(A) HSC-engrafted humanized mice were sensitized and then pretreated with 2 doses of acalabrutinib (1.5 or 15 mg/kg via gavage) or vehicle control at 16 and 4 hours before challenge with 20 μg NP-BSA to elicit moderate PSA. Core temperature drop from baseline (left) and clinical scores (right) are shown as measures of clinical response during PSA. Body temperature measurements were ceased after death; therefore, only the surviving mice at each time point are included in averages. Data are pooled from 3 independent experiments; n = 6–9 total per group. (B and C) To investigate the duration of acalabrutinib’s protection, engrafted humanized mice were sensitized and pretreated with acalabrutinib as described in A, except that NP-BSA challenge was performed either 2 days (B) or 7 days (C) after the last oral dose of acalabrutinib. Data are pooled from 3 separate experiments; n = 11–17 total per group. (D) To investigate acalabrutinib’s ability to prevent fatal anaphylaxis, engrafted humanized mice were sensitized and pretreated with 15 mg/kg acalabrutinib or vehicle, except a higher challenge dose (500 μg NP-BSA) was given to elicit a more severe PSA response. Data shown are pooled from 6 separate experiments; N = 24–27 total per group. (E) The Kaplan-Meier survival curve from experiments in D is shown for both the acalabrutinib- and vehicle-treated groups. All data were analyzed using 2-way ANOVA with repeated measures with the exception of the mortality rate in E, which was analyzed using χ² analysis. All data are displayed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with PBS control group.