Plenary Paper
`
`LYMPHOID NEOPLASIA
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Ibrutinib is an irreversible molecular inhibitor of ITK driving a
`Th1-selective pressure in T lymphocytes
`Jason A. Dubovsky,1 Kyle A. Beckwith,1,2 Gayathri Natarajan,3 Jennifer A. Woyach,1 Samantha Jaglowski,1 Yiming Zhong,1
`Joshua D. Hessler,1 Ta-Ming Liu,1 Betty Y. Chang,4 Karilyn M. Larkin,1 Matthew R. Stefanovski,1 Danielle L. Chappell,1
`Frank W. Frissora,1 Lisa L. Smith,1 Kelly A. Smucker,1 Joseph M. Flynn,1 Jeffrey A. Jones,1 Leslie A. Andritsos,1
`Kami Maddocks,1 Amy M. Lehman,5 Richard Furman,6 Jeff Sharman,7 Anjali Mishra,1 Michael A. Caligiuri,1
`Abhay R. Satoskar,8 Joseph J. Buggy,4 Natarajan Muthusamy,1 Amy J. Johnson,1,9 and John C. Byrd1,9
`
`1Department of Internal Medicine, Division of Hematology, 2Medical Scientist Training Program, and 3Department of Microbiology, The Ohio State
`University, Columbus, OH; 4Pharmacyclics, Inc., Sunnyvale, CA; 5Center for Biostatistics, The Ohio State University, Columbus, OH; 6Department of
`Medicine, Division of Hematology-Oncology, Weill Cornell Medical College, New York, NY; 7Willamette Valley Cancer Institute/US Oncology, Springfield,
`OR; and 8Department of Pathology, and 9Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH
`
`Key Points
`
`Ibrutinib is the first clinically
`viable irreversible ITK
`inhibitor.
`Ibrutinib inhibits the formation
`of Th2 but not Th1 immunity.
`
`•
`
`•
`
`Given its critical role in T-cell signaling, interleukin-2–inducible kinase (ITK) is an ap-
`pealing 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. (Blood.
`2013;122(15):2539-2549)
`
`Introduction
`
`The interplay between antigen-presenting cells and T lymphocytes
`forms an indispensable component of adaptive immunity, yet certain
`neoplastic, autoimmune, parasitic, and infectious diseases subvert
`adaptive immunity by specifically misdirecting helper T-cell polar-
`ity.1,2 A common mechanism of immune subversion is the aberrant
`recruitment of a Th2-dominant response that promotes B-cell antibody
`production and interferes with direct effector cell cytotoxicity. In
`contrast, a Th1-dominant response evokes cytotoxic effects with the
`production of interferon g (IFN-g) and interleukin 2 (IL-2), which
`contribute to effector-cell–based immune surveillance. Clearance of
`certain intracellular bacterial pathogens such as Listeria and parasites
`such as Leishmania, as well as tumor immune surveillance, hinge
`upon the capacity to elicit robust Th1 and CD8 T-cell responses.
`IL-2–inducible kinase (ITK) is a T-cell–dominant member of the
`TEC-kinase family that drives proximal T-cell receptor (TCR)
`signaling.3 Upon TCR ligation in Th1 and CD8 T cells, ITK and re-
`dundant resting lymphocyte kinase (RLK or TXK) activate phospho-
`lipase Cg (PLCg), launching a signaling cascade that includes the
`
`nuclear factor of activated T cells (NFAT), nuclear factor kB, and
`mitogen-activated protein kinase pathways, resulting in cellular
`activation, cytokine release, and rapid proliferation.4 In cancer, ITK
`is a critical signaling motif important to acute lymphoblastic T-cell
`leukemia and S´ezary syndrome/cutaneous T-cell lymphoma due
`to aberrant activation and heightened expression.5 In healthy Th1-
`polarized and CD8 effector cells, ITK plays a supportive yet
`dispensable role to RLK. However, the epigenetic evolution of Th2
`cells conserves a singular dominant role for ITK, pinning it as the
`Achilles’ heel of Th2 T cells.6-9
`Clinically applicable ITK inhibitors are sought by the medical
`community given their potential to inhibit a number of Th2-
`dominant autoimmune, inflammatory, and infectious diseases ranging
`from cancer immunosuppression and atopic dermatitis to inflam-
`matory bowel disease and even HIV/AIDS.10,11 Moreover, a viable
`ITK inhibitor would be a promising therapeutic advancement for
`many T-cell malignancies that are currently difficult to man-
`age.12,13 Although multiple chemical analogs have been reported,
`
`Submitted June 10, 2013; accepted July 18, 2013. Prepublished online as
`Blood First Edition paper, July 25, 2013; DOI 10.1182/blood-2013-06-507947.
`
`N.M., A.J.J., and J.C.B. contributed equally to this study.
`
`Presented in part at the 54th annual meeting of the American Society of
`Hematology, Atlanta, GA, December 9, 2012.
`
`There is an Inside Blood commentary on this article in this issue.
`
`The publication costs of this article were defrayed in part by page charge
`payment. Therefore, and solely to indicate this fact, this article is hereby
`marked “advertisement” in accordance with 18 USC section 1734.
`
`The online version of this article contains a data supplement.
`
`© 2013 by The American Society of Hematology
`
`BLOOD, 10 OCTOBER 2013 x VOLUME 122, NUMBER 15
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`2539
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`2540
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`DUBOVSKY et al
`
`BLOOD, 10 OCTOBER 2013 x VOLUME 122, NUMBER 15
`
`no ITK inhibitors have successfully transitioned into clinical
`trials.14
`Ibrutinib is an irreversible inhibitor of Bruton tyrosine kinase
`(BTK) that blocks downstream B-cell receptor activation.15,16
`Numerous in vitro and in vivo studies confirm the specific activity
`of ibrutinib against BTK-restricted targets.17,18 Ibrutinib has dem-
`onstrated clinical activity in phase 1 and 2 clinical trials, with
`durable remissions against a variety of B-cell malignancies including
`mantle cell lymphoma, follicular lymphoma, and chronic lympho-
`cytic leukemia (CLL).19-22 Intriguingly, ITK shares significant
`sequence and functional homology with BTK and both contain an
`ibrutinib inhibition motif consisting of an SH3 autophosphorylatable
`tyrosine (Tyr) and a covalent binding cysteine (Cys) residue within the
`hinge region connecting the C and N lobes of the active site.23 ITK had
`previously been discounted as a relevant target of ibrutinib given a lack
`of sufficient in vitro evidence; not long after, however, Herman et al
`noted effects on T-cell cytokine production, reigniting the inquiry.16,18
`The striking homology between BTK and ITK combined with
`intriguing in silico docking led to the hypothesis that ibrutinib is the
`first clinically viable ITK inhibitor. This was explored using healthy
`human T cells and human and murine CLL as a model system of
`dysregulated Th2-biased immunosuppression. In CLL, an increas-
`ingly defective immune synapse enables malignant B cells to evade
`immune detection by inducing T-cell anergy as well as improper Th2
`polarization.24,25 In addition to being incapable of responding to
`environmental pathogens, these improperly polarized T cells con-
`tribute both cytokine and direct signaling support to malignant
`B cells.26,27 The end result of this immunosuppression is a high
`incidence of severe infections, which is the leading cause of patient
`mortality.28,29
`Our molecular analysis confirms that ibrutinib irreversibly binds
`ITK and inhibits activation of Th2 cells after TCR stimulation. This
`inhibition is specific to Th2-polarized CD4 T cells, because RLK
`remains functional, thus providing a compensatory platform for
`activation of Th1 and CD8 T cells. These data demonstrate that CD4
`T-cell populations isolated from CLL patients are skewed toward a
`Th1 profile after exposure to ibrutinib. Findings were validated using
`mouse models of leukemia, cutaneous leishmaniasis, and Listeria
`monocytogenes infection. Ibrutinib’s immunomodulatory activity and
`ITK inhibition in humans were confirmed using irreversible ITK
`binding, cytokine, and T-cell signaling analysis from CLL patients
`treated with ibrutinib in 2 separate clinical trials. Together, these
`results confirm that ibrutinib is the first potent and irreversible
`inhibitor of ITK to achieve clinical viability, potentially repurposing
`the drug for a multitude of novel therapeutic applications.
`
`Methods
`
`Subject populations
`
`Sera and peripheral blood mononuclear cells (PBMCs) were obtained from
`normal donors or patients with CLL in accordance with the Declaration of
`Helsinki. All subjects gave written informed consent for their blood products
`to be used for research under an institutional review board–approved
`protocol. Blood was collected at The Ohio State University Wexner Medical
`Center (Columbus, OH). For additional information, see supplemental
`Methods.
`
`Cell culture, drug treatments, and T-cell polarization
`
`Primary T cells were isolated using RosetteSep or EasySep T-cell enrichment
`kits (STEMCELL Technologies, Vancouver, BC, Canada). Cells were pretreated
`
`for 30 minutes with ibrutinib, washed 2 times, then stimulated with plate-
`bound anti-CD3 and soluble anti-CD28 (eBiosciences, San Diego, CA). For
`additional details, see supplemental Methods.
`
`Calcium flux analysis
`
`Jurkat cells were stained with Fluo4-AM (Invitrogen), washed twice, and
`resuspended in phenol-red free RPMI. Fluo4 fluorescence was measured
`using a plate reader at 535 nm. For additional information, see supplemental
`Methods.
`
`Flow cytometry and cytokine bead array
`
`Flow-cytometric analysis was performed using fluorochrome-labeled anti-
`bodies using conventional methods. For specific antibodies and experimental
`design, see supplemental Methods.
`
`Ibrutinib probe assay
`
`Protein lysates were labeled with a biotinylated derivative of ibrutinib and
`added to a Streptavidin-coated plate, washed 3 times, and incubated with
`mouse anti-ITK. After washing with SULFO-TAG–conjugated anti-mouse
`antibody (MSD, catalog #R32AC-5), lysates were read on an SI2400. For
`additional details, see supplemental Methods.
`
`Immunoblot analysis
`
`Experiments were conducted using conventional sodium dodecyl sulfate
`polyacrylamide gel electrophoresis methodology. For specific antibodies
`and densitometry, see supplemental Methods.
`
`Confocal immunofluorescence microscopy
`
`Cells were centrifugally concentrated on microscope slides and stained with
`monoclonal antibodies. Images were taken using a 360 objective and 34 digital
`zoom with Olympus Fluoview 1000 laser scanning confocal microscope at The
`Ohio State University Microscopy and Imaging Facility. See supplemental
`Methods for details.
`
`Mouse models
`
`All animal procedures were performed in accordance with Federal and
`Institutional Animal Care and Use Committee requirements. Detailed
`methods for mouse models are provided in supplemental Methods.
`
`ELISA
`
`An enzyme-linked immunoabsorbent assay (ELISA) assay was performed
`for each immunoglobulin G (IgG) subisotype using a clonotyping system
`(B6/C57J-AP-5300-04B; Southern Biotech, Birmingham, AL) according to
`the manufacturer’s instructions. For additional details, see supplemental
`Methods.
`
`Statistics
`
`Unless otherwise noted, a 2-tailed Student t test was used for normal data
`at equal variance. Significance was considered for P , .05. For detailed
`statistics, see supplemental Methods.
`
`Results
`
`Ibrutinib is an irreversible inhibitor of ITK
`
`As a Th2-critical TEC family kinase, ITK retains significant struc-
`tural and functional homology to ibrutinib’s known irreversible
`target BTK, including a Cys442 putative covalent binding moiety
`located within the hinge region of the active site and an auto-
`phosphorylatable Tyr180 in the SH3 domain (Figure 1A). In silico
`
`

`

`BLOOD, 10 OCTOBER 2013 x VOLUME 122, NUMBER 15
`
`IBRUTINIB IS AN IMMUNOMODULATORY ITK INHIBITOR
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`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 5 8). Error bars represent SEM. (E) Primary CD4 T cells isolated from healthy donors were pretreated with ibrutinib
`(1 mM) 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
`1
`1
`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] 5 P . .05). Error bars represent SEM.
`
`docking studies showed potential covalent binding of ITK at
`Cys442 and occupancy of the active site similar to that achieved
`when ibrutinib irreversibly binds BTK (Figure 1B). In vitro probe
`binding assays confirmed that ibrutinib was capable of irreversibly
`binding a significant percentage of endogenous ITK in the Jurkat
`T-cell leukemia cell line (Figure 1C).
`To confirm that ibrutinib irreversibly binds ITK in vivo, we
`conducted an ITK probe assay on PBMC samples obtained from
`CLL patients in a phase 2 clinical trial of ibrutinib. Samples were
`tested immediately prior to receiving ibrutinib and after 8 days of
`daily oral administration (420 mg/day). The data revealed between
`40% and 80% of ITK is covalently bound by ibrutinib, similar
`to that achieved in vitro (Figure 1D). Multimerization is an es-
`tablished ITK regulatory mechanism that sequesters inactive
`ITK within the cytoplasm, potentially blocking complete ibrutinib
`occupancy of ITK. To explore this, we disrupted the cytoplasmic
`architecture and observed near-complete occupancy of the ITK
`active site at drug concentrations nearing 0.3 mM (supplemental
`Figure 1).
`In their initial description of ibrutinib, Honigberg et al did not find
`inhibition of the T-cell activation marker CD69 after stimulation with
`
`anti-CD3 and anti-CD28.18 Based upon this evidence, a T-cell–
`specific target had been ruled out. One reason for these divergent
`results could be the fact that CD28 costimulation alone can induce
`CD69 surface expression in a TCR- and ITK-independent fashion.
`To explore this possibility, we examined the CD69 activation marker
`in CD4 T-cells isolated from healthy donors and stimulated with anti-
`CD3, anti-CD28, or anti-CD3/anti-CD28 (Figure 1E).30 Ibrutinib
`significantly attenuated anti-CD3–induced surface expression of
`CD69. However, we did not observe any significant alteration
`to CD69 in CD4 T cells stimulated via CD28 or via CD3/CD28,
`indicating that CD28 costimulation provides an additional non-
`inhibited pathway that elicits surface presentation of CD69.
`
`Ibrutinib inhibits ITK signaling and molecular characteristics of
`TCR-induced activation in primary CD4 T cells and Jurkat cells
`
`T-cell activation is predicated upon robust downstream nuclear
`factor kB, mitogen-activated protein kinase, and NFAT signaling;
`therefore, components of each pathway were examined to determine
`the T-cell–specific effects of ibrutinib. Ibrutinib treatment yielded
`a dose-dependent inhibition of ITK autophosphorylation at Y180
`
`

`

`2542
`
`DUBOVSKY et al
`
`BLOOD, 10 OCTOBER 2013 x VOLUME 122, NUMBER 15
`
`resulting in downstream inactivation of IkBa, JunB, and NFAT
`signaling in both primary CD4 and Jurkat T cells (Figure 2A-B;
`supplemental Figure 2). Notably, inhibition of both JunB and
`STAT6 was observed, both of which are critical components of the
`IL-4 pathway.31,32 Although JAK3 inhibition could explain some
`of our in vitro data, our target validation studies demonstrate that
`ibrutinib does not directly influence this kinase in cell-based assays
`(supplemental Figure 3).
`To confirm that TCR-induced activation events preceding
`ITK autophosphorylation were not altered, we examined the
`proximal pathway components using both primary CD4 and
`Jurkat T cells. Immunoblot data revealed that upstream phos-
`phorylation of LCK, ZAP70, and LAT remain unchanged (Figure 2C;
`supplemental Figure 4). Furthermore, we used the PKC activator,
`phorbol 12-myristate 13-acetate, and the calcium ionophore,
`ionomycin, to confirm that distal elements of TCR activation,
`including NFAT activity and IkBa phosphorylation, were engaged
`regardless of ibrutinib treatment in Jurkat cells (Figure 2D).
`To functionally confirm our molecular data set, we examined
`NFAT nuclear translocation via confocal microscopy. As expected,
`NFAT nuclear translocation was inhibited by ibrutinib (Figure 2E-F).
`Remnant populations of activated CD4 T cells were observed, indi-
`cating that not all CD4 T cells were inhibited. We also confirmed that
`TCR-induced proliferation as well as na¨ıve, central, effector, and
`terminal memory subsets were unaffected by in vitro ibrutinib
`treatment (supplemental Figures 5 and 6).
`We sought to confirm functional ITK inhibition in patients
`receiving oral ibrutinib. Because PLCg1 is directly phosphory-
`lated at Tyr783 by active ITK, we conducted pPLCg1-Tyr783
`phosphoflow analysis on CD3/CD28-stimulated CD4 T cells
`collected from CLL patients receiving ibrutinib as part of a
`phase 2 clinical trial. Results reveal a significant decrease in
`TCR-induced pPLCg1 activation, confirming inhibition of CD4
`T-cell ITK signaling in these patients (Figure 2G; supplemental
`Figure 7).
`It has been demonstrated in mice that loss of ITK attenuates,
`yet does not ablate, intracellular calcium flux in response to TCR
`signaling.33,34 Ibrutinib treatment of Jurkat cells yielded similar
`results (Figure 2H-I), demonstrating that ibrutinib-based ITK inhi-
`bition significantly reduces intracellular calcium flux in response to
`TCR stimulation.
`
`Ibrutinib-induced ITK-C442–irreversible inhibition provides
`a selective advantage to RLK-expressing Th1 and CD8 T cells
`
`To confirm that the primary molecular target of ibrutinib in CD4
`T cells was ITK, TCR-induced activation of NFAT, pSTAT6,
`pIkBa, and JunB was evaluated in primary CD4 T cells pretreated
`with ibrutinib or 1 of 2 alternative BTK inhibitors, AVL-292 and
`PCI-45292, which do not target ITK (50% inhibition/inhibitory
`concentration . 22.5 nM) (Figure 3A; supplemental Figures
`8-13). Only ibrutinib (ITK 50% inhibition/inhibitory concentra-
`tion 5 2.2 nM) was capable of inhibiting TCR downstream molecular
`activation.
`Ibrutinib presumably requires a cysteine residue within the
`hinge region to form an irreversible covalent bond and inhibit a
`kinase target. In ITK, that cysteine moiety resides at amino acid
`442. Therefore, as molecular confirmation that ITK is the sole
`irreversible target in CD4 T cells, a stably transduced Jurkat line
`was generated with ITK-C442A, a version of ITK that lacks the
`putative covalent binding site for ibrutinib (supplemental Figure 14).
`The ITK-C442A Jurkat line maintained NFAT activation to drug
`
`concentrations exceeding 8 mM, whereas the parental line and
`Jurkat cells transfected with wild-type ITK were inhibited at 2 to
`4 mM (Figure 3B). These data were confirmed by intracellular
`calcium flux showing that
`the ibrutinib-resistant ITK-C442A
`Jurkat line significantly and completely restored calcium response
`(Figure 3C; supplemental Figure 15).
`A key reason why ITK inhibitors retain clinical interest is the
`potential to selectively inhibit Th2 T cells, which do not contain the
`compensatory RLK kinase. To elucidate the differential inhibition of
`Th2-polarized T cells in relation to Th1, na¨ıve CD4 T cells were
`polarized in vitro to obtain enriched cultures of IFN-g–producing
`Th1 cells and IL-4–producing Th2 cells (Figure 3D). In contrast to
`Th1, Th2 cultures were sensitive to pharmacologically relevant
`levels of ibrutinib as demonstrated by reduced IL-4 production
`(Figure 3E). Additionally, ibrutinib inhibited NFAT and IkBa
`activation in Th2 T cells, whereas Th1-polarized CD4 and CD8
`T cells were resistant (Figure 3F).
`In Th1 CD4 and CD8 T cells, RLK plays a redundant role to ITK;
`however, neither Th2 polarized CD4 T cells nor Jurkat cells express
`RLK.35 To test the hypothesis that RLK expression protects Th1
`T cells from ibrutinib inhibition, Jurkat cells stably transduced to
`express RLK were tested (supplemental Figure 16). TCR downstream
`activation of NFAT was protected in the Jurkat-RLK cell line,
`whereas both the parental and empty vector stable transfectants
`were susceptible to ibrutinib inhibition (Figure 3G). Confirmatory
`intracellular calcium release experiments demonstrate a significant
`restoration of calcium flux in Jurkat cells stably expressing RLK
`(Figure 3H; supplemental Figure 17). This result demonstrates that
`RLK can compensate for ibrutinib-inhibited ITK, thereby pro-
`viding an alternate activation platform for specific RLK-expressing
`T-cell subsets.
`
`Ibrutinib selectively limits Th2 activation, thereby initiating
`a Th1-selective pressure in a mixed population of CD4 T cells in
`vitro, in vivo, and in human CLL patients receiving ibrutinib
`
`To evaluate the effects of ibrutinib on the Th1/Th2 polarization
`of a CD4 T-cell population over time, CD4 T cells isolated from
`healthy donors were cultured for 3 days following ibrutinib pre-
`treatment. Outgrowth of IFN-g–positive T cells was confirmed
`by intracellular staining analysis (Figure 4A). This outgrowth
`correlated with a decrease in the Th2-dominant transcription factor
`JunB and an increase in the Th1-specific transcription factor T-bet
`(Figure 4B).
`To confirm the functional relevance of these results in the setting
`of CLL, intracellular staining was performed for IFN-g and IL-4 in
`ibrutinib-treated, TCR-stimulated CD4 T-cell cultures purified from
`CLL patients not previously treated with ibrutinib. Following stim-
`ulation, a significant decrease was identified in the IL-4–producing
`Th2 population of CD4 T cells, whereas IFN-g–producing Th1 cells
`were largely unaffected (Figure 4C). These results very closely
`matched data obtained on primary CD4 T cells from healthy donors
`(supplemental Figure 18). These data confirm that a significant
`divergence of the 2 populations can be achieved in a purified T-cell
`culture at ibrutinib doses ranging from 0.1 to 1 mM. This dose range
`is consistent with serum concentrations observed in vivo during
`pharmacokinetic studies of ibrutinib in both mouse and human
`trials.18,36
`To validate these findings in human patients, serial serum cytokine
`levels were investigated in relapsed or refractory CLL patients
`receiving ibrutinib as part of a phase 1 study. The data demonstrated
`a decrease in serum Th2-type cytokines, including IL-10, IL-4, and
`
`

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`
`BLOOD, 10 OCTOBER 2013 x VOLUME 122, NUMBER 15
`
`IBRUTINIB IS AN IMMUNOMODULATORY ITK INHIBITOR
`
`2543
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`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 CD41 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, pIkBa-S32/36, total IkBa, JunB, and actin. Densitometry analysis normalized to dimethylsulfoxide (DMSO)-treated (0 mM) sample. (B) Immunoblot analysis of freshly isolated
`1
`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 mM) sample. (C) Immunoblot analysis of freshly isolated
`1
`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, pIkBa-S32/36, total IkBa, and actin. Densitometry analyses are normalized to the DMSO-treated (0 mM) 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 pIkBa-S32/36, total IkBa, and actin (cellular lysates). (E) Immunofluorescent microscopy of ibrutinib-pretreated, freshly
`1
`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 5 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 pPLCg1-Tyr783 in 1hr anti-CD3/anti-CD28–stimulated cryopreserved PBMCs obtained immediately predose or after 8 days of receiving ibrutinib therapy for
`1
`1
`2
`1
`CLL (n 5 11). A minimum of 400 000 events were collected. Graph displays the overall percent of live CD3
`pPLCg1
`CD4
`Tyr783
`events in each sample. Error bars represent
`SEM. (H) Calcium flux analysis of ibrutinib (n 5 8), vehicle (n 5 24), or BAPTA-AM (n 5 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.
`
`IL-13, from pretreatment to day 28 of ibrutinib therapy (Figure 4D).
`This was in sharp contrast to an increase in the Th1 cytokine IFN-g.
`To rule out the potential contribution of B cells to the observed Th1
`
`cytokines, we analyzed peripheral CD191 B-cell and CLL messenger
`RNA levels and found no such alteration in B-cell cytokine ex-
`pression (supplemental Figure 19).
`
`

`

`2544
`
`DUBOVSKY et al
`
`A
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`lbrutinib
`
`AVL-292
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`BLOOD, 10 OCTOBER 2013 x VOLUME 122, NUMBER 15
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`Figure 3. Ibrutinib irreversibly binds to ITK-C442 and RLK expression provides compensatory kinase activity, which protects Th1 and CD8 T cells. (A) Immunoblot
`1
`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, pIkBa-S32/36, total IkBa, 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-g (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-g)– 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