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.

Biological screening of a unique drug library targeting MRGPRX2.

Background: Allergic drug reaction or drug allergy is an immunologically mediated drug hypersensitivity reaction (DHR). G-protein coupled receptors (GPCRs) are common drug targets and communicate extracellular signals that initiate cellular responses. Recent evidence shows that GPCR MRGPRX2 is of major importance in IgE-independent pseudo-allergic DHRs based on the suspected interactions between many FDA-approved peptidergic compounds and MRGPRX2. Objective: Our aim was to uncover novel MRGPRX2-selective and -potent agonists as drug candidates responsible for clinical features of pseudo-allergic DHRs. Methods: We conducted a primary high-throughput screening (HTS), coupled with mutagenesis targeting the MRGPRX2 N62S mutation, on a panel of 3,456 library compounds. We discovered pharmacologically active hit compounds as agonists of the MRGPRX2 protein according to high degrees of potency evaluated by the calcium response and validated by the degranulation assay. Using the molecular tool Forge, we also characterized the structure-activity relationship shared by identified hit compounds. Results: The alternative allele of single nucleotide polymorphism rs10833049 (N62S) in MRGPRX2 demonstrated loss-of-function property in response to substance P and antineoplastic agent daunorubicin hydrochloride. We applied a unique assay system targeting the N62S mutation to the HTS and identified 84 MRGPRX2-selective active hit compounds representing diverse classes according to primary drug indications. The top five highly represented groups included fluoroquinolone and non-fluoroquinolone antibiotics; antidepressive/antipsychotic; antihistaminic and antineoplastic agents. We classified hit compounds into 14 clusters representing a variety of chemical and drug classes beyond those reported, such as opioids, neuromuscular blocking agents, and fluoroquinolones. We further demonstrated MRGPRX2-dependent degranulation in the human mast cell line LAD2 cells induced by three novel agonists representing the non-fluoroquinolone antibiotics (bacitracin A), anti-allergic agents (brompheniramine maleate) and tyrosine-kinase inhibitors (imatinib mesylate). Conclusion: Our findings could facilitate the development of interventions for personalized prevention and treatment of DHRs, as well as future pharmacogenetic investigations of MRGPRX2 in relevant disease cohorts.

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.

Response of peripheral blood basophils in subjects with chronic spontaneous urticaria during treatment with omalizumab.

BACKGROUND: Treatment of patients with asthma or food allergy with omalizumab results in several consistent changes in circulating basophils. The multiple basophil phenotypes observed in patients with chronic spontaneous urticaria (CSU) present some unique attributes that may not respond in a similar fashion to patients with asthma or food allergy. As part of a clinical study on the therapeutic outcomes of omalizumab treatment in CSU, the basophil compartment was examined for changes in characteristics predicted by prior studies. OBJECTIVE: This study sought to examine the changes in basophil function and its relationship to auto-antibodies in serum during treatment with omalizumab. METHODS: At multiple time points before and during omalizumab treatment of patients with CSU, basophil surface IgE and FcεRI expression, cellular spleen tyrosine kinase (SYK) expression, IgE-mediated histamine release (HR), and the presence of auto-antibodies in serum were determined. RESULTS: Three basophil phenotypes were enumerated in the clinical study and used to group results in this basophil study: subjects with (1) basopenia, (2) normal basophil numbers with normal IgE-mediated HR, and (3) normal basophil numbers with poor HR. Basopenia was highly associated with the presence of auto-antibodies to unoccupied FcεRI and basophil numbers did not change during treatment. Likewise, subjects who are basopenic showed no changes in SYK expression or HR during treatment. In basophils of subjects who are nonbasopenic, increases in SYK expression and HR showed the expected inverse relationship to starting SYK and HR levels. Treatment with omalizumab resulted in similar kinetics for decreases in surface FcεRI and IgE in all 3 groups. CONCLUSIONS: A unifying interpretation of the results revolves around the presence of auto-antibodies to FcεRI in CSU. If present, basopenia and an absence of changes in basophils during omalizumab treatment are observed. If auto-antibodies are absent, the changes in the basophil compartment are consistent with prior studies of asthma and food allergy. These group differences also are related to efficacy of the treatment for clinical outcomes, as found in the parent clinical study.

The relationship between released soluble FceRI-alpha and its cell surface density on human basophils.

BACKGROUND: The IgE-mediated activation of mast cells and basophils results in the secretion of many substances, including the release of FceRI-alpha subunit. This released alpha subunit can bind IgE and it may act as a down-regulator of subsequent IgE-dependent reactions. However, previous studies do not observe loss of the mass of FceRI-alpha associated with the cells, at least not for human basophils. This study was designed to understand the basis for the discordant observations. METHODS: Purified human basophils were stimulated with multiple activating secretagogues and supernatants were examined for histamine and released FceRI-alpha. In addition, cell surface IgE densities (occupied and unoccupied) were measured by flow cytometry and total cellular content of mature and immature FceRI-alpha determined with Western blots. RESULTS: Released FceRI-alpha, on average, represented 7% of the total surface FceRI before the reaction. The molecular weight of the soluble FceRI-alpha was approximately 54 kD, larger than immature subunit and somewhat smaller than surface subunit. In addition, 1) release ceased long before internalized FceRI-alpha was processed, 2) release was insensitive to Bafilomycin A, 3) release was independent of the starting density of FceRI and 4) release occurred more effectively with non-IgE-dependent stimuli, FMLP or C5a. CONCLUSIONS: There appears to be relatively constant amount of nearly mature FceRI-alpha that is susceptible to secretion events induced by any form of stimulation. The amount, on average, represents about 7% of the mature form of FceRI-alpha.

Modulating the Human Basophil Phenotype During Its Development and Maturation: Basophils Derived from In Vitro Cultures of CD34+ Progenitor Cells.

Historically, the human basophil that is studied experimentally comes from peripheral blood. But there is evidence that only a short portion of the basophil life cycle related to IgE-mediated function occurs in the blood. The same evidence suggests that IgE-mediated functionality is present for 5-7 days in the bone marrow (or other tissues) during which the cell modulates its phenotype according to local conditions. It is suggested that to properly understand the nature of basophil behavior, a better understanding of its biology during maturation would be helpful. For example, one highly suggestive line of evidence for the relevance of understanding the maturation period is related to the change in basophil phenotype that occurs during treatment of patients with omalizumab. During this treatment, the intrinsic reactivity or sensitivity of the basophils is significantly increased despite, or perhaps because of, the dramatic reduction in FcεRI expression that accompanies this treatment. One of the critical signaling enzymes to increase expression selectively in basophils during treatment is SYK, which is one of the earliest signaling tyrosine kinases involved in translating the aggregation of FcεRI into secretion from the cell. Treatment with omalizumab increases SYK expression, and this observation focuses some attention of how SYK expression is regulated. It is possible that the key regulation occurs during maturation of the basophil. Regardless of the mechanisms operative in this particular treatment, understanding the process of maturation and the extrinsic factors that influence it may lead to better understanding of disease processes. Therefore, this chapter will discuss and present techniques to work with maturing human basophils.

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.

Identifying regulatory pathways of spleen tyrosine kinase expression in human basophils.

BACKGROUND: Expression levels of spleen tyrosine kinase (SYK), a critical signaling tyrosine kinase in basophils, are uniquely low relative to all other circulating leukocytes, and levels are highly variable in the population. OBJECTIVE: We sought to determine whether transcriptional regulation of SYK through unique silencing of the SYK gene determines its basophil-specific expression patterns. METHODS: Culture-derived basophils (CD34B cells) were derived from cultures of CD34+ progenitor cells by using 2 methods (G1 or G3). Peripheral blood basophils (PBBs; relative SYK protein level = 1), B cells (SYK = 8), CD34B-G1 cells (SYK = 11), and CD34B-G3 cells (SYK = 5) were examined by using assay for transposase-accessible chromatin sequencing (ATAC-seq) methods. In addition, the transcriptomes of 6 cell types, PBBs, peripheral blood eosinophils (SYK = 11), plasmacytoid dendritic cells (SYK = 30), CD34+ progenitors (SYK = 11), CD34B-G1 cells, and CD34B-G3 cells, were analyzed for patterns that matched patterns of SYK expression in these cells, with a focus on transcription factors. RESULTS: ATAC-seq showed that PBBs have multiple open regions in the SYK gene, suggesting a nonsilenced state with 1 region unique to PBBs (low SYK expression), 1 region unique to both PBBs (low SYK expression) and both G1 and G3 CD34B cells (high and moderate SYK expression, respectively), and 5 regions unique to B cells (high SYK expression). SYK expression across the 6 cell types explored showed a unique pattern that was matched to expression patterns of 3 transcription factors: Kruppel-like factor 5 (KLF5), zinc-finger protein 608 (ZNF608), and musculoaponeurotic fibrosarcoma protein (c-MAF). CONCLUSIONS: Two new potential regulatory pathways for SYK expression were identified. One appears independent of transcriptional regulation, and one appears to be dependent on transcriptional control in the SYK gene.

Autoantibodies to IgE and FcεRI and the natural variability of spleen tyrosine kinase expression in basophils.

BACKGROUND: Secretion from human basophils and mast cells requires spleen tyrosine kinase (SYK) activity, but SYK expression is highly variable in the general population, and this variability predicts the magnitude of IgE-mediated secretion. One known mechanism of modulating SYK expression in human basophils is aggregation of FcεRI. OBJECTIVE: This study examines the possibility that functional autoantibodies are present in a wide variety of subjects and, in particular, subjects whose basophils poorly express SYK. It also tests whether any found antibodies could modulate SYK expression in maturing basophils and whether interaction with FcγRIIb/CD32b modulates the effect. METHODS: An experimental algorithm for classifying the nature of histamine release induced by serum from 3 classes of subjects was developed. RESULTS: The frequency of functional autoantibodies that produce characteristics concordant with FcεRI-mediated secretion was zero in 34 subjects without chronic spontaneous urticaria (CSU). In patients with CSU, the frequency was lower than expected, approximately 7%. For the 5 of 68 unique sera from patients with CSU tested that contained anti-FcεRI or anti-IgE antibodies, these antibodies were found to induce downregulation of SYK in both peripheral blood basophils and basophils developed from CD34+ progenitors. Blocking interaction of these antibodies with CD32b did not alter their ability to downregulate SYK expression. CONCLUSIONS: This study establishes that functional autoantibodies to IgE/FcεRI do not provide a good explanation for the variability in SYK expression in basophils in the general population. They do show that if antibodies with these characteristics are present, they are capable of modulating SYK expression in developing basophils.

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.

FceRI density and spontaneous secretion from human basophils.

Mast cells and basophils depend on aggregation of the high-affinity IgE receptor, FceRI, to initiate secretion. A variety of studies have shown that FceRI densities vary 100 fold among subjects' basophils and it has been speculated that high densities might be responsible for unusual behaviors of the cells, notably sensitivity to certain monomeric IgE antibodies or spontaneous release. These studies experimentally examined the density dependence of spontaneous release and signaling element expression in subjects' basophils with FceRI densities ranging from approximately 6000 to 600,000 per cell. Through the use of numerical simulation, this study examined the expectations for spontaneous receptor aggregation and aggregate persistence at densities of FceRI ranging from 5000 to 500,000. Experimentally, FceRI density was not associated with greater spontaneous histamine release even when secretion was enhanced by the inclusion of deuterium oxide in the buffers. There was also no association of 15 activating or de-activating signaling elements with FceRI density. The numerical simulations demonstrated that at densities of ≈500,000 receptors, 13% of receptors were involved in transient aggregates at any given moment but that these aggregates rarely persisted for greater than 10 milliseconds. In contrast, a weak linear antigen aggregator, with ligand affinities known to induce secretion, would generate aggregates persisting for an average of ≈60 milliseconds. These results suggest that although a high density of FceRI likely produces a large number of transient aggregates, these aggregates do not persist long enough to induce signaling that results in secretion and do not induce the cells to alter their expression of several signaling elements known to be important in regulating secretion from human basophils. The results set some boundaries on the aggregation requirements for inducing histamine release from human basophils.

Pros and Cons of Clinical Basophil Testing (BAT).

PURPOSE OF REVIEW: We review basophil testing by flow cytometry with an emphasis on advantages and disadvantages. RECENT FINDINGS: There are many tools available to assess the presence and severity of allergic diseases in patients. For 50 years, peripheral blood basophils have been used as tools to study these diseases. It is a very accessible cell that binds IgE antibody and secretes the classical mediators responsible for the symptoms of allergic reactions. In the last decade, an even more accessible methodology, using flow cytometry, has been developed to enhance the ability to use basophils for both mechanistic and clinical diagnostics. Basophil testing has been included in diagnostics for different forms of allergies as well as to monitor disease status. A variety of studies have begun to establish both precise methods and their clinical relevance for disease diagnosis, but there remain some important questions on how to take optimal advantage of the behaviours of basophils.