include the measurement of in vivo allergen challenge responses, serologic measures, or in vitro studies of cells that participate in the allergic reaction. Several decades of work support that measures of allergen responses of IgE-bearing peripheral blood basophils can reflect clinical expression of allergic disease. In the last decade, an immune-based therapy targeting IgE, omalizumab, has emerged as an adjunct treatment for a variety of allergic diseases. This monoclonal humanized IgG antibody specifically binds circulating IgE at a region in the Fc tail that prevents IgE attachment to high affinity IgE receptor (FcεRI) bearing cell types such as tissue mast cells and blood basophils. This review focuses on methods to monitor changes of basophil allergen reactivity with a focus on omalizumab therapy and the implications for clinical disease management.

Glycomic analysis of human mast cells, eosinophils and basophils.

In allergic diseases such as asthma, eosinophils, basophils and mast cells, through release of preformed and newly generated mediators, granule proteins and cytokines, are recognized as key effector cells. While their surface protein phenotypes, mediator release profiles, ontogeny, cell trafficking and genomes have been generally explored and compared, there has yet to be any thorough analysis and comparison of their glycomes. Such studies are critical to understand the contribution of carbohydrates to the induction and regulation of allergic inflammatory responses and are now possible using improved technologies for detecting and characterizing cell-derived glycans. We thus report here the application of high-sensitivity mass spectrometric-based glycomics methodologies to the analysis of N-linked glycans derived from isolated populations of human mast cells, eosinophils and basophils. The samples were subjected to matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) screening analyses and MALDI-TOF/TOF sequencing studies. Results reveal substantive quantities of terminal N-acetylglucosamine containing structures in both the eosinophil and the basophil samples, whereas mast cells display greater relative quantities of sialylated terminal epitopes. For the first time, we characterize the cell surface glycan structures of principal allergic effector cells, which by interaction with glycan-binding proteins (e.g. lectins) have the possibility to dictate cellular functions, and might thus have important implications for the pathogenesis of inflammatory and allergic diseases.

Regulation of Syk kinase and FcRbeta expression in human basophils during treatment with omalizumab.

BACKGROUND: In human basophils from different subjects, maximum IgE-mediated histamine release and the level of Syk protein expression correlate well. Recent studies suggest that in some patients treated with omalizumab, the response to stimulation with anti-IgE antibody increases. In unrelated studies there is also evidence that the composition of FcepsilonRI in basophils differs among subjects. This observation raised the possibility that the stoichiometry of FcRbeta/FcepsilonRIalpha is not fixed to a 1:1 ratio and might be modifiable during changes in the basophil's environment. OBJECTIVE: We sought to determine whether treatment with omalizumab results in increases in Syk expression and anti-IgE-mediated histamine release and disproportionately alters the relative presence of FcRbeta and FcepsilonRIalpha. METHOD: Syk, FcepsilonRIalpha, and FcRbeta expression was monitored during the treatment of subjects with omalizumab. RESULTS: Treatment with omalizumab reduced histamine release from peripheral blood leukocytes stimulated with cat allergen in vitro, but histamine release stimulated with anti-IgE antibody increased 2-fold. Expression of Syk increased 1.86-fold. There was no change in the expression of c-Cbl, a signaling element that is sensitive to the presence of IL-3, and no increase in response to formyl-met-leu-phe (tripeptide), a response that also increases in the presence of IL-3. There was a 60% decrease in the FcRbeta/FcepsilonRIalpha ratio in patients treated with omalizumab. CONCLUSIONS: In the context of previous studies, these studies provide support for a proposal that Syk expression is modulated in vivo through an IgE-dependent mechanism and that the ratio of FcepsilonRI alpha and beta subunits in basophils is influenced by factors extrinsic to the cell.

Regulation of Fc epsilon RI expression during murine basophil maturation: the interplay between IgE, cell division, and Fc epsilon RI synthetic rate.

Expression of Fc epsilonRI on basophils and mast cells is modulated by IgE Ab. Previous studies have noted in vivo receptor expression dynamics that are discordant with expectations derived from in vitro studies. The current study presents a formal hypothesis to explain the discordant observations and tests two assumptions that underlie a proposed model of receptor dynamics. After first showing that a murine model of receptor expression on basophils recapitulates observations made using human basophils, the effect of changes in IgE on basophil egress rates was examined. In the proposed model, egress rates from bone marrow (BM) were assumed to be unaffected by changes in IgE concentration. Egress was tested by examining the labeling of BM and peripheral blood (BL) basophils at various times after injection of BrdU with and without injection with IgE. The IgE Ab did not alter the appearance of BrdU label in peripheral BL basophils. In addition, BM and BL basophils were responsive to the elevations in IgE, with receptor expression increasing on BM basophils before BL basophils. It was also noted that BL basophils expressed approximately 50% of the receptor density of BM basophils. There was a 3-fold greater synthetic rate of Fc epsilonRI on BM basophils that readily explained the difference. These results provide support for the proposed hypothesis of rapid changes in receptor expression being controlled by cell replacement. The studies also support a model whereby receptor expression is limited by cell division and that basophils, once mature, slow their rate of receptor synthesis.

IgE antibody-specific activity in human allergic disease.

IgE antibody concentration, affinity, clonality and specific activity (also known as the allergen-specific IgE to total IgE ratio) influence the translation of IgE responses into clinically evident allergic symptoms following allergen exposure. Reported IgE-specific activity levels >3-4% place allergic individuals undergoing anti-IgE (Omalizumab((R))) therapy at a disadvantage for poor resolution of their allergy symptoms following manufacturer's recommended dosing schemes. We investigated the hypothesis that the specific activity of the IgE antibody response is highly variable with respect to age, allergen specificity and an individual's total serum IgE level. Second, we investigated whether the IgE-specific activity level influences the extent and rate of loss of effector cell mediator release. IgE-specific activity distributions were plotted against age, allergen specificity and total serum IgE using 18,950 paired total IgE and allergen-specific IgE antibody data obtained from the analysis of sera from 3,614 allergic subjects and covering 182 allergen specificities. The fraction of specific IgE antibody of the total serum IgE was dependent on age of the individual, epitope specificity (clonality) and total serum IgE. The youngest group of allergic individuals with the lowest total serum IgE levels tended to have the highest allergen-specific IgE to total IgE ratios. Hymenoptera venom (54%), peanut (33%) and milk (27%) were the three allergen specificities that elicited the highest frequency of IgE-specific activities >4% among sensitized individuals. A prospective double-blind, placebo-controlled clinical study involving anti-IgE treatment of cat-allergic subjects showed IgE-specific activity was remarkably constant over the 16-week course of treatment, despite the up to 8-fold rise in total serum IgE following repetitive Omalizumab administration. Changes in specific and total IgE levels paralleled each other in patients receiving anti-IgE therapy. The fastest rate of reduction in cat allergen-induced basophil histamine release following anti-IgE therapy was observed when the cat-specific IgE to total IgE ratio was <2.5%. This reflected the more rapid loss of surface cat-specific IgE antibody with anti-IgE therapy in allergic individuals who displayed a more diverse IgE antibody repertoire. We conclude that IgE-specific activity is an age-, IgE heterogeneity- and total serum IgE-dependent variable that influences the magnitude of effector cell mediator release, and by inference, ultimate allergic symptom induction.

Effects of omalizumab on basophil and mast cell responses using an intranasal cat allergen challenge.

BACKGROUND: Omalizumab treatment suppresses FcepsilonRI expression faster on blood basophils than skin mast cells. OBJECTIVE: We used omalizumab to elucidate the relative contributions of basophil versus mast cell FcepsilonRI activation in a nasal allergen challenge (NAC) model. METHODS: Eighteen subjects with cat allergy were enrolled in a 3.5-month, double-blind, randomized (3.5:1), placebo-controlled trial of omalizumab using standard dosing. At baseline, subjects underwent NAC with lavage for prostaglandin D(2) measurement, skin prick test titration (SPTT), and blood sampling for basophil histamine release (BHR) and basophil IgE/FcepsilonRI measurements. Basophil studies were repeated at day 3 and then weekly until cat allergen-induced BHR was <20% of baseline or until day 45. Baseline visit procedures were repeated after the BHR reduction (midstudy NAC) and at the treatment period's completion (final NAC). RESULTS: Subjects treated with omalizumab who completed all NACs (n = 12) demonstrated significant mean reduction in BHR to an optimal dose of cat allergen by midstudy NAC compared with baseline (74% decrease; P = .001). In addition, these subjects demonstrated significant decreases in mean combined nasal symptom scores (50% decrease; P = .007) and total sneeze counts (59% decrease; P = .01) by midstudy NAC relative to baseline NAC. In contrast, measures of mast cell response (SPTT and nasal lavage prostaglandin D(2)) were only significantly reduced by the final NAC. Subjects on placebo (n = 4) did not experience a shift in basophil, NAC symptom, or mast cell measures. CONCLUSION: Reduction in nasal symptom scores occurred when the basophil, but not mast cell, response was reduced on omalizumab, implicating a role for basophils in the acute NAC response.