Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes

Abstract

Given its critical role in T-cell signaling, interleukin-2-inducible kinase (ITK) is an appealing therapeutic target that can contribute to the pathogenesis of certain infectious, autoimmune, and neoplastic diseases. Ablation of ITK subverts Th2 immunity, thereby potentiating Th1-based immune responses. While small-molecule ITK inhibitors have been identified, none have demonstrated clinical utility. Ibrutinib is a confirmed irreversible inhibitor of Bruton tyrosine kinase (BTK) with outstanding clinical activity and tolerability in B-cell malignancies. Significant homology between BTK and ITK alongside in silico docking studies support ibrutinib as an immunomodulatory inhibitor of both ITK and BTK. Our comprehensive molecular and phenotypic analysis confirms ITK as an irreversible T-cell target of ibrutinib. Using ibrutinib clinical trial samples along with well-characterized neoplastic (chronic lymphocytic leukemia), parasitic infection (Leishmania major), and infectious disease (Listeria monocytogenes) models, we establish ibrutinib as a clinically relevant and physiologically potent ITK inhibitor with broad therapeutic utility. This trial was registered at www.clinicaltrials.gov as #NCT01105247 and #NCT01217749.

Figures

Figure 1

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.

Figure 2

In T cells, ibrutinib specifically targets ITK, inhibiting TCR-induced cellular signaling and activation. (A) Immunoblot analysis of freshly isolated ibrutinib pretreated primary CD4+ cells from a healthy donor, anti-CD3/anti-CD28 stimulated (or unstimulated), whole-cell lysates. Blot probed for pITK-Y180, total ITK, pSTAT6-Y641, total STAT6, pIkBα-S32/36, total IkBα, JunB, and actin. Densitometry analysis normalized to dimethylsulfoxide (DMSO)-treated (0 µM) sample. (B) Immunoblot analysis of freshly isolated ibrutinib pretreated primary CD4+ cells from a healthy donor, anti-CD3/anti-CD28–stimulated (or unstimulated), cytoplasmic, and nuclear lysates. Blots probed for NFAT (and activated hyperdephosphorylated NFAT), Brg1, and actin. Densitometry analyses are normalized to the DMSO-treated (0 µM) sample. (C) Immunoblot analysis of freshly isolated ibrutinib-pretreated primary CD4+ cells from a healthy donor, anti-CD3/anti-CD28–stimulated (or unstimulated), whole-cell lysates. Blots were probed for pZAP70-Y319, total ZAP70, pLAT-Y191, total LAT, pLCK-Y505, total LCK, pIkBα-S32/36, total IkBα, and actin. Densitometry analyses are normalized to the DMSO-treated (0 µM) sample. (D) Nuclear or whole-cell lysate immunoblot analysis of Jurkat cells pretreated with ibrutinib and stimulated with either anti-CD3/anti-CD28 or phorbol 12-myristate 13-acetate/ionomycin for 45 minutes. Blots were probed with Brg1, NFAT1, and actin (nuclear lysates) or pIkBα-S32/36, total IkBα, and actin (cellular lysates). (E) Immunofluorescent microscopy of ibrutinib-pretreated, freshly isolated, primary CD4+ cells from healthy donors (panels A and B) were stimulated for 45 minutes with anti-CD3/anti-CD28 (or unstimulated), fixed, and stained for NFAT (green) and nuclei (4,6 diamidino-2-phenylindole [DAPI], blue). Activated cells are characterized by influx of NFAT into nuclear region (green overlay with blue = cyan) and are denoted by white arrows. (F) Percent relative NFAT1/DAPI colocalization derived from Pearson correlation analysis of 10 independent immunofluorescent microscopy fields (different donors than pictured in panel E and normalized to the average unstimulated value. Cyclosporin A (CSA) treatment was used as an additional negative control. Error bars represent SEM. (G) Phosphoflow analysis of pPLCγ1-Tyr783 in 1hr anti-CD3/anti-CD28–stimulated cryopreserved PBMCs obtained immediately predose or after 8 days of receiving ibrutinib therapy for CLL (n = 11). A minimum of 400 000 events were collected. Graph displays the overall percent of live CD3+CD4+pPLCγ1−Tyr783+ events in each sample. Error bars represent SEM. (H) Calcium flux analysis of ibrutinib (n = 8), vehicle (n = 24), or BAPTA-AM (n = 8) pretreated Jurkat cells after TCR stimulation by anti-CD3. Area under the curve (AUC) is presented for each dataset in the center. All data were normalized to baseline and BAPTA-treated fluorescent averages. Time points depicted on horizontal axis are relative to stimulation with anti-CD3. (I) AUC for calcium flux of various concentrations of ibrutinib. Each symbol indicates a single replicate experiment. Statistical analysis represented on graph is relative to DMSO treatment. PMA/Iono., phorbol 12-myristate 13-acetate and ionomycin; Unstim, unstimulated.

Figure 3

Ibrutinib irreversibly binds to ITK-C442 and RLK expression provides compensatory kinase activity, which protects Th1 and CD8 T cells. (A) Immunoblot analysis of 45-minute nuclear and 2-hour whole-cell extracts from ibrutinib or alternate BTK inhibitor–pretreated, freshly purified healthy donor primary CD4+ cells stimulated with anti-CD3/anti-CD28. Nuclear extracts were probed for NFAT1 and Brg1; whole-cell extracts were probed for pSTAT6-Y641, total STAT6, pIkBα-S32/36, total IkBα, JunB, and actin. (B) Immunoblot analysis of Jurkat parental, Jurkat-ITKwt, and Jurkat-ITKC442A nuclear lysates after ibrutinib pretreatment and anti-CD3/anti-CD28 stimulation. Blots were probed for NFAT1 and Brg1. (C) AUC for Fluo4-AM calcium release analysis of Jurkat-ITK and Jurkat-ITKC442A cell lines after pretreatment with ibrutinib or DMSO and stimulation with anti-CD3. Each symbol represents a single replicate experiment. Error bars represent SEM. (D) Cytokine analysis of IL-4 (black bars and right y-axis) and IFN-γ (open bars and left y-axis) media levels in anti-CD3/anti-CD28–stimulated Th1- and Th2-polarized cell cultures. These are the same cell cultures used in panels E and F. (E) Intracellular cytokine analysis of Th1(IFN-γ)– and Th2(IL-4)–polarized T-cell cultures pretreated with the indicated concentration of ibrutinib or DMSO and stimulated for 6 hours via anti-CD3/anti-CD28. Cytokine measurements were taken on separately cultured subsets of cells after 3 weeks of polarizing cell culture with weekly anti-CD3/anti-CD28 stimulation. Error bars represent SEM. (F) Th1-, Th2-, and CD8-purified primary cells were stimulated with anti-CD3/anti-CD28 after pretreatment with ibrutinib. Immunoblot analysis was conducted probing for NFAT and Brg1 as well as pIkBα-S32/36, total IkBα, and actin. (G) Immunoblot analysis of Jurkat parental, Jurkat-RLK, and Jurkat-EV (empty vector) nuclear lysates after ibrutinib pretreatment and anti-CD3/anti-CD28 stimulation. Blots were probed for NFAT1 and Brg1. (H) AUC for Fluo4-AM calcium release analysis of Jurkat-EV (empty vector) and Jurkat-RLK cell lines after pretreatment with ibrutinib or DMSO and stimulation with anti-CD3. Each symbol represents a single replicate experiment. Error bars represent SEM. Unstim, unstimulated.

Figure 4

Ibrutinib can limit Th2 activation, thereby selectively promoting Th1 expansion and skewing Th1/Th2 cytokines in human CLL patients and IgG subisotypes in TCL1 leukemic mice. (A) Intracellular staining analysis of IFN-γ in 5-day anti-CD3/anti-CD28–stimulated CD4+ T-cell cultures pretreated with ibrutinib or vehicle. Experiment was repeated 5 times; isotype staining control is provided. (B) Immunoblot analysis of JunB (top) and T-bet (bottom) levels in bulk CD4+ cultures pretreated with ibrutinib and anti-CD3/anti-CD28 stimulated (or unstimulated) for 3 days in vitro. Actin is used as loading control. Densitometry analyses are normalized to the DMSO-treated (0 µM) sample. (C) Normalized intracellular staining analysis of IL-4 (open bars n = 6) and IFN-γ (closed bars n = 9) CD4+ cells derived from CLL patients pretreated with ibrutinib and stimulated with anti-CD3/anti-CD28. Error bars represent SEM. (D) Percent relative alteration in plasma cytokine levels from pretreatment to day 28 of therapy in relapsed refractory CLL patients enrolled in a phase 1 trial of oral ibrutinib. (E) Plasma IgG1 (Th2) and IgG2c (Th1) subisotype analysis of C57BL/6 EµTCL1 mice at 8 months of age after 7 consecutive months of ibrutinib (25 mg/kg/day) (n = 12) or vehicle (n = 13) administration via drinking water. IFNG, interferon γ; Unstim, unstimulated.

Figure 5

Ibrutinib drives Th1-mediated L major immunity in an in vivo model of Th2-dominant cutaneous leishmaniasis. (A) Schematic representation of the L major mouse experiment time course. Mice were initiated on ibrutinib (25 mg/kg/day) or vehicle 2 days prior to being infected with 2 × 106 stationary-phase L major promastigotes. Lesion size was tracked for 9 weeks and immune correlates were collected upon sacrifice at week 9. (B) Lymphocytes isolated from draining lymph nodes were stimulated with L major antigens for 72 hours and culture supernatant was analyzed by ELISA for IL-4 and IL-10. Error bars represent SEM. (C) Lymphocytes isolated from draining lymph nodes were stimulated with L major antigens for 72hr and culture supernatant was analyzed by ELISA for IFN-γ. IFN-γ responses are displayed as a ratio with IL-4 (left panel) or IL-10 (right panel) to compare relative Th1 and Th2 immunity in ibrutinib- or vehicle-treated groups. (D) Whole-mount gross histologic preparations of vehicle- and ibrutinib-treated L major–infected footpads are depicted along with a centimeter ruler for size comparison. Cutaneous lesions are visible on the underside of the footpad as indicated by arrows. (E) Log dilution of parasites obtained from footpad lesions are displayed. Error bars represent SEM. (F) Time course analysis of cutaneous lesion size over the 9-week period of L major infection. Measurements were taken at weekly intervals. Error bars represent SEM.

Figure 6

Ibrutinib functionally restores immunity in a leukemia/listeriosis mouse model. (A) Schematic representation of the leukemia/listeriosis mouse experiment time course. Mice were engrafted via intravenous injection with leukemic cells purified from the spleen of a EµTCL1 transgenic animal. Engrafted mice were randomly divided between vehicle and ibrutinib (25 mg/kg/day) groups on day 7. IV L monocytogenes-OVA inoculation (5000 CFU) was conducted 14 days after engraftment. Rechallenge began 14 days after initial inoculation and consisted of a single 5000 CFU L monocytogenes OVA intravenous injection. Mice were sacrificed at day 32 and tissues were collected for memory cell analysis. (B) Time course analysis of OVA major histocompatibility complex I tetramer-positive peripheral CD8 T cells from leukemia/listeriosis mouse study. A total of 5000 CFU of OVA-expressing L monocytogenes was injected at day 0. Statistical analysis for day 8 mean is presented (**P = .0052; *′P = .0438 for repeat experiment). Error bars represent SEM. (C) Analysis of OVA-tetramer+ CD8+ T cells within the spleen of animals killed on day 32 of the leukemia/listeria infection experiment. Data are displayed as percentage of CD8+ T cells. Error bars represent SEM. (D) Analysis of CD8+CD62L+ central memory cells within the spleen of mice killed at day 32 of the leukemia/listeria infection experiment. Data are presented as the average of the total CD8+ population. Error bars represent SEM. (E) Analysis of CD4+CD62L+ central memory cells within the spleen of mice killed at day 32 of the leukemia/listeria infection experiment. Data are presented as the average of the total CD4+ population. Error bars represent SEM.