Ibrutinib is an irreversible molecular inhibitor of ITK, displaying BTK-independent antileukemic potential. (A) A graphical depiction of the sequence and domain homology between ITK and BTK. The relevant phosphorylation sites as well as ibrutinib irreversible covalent binding sites are labeled. (B) In silico representation of docked ibrutinib within the active site of crystallized ITK (top panel) (Protein Data Bank code 3QGW) or BTK (bottom panel) showing close approximation of Cys442 or Cys481 to reactive moiety of ibrutinib. Shape and chemical complementarity of ibrutinib are shown in surface representation. (C) A molecular probe assay was used to calculate the percent irreversible occupancy of total ITK in Jurkat whole-cell lysates irreversibly bound by ibrutinib. Error bars represent standard error of the mean (SEM). (D) A molecular probe assay was used to calculate the percent irreversible occupancy of ITK by ibrutinib in cryopreserved PBMCs obtained from patients immediately prior to (predose) and 8 days into (ibrutinib) daily oral ibrutinib therapy for CLL (n = 8). Error bars represent SEM. (E) Primary CD4 T cells isolated from healthy donors were pretreated with ibrutinib (1 µM) or vehicle and subjected to stimulation with anti-CD3, anti-CD28, or anti-CD3/anti-CD28 for 6 hours and analyzed via fluorescence-activated cell sorter for CD69 surface expression. Baseline (unstimulated) CD69 percentage was subtracted and data are represented in log percent CD69⁺CD4⁺ T cells. A 2-tailed paired Student t test was used for statistical analysis (nonsignificant [ns] = P > .05). Error bars represent SEM.