Short-term ibrutinib therapy suppresses skin test responses and eliminates IgE-mediated basophil activation in adults with peanut or tree nut allergy

To the Editor:

There is an obvious unmet need for treatments capable of preventing anaphylaxis, especially in the case of food allergy, where avoidance of triggers is not always possible. Bruton’s tyrosine kinase (BTK) is a key component of B-cell receptor signaling, and is also essential for FcεRI signaling and triggering of mast cells and basophils.^1–3 Ibrutinib (brand name Imbruvica; Janssen Pharmaceuticals [Horsham, Pa] and Pharmacyclics, Inc [Sunnyvale, Calif]), an irreversible BTK inhibitor that is Food and Drug Administration–approved for the treatment of B-cell malignancies, has been shown to be generally well tolerated, even with chronic use.⁴ Thus, ibrutinib could potentially be used to prevent allergic reactions including anaphylaxis.⁵ Our laboratory recently completed a pilot study demonstrating that ibrutinib completely eliminated skin test reactivity and IgE-mediated basophil activation test (BAT) responses to aeroallergens in 2 cancer patients within 1 week of starting treatment for chronic lymphocytic leukemia.⁶ We therefore hypothesized that short-term use of ibrutinib would inhibit allergic responses in food-allergic adult subjects. We performed an institutional review board–approved open-label study in which peanut- and/or tree nut–allergic subjects took a standard Food and Drug Administration–approved dose (420 mg daily) of ibrutinib daily for up to 7 days. We performed skin prick tests (SPTs) and BATs before, during, and after ibrutinib treatment (see this article’s Methods section in the Online Repository at www.jacionline.org).

Six subjects with a history of IgE-mediated allergy to peanut and/or tree nuts were enrolled (see Table E1 in this article’s Online Repository at www.jacionline.org; demographic information can be found in this article’s Online Repository at www.jacionline.org). The most common previous reaction to peanut and/or tree nuts was anaphylaxis, in many cases requiring administration of 1 or more doses of intramuscular epinephrine. With the exception of subject 1, who took only 2 doses, all subjects were instructed to take ibrutinib for 7 consecutive days. Subject 6 was able to take ibrutinib for only 6 days because of her seventh dose being accidentally destroyed; thus, her “day 7” BAT and SPT were performed 36 hours after her last ibrutinib dose. After only 2 doses of ibrutinib, SPT wheal and flare area decreased significantly in all subjects for all nuts tested (average area reduction 76.6% for wheal and 86.0% for flare; P < .0001 for both, n = 25; Fig 1). Overall, 44% of all SPT results became negative (wheal diameter < 3 mm) after 2 days. Additional doses of ibrutinib for 4 or 7 days maintained suppression of SPT wheal (P = .0004 at 4 days, P < .0001 at 7 days) and flare (P < .0001 at 4 days, P = .0003 at 7 days), but neither was superior to 2 days of treatment. Interestingly, we observed that the magnitude of SPT area reduction by ibrutinib varied within individuals. The reason for this is unknown, but it is possible that the degree of clinical reactivity to each allergen may determine the degree of SPT area suppression by ibrutinib. It is also possible that larger SPTs at baseline are less likely than smaller SPTs to become completely negative during ibrutinib treatment. We observed a weak inverse correlation between the baseline wheal size and the degree of suppression by ibrutinib; however, this was not significant, possibly due to low sample size. No changes were observed in serum-specific IgE to foods during the study (P = .20, .22, and .07 at 2, 4, and 7 days of ibrutinib treatment, respectively; see Fig E2 in this article’s Online Repository at www.jacionline.org), which supports our hypothesis that ibrutinib is working directly in mast cells to prevent signaling downstream of IgE cross-linking by antigen. As expected, histamine control SPT results were unchanged with 2, 4, and 7 days of ibrutinib treatment (P = .13, .25, and .25 for wheal, and P = .22, .25, and .19 for flare, respectively).

FIG 1.

Ibrutinib reduces or eliminates SPT size in as few as 2 doses. Graphs depict the area for the wheal (A; n = 25) and flare (B; n = 25) of SPTs to all foods for all subjects. Bars indicate mean ± SD. Red circles are SPTs performed while subjects were taking ibrutinib, and black circles represent SPTs that were performed before (at baseline) or after ibrutinib treatment. Histamine control SPT results are shown for all subjects in C (wheal; n = 6) and D (flare; n = 6). F/u, Follow-up. Data were analyzed using Friedman tests and Wilcoxon signed rank tests. ***P < .001; ****P < .0001.

IgE-mediated BAT responses were completely abrogated in all subjects after just 2 doses of ibrutinib (P = .0002), and abrogation was sustained with 4 and 7 days of ibrutinib therapy (P = .0002 and P < .0001 compared with baseline, respectively; Fig 2). As expected, ibrutinib did not affect N-formyl-L-methionyl-L-leucyl-phenylalanine–mediated basophil activation at 2, 4, or 7 days (P = .32, .34, and .80, respectively), although several subjects had poor N-formyl-L-methionyl-L-leucyl-phenylalanine BAT responses throughout the study.

FIG 2.

Ibrutinib abrogates IgE-mediated, but not non–IgE-mediated, basophil activation after just 2 doses. BATs using anti-FcεRI antibody (top, n = 6) or fMLP (bottom; n = 6) stimulation are shown forall subjects at all time points. Red circles are BATs performed while subjects were taking ibrutinib, and black circles represent BATs that were performed before (at baseline) or after ibrutinib treatment. FMLP, N-formyl-L-methionyl-L-leucyl-phenylalanine; f/u, follow-up. Data were analyzed using repeated-measures ANOVA and paired Student t tests. ***P < .001; ****P < .0001.

Given the irreversible inhibitory pharmacology of ibrutinib, it was of interest to study the duration of SPT and BAT suppression. Most SPT areas returned to within 80% of baseline diameter within 1 week after the cessation of treatment, though some SPT areas took up to 3 weeks after treatment to return to baseline size. In contrast, IgE-mediated BAT responses began to recover within days after cessation of ibrutinib treatment, and all were back to baseline within 1 week for all subjects, suggesting that ibrutinib’s suppressive effects last longer in mast cells than in basophils in vivo. Because the lifespan of tissue mast cells is long compared with that of basophils, ibrutinib’s varied duration of action is likely due to differing rates of BTK turnover and de novo synthesis in mast cells and basophils. Whether ibrutinib’s inhibitory effects will last for similar durations on mast cells in other organs besides the skin is unknown.

Safety monitoring studies were determined by potential adverse events previously reported in cancer studies. Subject 2 experienced transient mild nausea on days 1 and 3 of the ibrutinib course but did not experience any adverse effects on any other day. No other subjects experienced any adverse events or illness during the study. Safety monitoring in all 6 subjects showed no significant changes in quantitative levels of IgE, IgG, IgA, or IgM (P = .31, .22, .23, and .11, respectively; see Fig E3 in this article’s Online Repository at www.jacionline.org); white blood cell count; hematocrit; platelet count; absolute numbers of circulating neutrophils, monocytes, eosinophils, or basophils; kidney function; or liver function (see Table E2 in this article’s Online Repository at www.jacionline.org). Absolute lymphocyte count was increased on days 2 (P = .02) and 7 (P = .03) of ibrutinib treatment compared with baseline, but not on other days, and total bilirubin was increased (but remained within normal ranges) on days 2 (P = .03) and 4 (P = .02) only (see Fig E4 in this article’s Online Repository at www.jacionline.org) and was entirely reversible after cessation of treatment. Therefore, the clinical significance of these changes is likely minimal, because both these parameters were reversible and always remained within the normal range for adults. No changes on electrocardiogram were detected (data not shown). To date, no previous studies have profiled the potential side effects or toxicity of ibrutinib in healthy patients without lymphoproliferative disorders, nor have any previous studies investigated the safety of courses of ibrutinib as short as 2 to 7 days. Our study is the first to show that short-term ibrutinib treatment in healthy adults without cancer appears to be well tolerated. Other ongoing studies with chronic use of next-generation BTK inhibitors are exploring safety in normal subjects and both safety and efficacy in those with rheumatoid arthritis.

To our knowledge, no other known therapies have the capability to effectively reduce both mast cell and basophil activation. Emerging evidence suggests that both mast cells and basophils are involved in anaphylaxis pathogenesis⁷; therefore, an optimal prevention strategy should inhibit both. Although antihistamines can inhibit SPT to allergens, they target only 1 mediator and cannot prevent anaphylaxis, presumably because additional mast cell and basophil mediators other than histamine (such as platelet-activating factor) are involved, especially for more serious complications such as hypotension and shock.⁸ Omalizumab has been shown to increase the threshold dose of allergen consumption in food-allergic subjects, but it takes weeks to months of omalizumab therapy to gain efficacy.⁹ Because we have shown here that BTK inhibitors such as ibrutinib can be used for rapid inhibition of IgE-dependent activation of both mast cells and basophils, additional studies will soon be underway to determine its ability to increase the threshold dose of allergen in food-allergic subjects. Notably, ibrutinib has inhibitory activity on several kinases other than BTK, including many in the FcεRI pathway such as IL-2–inducible T-cell kinase, proto-oncogene tyrosine-protein kinase Fyn, and tyrosine-protein kinase Lyn, and studies with human basophils show that ibrutinib inhibits multiple secretion pathways downstream of FcεRI, not just histamine release.³ The relative contribution of these kinases to ibrutinib’s mechanism of action in vivo is unknown. Future studies may help to answer this by comparing ibrutinib to the more selective BTK inhibitors in development. We have previously shown that acalabrutinib can inhibit IgE-mediated BAT responses in vitro with an IC₅₀ of 105 nM.⁶ Therefore, it seems that ibrutinib’s off-target effects are not necessary for its action on basophils.

Limitations of our study include a low number of subjects (n = 6), which was partially mitigated by the fact that most subjects were allergic to multiple foods, and so we were able to perform skin testing to all clinically relevant nuts (n = 25) for each subject. Even though SPTs and BAT responses were maximally suppressed after 2 doses, whether a single dose of ibrutinib would have the same effects was not determined. In addition, for most subjects, the first time point examined after stopping drug was at 1 week, by which time the vast majority of SPT responses had returned to baseline. Whether this loss of protection waned within 7 days after discontinuation cannot be ascertained. Finally, SPTs and BAT are imperfect surrogate indicators of clinical reactivity to foods. Future studies will need to use oral food challenges, the gold standard to test for clinical reactivity, to see whether ibrutinib can alter allergic food reactions and potentially prevent anaphylaxis.

In conclusion, we have demonstrated that short-term ibrutinib therapy (as few as 2 doses) is well tolerated and can eliminate or drastically reduce mast cell and basophil reactivity to food allergens. No other known therapies are capable of inducing such a significant reduction of both mast cell and basophil activation. Episodic use of a BTK inhibitor deserves further evaluation for its potential to reduce the risk and severity of anaphylactic reactions, but it is premature to consider the use of ibrutinib as a chronic treatment for any allergic disease.

Special thanks to Angela Baumgartner, Senior Regulatory Coordinator, Center for Clinical Research for Northwestern Medicine, and the staff in the Lurie Children’s Hospital Clinical Research Unit, as well as to Dr Alfred Rademaker at Northwestern University for statistical advice. This study is listed on ClinicalTrials.gov under identifier NCT03149315.

METHODS

Subject screening and enrollment

After obtaining an investigational new drug approval from the Food and Drug Administration for off-label use of ibrutinib, joint institutional review board approval was obtained through the Northwestern Memorial Hospital and Ann & Robert H. Lurie Children’s Hospital of Chicago (herein referred to as Lurie Children’s). Subjects older than 18 years and allergic to peanuts and tree nuts were recruited through Northwestern Memorial Hospital and Lurie Children’s allergy clinics. Inclusion criteria were as follows: symptoms consistent with an IgE-mediated systemic reaction (urticaria, angioedema, wheezing, gastrointestinal symptoms, and/or hypotension) within 2 hours of ingestion of peanut and/or tree nuts plus confirmation of IgE-mediated sensitivity via the presence of serum food-specific IgE (>0.35 kU/L) and/or positive SPT result (wheal diameter ≥ 3 mm). At screening, data were collected on subjects’ previous allergic reactions to peanut and/or tree nuts, previous food allergy testing, medical history, and current medications. Subjects were excluded if they had taken antihistamines within the past 5 days or systemic immunosuppressive agents within the last 4 weeks, although use of intranasal and/or inhaled corticosteroids was allowed; history of severe anaphylaxis resulting in intubation, prolonged hypotension, or neurological sequalae; idiopathic urticaria, angioedema, and/or anaphylaxis; eosinophilic esophagitis or other non–IgE-mediated adverse reaction to foods; immunodeficiency or history of recurrent and/or severe infections; current cardiac arrhythmia; congestive heart failure; current use of anticoagulants or antiplatelet agents; history of bleeding disorder, low white blood cell, neutrophil, or platelet counts; or abnormal liver or kidney function at the baseline visit. Pregnant and breast-feeding subjects were also excluded, and female subjects of child-bearing age were required to use at least 2 forms ofbirth control while taking ibrutinib and for at least 1 month after cessation of treatment. Subjects with asthma were required to have good asthma control at the time of skin testing. All study visits were performed in the Lurie Children’s Clinical Research Unit.

Ibrutinib treatment

After screening, subjects took ibrutinib 420 mg orally (three 140-mg capsules) once daily each morning for at least 2 days, and the drug was continued until either all skin test results were reduced by at least 80% in diameter or for a maximum of 7 days of treatment. Return visits were standardized to occur 4 to 6 hours after each morning’s ibrutinib dose. Safety and other outcomes were monitored at each visit using review of systems, physical examinations, complete blood cell counts with differentials, complete metabolic profiles, quantitative immunoglobulin levels, and serum-specific IgE (Phadia Immunocap) to relevant foods. In addition, electrocardiograms were performed at baseline and again after ibrutinib treatment.

Skin prick testing

SPTs to clinically relevant foods were performed at baseline and repeated on days 2, 4, and 7 of ibrutinib treatment, and then at 1-week intervals after cessation of ibrutinib treatment until SPT areas returned to within 80% of baseline diameter. All skin tests were performed by the same investigator (M.C.D.) on the volar aspect of the forearm using Quintip devices and commercially purchased extracts for peanut, almond, brazil nut, cashew, hazelnut/filbert, pecan, pistachio, and English walnut (all purchased from Greer [Lenoir, NC] except for pistachio, which was purchased from ALK-Abello [Round Rock, Tex]). Extracts for macadamia and pine nut were produced in the allergy research lab at Northwestern. Controls included histamine (ALK-Abello) and saline (Greer). Skin test results were measured 15 minutes after prick, and the area (πr²) was calculated on the basis of the largest diameter of the wheal and flare. Skin test results were considered positive if the wheal was greater than 3 mm in diameter, and only positive skin test results at baseline were repeated at subsequent visits. Skin test sites were rotated between arms at subsequent visits.

Basophil activation testing

Peripheral blood samples obtained at each visit were activated with anti-FcεRI mAb and N-formyl-L-methionyl-L-leucyl-phenylalanine ex vivo and then stained using FlowCAST Basophil Activation Test kits (Bühlmann Laboratories, Schönenbuch, Switzerland) before analysis on a CytoFLEX flow cytometer (Beckman Coulter, Indianapolis, Ind). After gating on side scatter and CCR3 expression, basophil surface CD63 expression was used as a marker for activation.

Statistics

Values for grouped data were reported as mean ± SD and were calculated using Prism 7 software (GraphPad Software, La Jolla, Calif), which was also used for generating figures and for statistical analyses. Changes in skin test area (wheal and flare) were analyzed using Friedman tests for grouped analysis and Wilcoxon signed rank tests between pairs. Changes in BAT and clinical laboratory values were analyzed using repeated-measures ANOVA for grouped analysis and paired Student t tests between pairs. A P value of less than .05 was considered significant.

RESULTS

Patient demographic characteristics

The patients’ average age was 32.8 ± 12.5 years, and 50% were male. Five of 6 subjects were allergic to multiple nuts; consequently, SPTs were performed to all nuts that were clinically relevant. The average number of SPTs performed per subject at each visit was 4.2 ± 2.7. The most common nuts tested were peanut, cashew, and hazelnut (n = 4 for each), followed by almond and pistachio (n = 3), macadamia and pine nut (n = 2), and finally brazil nut, pecan, and walnut (n = 1). Four of 6 subjects had a history of other atopic diseases, such as allergic rhinitis, asthma or exercise-induced bronchospasm, or allergy to other foods such as sesame or shellfish. All subjects had positive SPT results at baseline to clinically relevant foods. All saline control SPT results were negative without wheals at all time points.