Purpose: Responses to therapy with chimeric antigen receptor T cells recognizing CD19 (CART19, CTL019) may vary by histology. Mantle cell lymphoma (MCL) represents a B-cell malignancy that remains incurable despite novel therapies such as the BTK inhibitor ibrutinib, and where data from CTL019 therapy are scant. Using MCL as a model, we sought to build upon the outcomes from CTL019 and from ibrutinib therapy by combining these in a rational manner.

Experimental Design: MCL cell lines and primary MCL samples were combined with autologous or normal donor-derived anti-CD19 CAR T cells along with ibrutinib. The effect of the combination was studied in vitro and in mouse xenograft models.

Results: MCL cells strongly activated multiple CTL019 effector functions, and MCL killing by CTL019 was further enhanced in the presence of ibrutinib. In a xenograft MCL model, we showed superior disease control in the CTL019- as compared with ibrutinib-treated mice (median survival not reached vs. 95 days, P < 0.005) but most mice receiving CTL019 monotherapy eventually relapsed. Therefore, we added ibrutinib to CTL019 and showed that 80% to 100% of mice in the CTL019 + ibrutinib arm and 0% to 20% of mice in the CTL019 arm, respectively, remained in long-term remission (P < 0.05).

Conclusions: Combining CTL019 with ibrutinib represents a rational way to incorporate two of the most recent therapies in MCL. Our findings pave the way to a two-pronged therapeutic strategy in patients with MCL and other types of B-cell lymphoma. Clin Cancer Res; 22(11); 2684–96. ©2016 AACR.

Translational Relevance

Most patients with relapsed mantle cell lymphoma can now be treated with the BTK inhibitor ibrutinib. However, up to 30% of these patients do not respond to ibrutinib and the majority of responders eventually relapse. Recent reports highlight potent activity of anti-CD19 chimeric antigen receptor T cells (CART19, CTL019) in B-cell malignancies. In this study, we illustrate for the first time that ibrutinib can be added to CTL019 and that only the combined approach leads to profound, durable responses in xenograft models of MCL. These findings set the stage for future clinical trials evaluating this combination in B-cell neoplasms.

Mantle cell lymphoma (MCL) accounts for up to 10% of all lymphomas (1) and typically presents in advanced stage (2). For most patients with MCL, the prognosis is poor with a median survival of 4 years (3). Currently, there is no curative treatment for MCL and, therefore, novel therapies for this type of lymphoma are urgently needed.

The B-cell receptor (BCR) complex is critical for antigen-induced activation of normal B lymphocytes and plays a key role in the pathogenesis of certain types of B-cell lymphoma. BCR engagement activates several kinases including LYN, SYK, and BTK (4,5). BTK recently gained particular attention, as the potent BTK inhibitor ibrutinib demonstrated therapeutic efficacy in several types of B-cell lymphoma including MCL (6–8). However, up to one third of MCL patients do not respond to ibrutinib and among the responders only a third achieve complete remission (CR). Furthermore, the therapy usually leads to drug resistance as the median duration of response is only 17.5 months with a 24-month PFS of 31% (8,9). The mechanisms of resistance are currently poorly understood but are thought to involve mutations in BTK that impair ibrutinib binding, or activating mutations of the enzyme PLCγ2 resulting in constitutive BTK-independent cell signaling (10,11). Furthermore, because blockade of BTK function is not directly cytotoxic, at least in some types of lymphoma (11), it may predispose to clonal evolution by conferring a selection pressure. Rationally designed combinations of ibrutinib with other antilymphoma modalities could potentially overcome this shortcoming and thereby improve patient outcomes.

Infusion of autologous T cells transduced with chimeric antigen receptors (CAR) against the B-cell–specific CD19 antigen (CART19, CTL019) leads to dramatic clinical responses in many patients with various types of B-cell neoplasms but CTL019 efficacy against MCL specifically has not yet been established (12–17). The presence of bulky masses may hinder T-cell infiltration with consequent impairment of antitumor activity (18). Conversely, bulky lymphadenopathy does not appear to impair the response to ibrutinib and the drug actually triggers mobilization of the malignant cells to peripheral blood, potentially making them more accessible to CTL019 cells (8).

In addition to BTK, ibrutinib irreversibly inhibits the TEC family kinase ITK (IL2-inducible T-cell kinase). ITK activates PLCγ upon T-cell receptor (TCR) ligation and leads to a signaling cascade that culminates in the activation of T lymphocytes (19). Recent preclinical data suggest that ibrutinib preferentially inhibits Th2-polarized CD4 T cells thus skewing T cells towards Th1 antitumor immune response (20). However, another recent study shows that ibrutinib can antagonize rituximab-dependent NK cell–mediated cytotoxicity and reduce cytokine production, indicating that ITK inhibition may also lead to reduced tumor killing (21). In this context, it is important to discover whether stimulation of the chimeric antigen receptor in CTL019 cells would lead to activation of ITK and, if so, whether inhibition of ITK by ibrutinib would have an advantageous or deleterious effect on CTL019 function.

In principle, the combination of the BTK inhibitor ibrutinib with CTL019 brings together two leading novel approaches to the treatment of B-cell lymphoma and taking advantage of their vastly different mechanisms of action may prove particularly effective. Using in vitro and in vivo models of MCL, including a novel cell line highly sensitive to ibrutinib, we demonstrate here that CTL019 is more effective than ibrutinib as monotherapy, and that the addition of ibrutinib to CTL019 further augments the antitumor effect and leads to prolonged remissions.

Cell lines and primary samples

Cell lines were originally obtained from ATCC (K-562, Mino and JEKO-1) or DSMZ (MOLM-14 and NALM-6); cell lines were obtained more than 6 months prior experiments and authentication was performed by cell banks utilizing short tandem repeat profiling. MCL-RL was generated in our laboratory from a pleural effusion of a MCL patient (the presence of the t(11, 14) characteristic of MCL was tested by FISH). All cell lines were tested for the presence of mycoplasma contamination (MycoAlert Mycoplasma Detection Kit, LT07-318, Lonza). For some experiments, MCL-RL and JEKO-1 cells were transduced with firefly luciferase/eGFP and then sorted to obtain >99% positive population. Cell lines MOLM-14, K562, and NALM-6 were used as controls as indicated in the relevant figures. The cell lines were maintained in culture with RPMI1640 (Gibco, 11875–085, LifeTechnologies) supplemented with 10% FBS (Gemini, 100–106) and 50 U/mL penicillin/streptomycin (Gibco, Life Technologies, 15070–063). Deidentified primary human MCL bone marrow and peripheral blood specimens were obtained from the clinical practices of University of Pennsylvania under an Institutional Review Board–approved protocol (UPCC #03409). For all functional studies, primary cells were thawed at least 12 hours before experiment and rested at 37°C.

FISH and IHC

The FISH analysis and IHC were performed according to the standard method and as described previously (22). Specifics of the experiment of this article are detailed in the Supplementary Methods section.

IHC

Thin-layer cell preparation was obtained by Cytospin (Thermo Scientific) and stained with Giemsa. For formalin-fixed paraffin-embedded tissues, immunohistochemical staining was performed on a Leica Bond-III instrument (Leica Biosystems) using the Bond Polymer Refine Detection System. Antibodies against CD2, SOX-11, Pax5, and CyclinD1 were used undiluted. Heat-induced epitope retrieval was done for 20 minutes with ER2 solution (Leica Microsystems, AR9640). Images were digitally acquired using the Aperio ScanScope (Leica Biosystems).

Generation of CAR constructs and CAR T cells

The murine anti-CD19 chimeric antigen receptor (CD8 hinge, 4-1BB costimulatory domain and CD3 zeta signaling domain) was generated as described previously (ref. 23; Supplementary Fig. S3A). Production of CAR-expressing T cells was performed as described previously (ref. 24; Supplementary Fig. S3B).

Ibrutinib

Ibrutinib (PCI-32765) was purchased from MedKoo (#202171) or Selleck Biochemicals (#S2680) as a powder or DMSO solution. The products obtained from the two companies were compared and proven to have equivalent activity (data not shown). For in vitro experiments, ibrutinib was dissolved in DMSO and diluted to 2, 10, 100, or 1,000 nmol/L in culture media. For in vivo experiment, ibrutinib powder was dissolved in a 10% HP-β-cyclodextrin solution (1.6 mg/mL) and administered to mice in the drinking water.

Multiparametric flow cytometry

Flow cytometry was performed as described previously (24,25) and detailed characteristics of the experiments are provided in Supplementary Methods.

MTT enzymatic conversion assay

The assay was performed as described previously (26). Specifications of this experiment are detailed in the Supplementary Methods section.

DNA fragmentation (TUNEL) assay

ApoAlert DNA fragmentation assay kit (Clontech, 630108) was used according to the manufacturer's protocol. In brief, cells were cultured at 0.5 × 106 cells/mL for 72 hours with DMSO (control) or ibrutinib at the listed doses. The cells were then washed, fixed, permeabilized, and incubated for 1 hour at 37°C with or without terminal deoxynucleotidyl transferase (TdT). After exposure to the stopping buffer and washing, the cells were analyzed by flow cytometry using the CellQuest PRO software v. 5 (BD Biosciences).

Western blot analysis

The assay was performed as described previously (27). Specifications of this experiment are detailed in the Supplementary Methods section.

Real-time PCR

CTL019 cells were screened by RT-PCR analysis for Fas ligand (Applied Biosystems, Life Technologies, Hs00181225_m1), granzyme B (Applied Biosystems, Hs01554355), perforin (Applied Biosystems, Hs00169473_m1), and TRAIL [Applied Biosystems, Hs00921974 mRNA expression at the end of expansion (day 10)]. RNA was extracted with RNAqueos-4PCR Kit (Ambion, Life Technologies, AM-1914) and cDNA was synthesized with iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad, 170-8841). The relative target cDNA copies were quantified by relative qPCR (qPCR) with ABI TaqMan-specific primers and probe set; TaqMan GUSB primers (AB, Hs00939627) and probe set were used for normalization.

In vitro T-cell effector function assays

CD107a degranulation, carboxyfluorescein diacetate succinimidyl ester proliferation (CFSE), cytotoxicity assays, and cytokine measurements were performed as described previously (24,28). Specifications of this experiment are detailed in the Supplementary Methods section.

Animal experiments

In vivo experiments were performed as described previously (24,25,29). Schemas of the utilized xenograft models are discussed in detailed in the relevant figure legends, Results, and the Supplementary Material section.

Statistical analysis

All statistical analyses were performed as indicated using GraphPad Prism 6 for Windows, version 6.04. Student t test was used to compare two groups; in analysis where multiple groups were compared, one-way ANOVA was performed with Holm–Sidak correction for multiple comparisons. When multiple groups at multiple time points/ratios were compared, the Student t test or ANOVA for each time points/ratios was used. Survival curves were compared using the log-rank test. In the figures, asterisks represent P values (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) and “ns” means “not significant” (P > 0.05). Further details of the statistics for each experiment are listed in figure legends.

Sensitivity of MCL cell lines to ibrutinib

Most MCL cell lines in existence have been immortalized and propagated for many generations in vitro and are poorly sensitive to ibrutinib (30,31). We harvested MCL cells from the pleural effusion of a patient with an advanced MCL and established a cell line (MCL-RL) that retained the primary cell polymorphic morphology, the characteristic MCL immunophenotype including CD19 and CD5 coexpression, and the classical t(11;14) translocation with 6 to 7 copies per cell of the IgH-Cyclin D1 fusion gene cell (Fig. 1A). Exposure of the MCL-RL cell line to increasing concentrations of ibrutinib led to a dose-dependent inhibition of cell growth with an IC50 of 10 nmol/L (Fig. 1B), including their apoptotic cell death (Supplementary Fig. S1A, top row). In contrast, the commonly used MCL cell lines Mino and JEKO-1 were relatively resistant to ibrutinib, with IC50 of 1 and 10 μmol/L, respectively (Fig. 1B) and showed no evidence of cell death (Supplementary Fig. S1A, bottom row). Of note, ibrutinib inhibited phosphorylation of BTK to a similar degree in both sensitive (MCL-RL) and resistant (JEKO-1) cell lines, indicating that the resistance in JEKO-1 cells is BTK-independent (Supplementary Fig. S1B). Non-MCL cell lines NALM-6 (B-cell acute lymphoid leukemia) and K562 (acute myeloid leukemia) were also tested for ibrutinib sensitivity showing IC50 of >1 and >10 μmol/L, respectively (Supplementary Fig. S1C) and hence served as additional negative controls throughout the study. To determine the suitability of MCL-RL cells for in vivo experiments, we injected immunodeficient NSG mice intravenously with 1 × 106 MCL-RL cells expressing firefly luciferase and monitored the mice for tumor burden by bioluminescence imaging and for survival. The MCL-RL cells engrafted in all mice and localized predominantly to the spleen and liver, followed by dissemination to bone marrow, blood, and other organs (Supplementary Fig. S2A). Histology and IHC of the tumors recapitulated the morphology and immunophenotype of the original MCL-RL cells (Supplementary Fig. S2B). Importantly, MCL-RL also demonstrated a response to ibrutinib treatment in this in vivo setting with a dose-dependent reduction in tumor growth (Fig. 1C, top) and improvement in overall survival (Fig. 1C, bottom).

Figure 1.

Establishment of an ibrutinib-sensitive MCL cell line. A, morphology, phenotype, and FISH analysis of the MCL-RL cell line and primary cells. Thin-layer cell preparation of the MCL-RL cell line was obtained by Cytospin. MCL-RL cells were stained with Giemsa and demonstrated blastoid morphology (top left). Flow cytometry analysis revealed that CD19 and CD5 coexpression, hallmark of MCL, is maintained (right). In FISH analysis, MCL-RL cells were analyzed by FISH using a dual color gene fusion probe against the IgH (green) and CCND1 (orange) genes, located on chromosomes 14 and 11, respectively. The isolated green color corresponds to the nontranslocated IgH gene locus and isolated orange to the CCND1 gene locus. The fused green and orange, typically blended together into a yellow color, mark the translocated, hybrid IgH/CCND1 gene (bottom left). B, MTT assay of MCL cell lines. JEKO-1, MINO, and MCL-RL were cultured for 48 hours with increasing doses of ibrutinib (0–10 μmol/L). MCL-RL cell line was the most sensitive to ibrutinib, with an IC50 of 10 nmol/L. The MCL cell lines MINO and JEKO-1 were more resistant. C, ibrutinib sensitivity of MCL-RL cell line in vivo. NSG mice were engrafted with luciferase-positive MCL-RL cells (1 × 106/mouse); at day 7 mice were randomized according to tumor burden (bioluminescence, BLI) to receive vehicle (HP-β-cyclodextrin), ibrutinib 25 mg/kg/day, or ibrutinib 125 mg/kg/day in the drinking water. A dose-related antilymphoma activity was observed using bioluminescence (top; ANOVA at day 70, P < 0.0001 for both doses). This antilymphoma activity was also reflected in an improved overall survival of mice treated with both doses compared with controls (log-rank test P = 0.0086 and 0.0017, respectively; bottom). Graphs are representative of two experiments with 4–5 animals per group.

Figure 1.

Establishment of an ibrutinib-sensitive MCL cell line. A, morphology, phenotype, and FISH analysis of the MCL-RL cell line and primary cells. Thin-layer cell preparation of the MCL-RL cell line was obtained by Cytospin. MCL-RL cells were stained with Giemsa and demonstrated blastoid morphology (top left). Flow cytometry analysis revealed that CD19 and CD5 coexpression, hallmark of MCL, is maintained (right). In FISH analysis, MCL-RL cells were analyzed by FISH using a dual color gene fusion probe against the IgH (green) and CCND1 (orange) genes, located on chromosomes 14 and 11, respectively. The isolated green color corresponds to the nontranslocated IgH gene locus and isolated orange to the CCND1 gene locus. The fused green and orange, typically blended together into a yellow color, mark the translocated, hybrid IgH/CCND1 gene (bottom left). B, MTT assay of MCL cell lines. JEKO-1, MINO, and MCL-RL were cultured for 48 hours with increasing doses of ibrutinib (0–10 μmol/L). MCL-RL cell line was the most sensitive to ibrutinib, with an IC50 of 10 nmol/L. The MCL cell lines MINO and JEKO-1 were more resistant. C, ibrutinib sensitivity of MCL-RL cell line in vivo. NSG mice were engrafted with luciferase-positive MCL-RL cells (1 × 106/mouse); at day 7 mice were randomized according to tumor burden (bioluminescence, BLI) to receive vehicle (HP-β-cyclodextrin), ibrutinib 25 mg/kg/day, or ibrutinib 125 mg/kg/day in the drinking water. A dose-related antilymphoma activity was observed using bioluminescence (top; ANOVA at day 70, P < 0.0001 for both doses). This antilymphoma activity was also reflected in an improved overall survival of mice treated with both doses compared with controls (log-rank test P = 0.0086 and 0.0017, respectively; bottom). Graphs are representative of two experiments with 4–5 animals per group.

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Mantle cell lymphoma cells are sensitive to CTL019 effector functions

To examine sensitivity of the MCL cells to killing by CTL019 cells, we transduced healthy donor T cells with the same anti-CD19 CAR construct that has been used in the clinical trials of our group (16) and used for the following experiments. The design of this CAR and the T-cell production schema are shown in Supplementary Fig. S3A and S3B. To test whether CTL019 cells could be manufactured also from the blood of patients with leukemic MCL, we expanded and transduced patient-derived T cells (Fig. 2A, top) and then performed a CD107a degranulation and cytokine production assay to demonstrate reactivity against that patient's own MCL (Fig. 2A, bottom). Given the recent interest (32) in tumor-infiltrating and marrow-infiltrating lymphocytes, we also performed a similar study using marrow-derived T cells from a patient with stage IV MCL (Fig. 2A and Supplementary Fig. S4A and S4B). A series of in vitro experiments showed that both the ibrutinib-sensitive MCL-RL and the ibrutinib-resistant JEKO-1 cell line induced comparably strong activation of CTL019 cells as determined by their degranulation, cytokine production, cytotoxic activity, and proliferation (Fig. 2B and C and Supplementary Fig. S4C). As shown in Fig. 2C, the MCL-RL cell line was less sensitive to CTL019 cytotoxicity as compared with JEKO-1. This was likely due to the increased activation-induced apoptosis of CTL in the presence of MCL-RL (Supplementary Fig. S4D). The CTL019 activation was strictly CAR-dependent, as the untransduced cells (UTD) from the same donors tested in parallel showed no, or very limited, activity in these assays. We next evaluated in vivo different doses of CTL019 cells and demonstrated a dose-dependent antitumor efficacy, with 2 × 106 CTL019 cells per mouse proving to be the most effective. Higher doses of T cells were associated with nonspecific alloreactivity (data not shown). Notably, the antilymphoma activity of CTL019 was observed in NSG mice engrafted with both ibrutinib-resistant (JEKO-1; Fig. 2D, top) and ibrutinib-sensitive (MCL-RL) MCL cell lines (Fig. 2D, bottom). These results indicate that MCL is sensitive to the effector functions of CTL019 cells.

Figure 2.

CTL019 cells exhibit potent in vitro and in vivo effector functions against diverse MCL cell lines. A, feasibility of CTL019 production in MCL patients and antilymphoma effector activity. Peripheral blood (PB; #001) or bone marrow (BM; #002) samples were obtained from patients with active MCL (infiltration 68% and 4%, respectively). CAR19 T cells were expanded according to the standard protocol used at our institution (see Materials and Methods). CTL019 expansion was feasible for both peripheral blood and bone marrow T cells, with a range of population doublings from 3.5 (BM#002) to 6.5 (PB#001). Expanded T cells included both CD8 and CD4 cells with a variable CAR19 expression (from 10% to 49%) similar to what is currently obtained in other CTL019 trials at our institution (right; ref. 28) As shown in the bottom panel, CTL019 and MCL cells from the same patient were cocultured for 6 hours and then harvested and analyzed by flow cytometry for CD107a degranulation or cytokine production. Autologous CTL019 but not control T cells (UTD) showed significant activation with CD107a degranulation and intracytoplasmic production of cytokines, including IL2, TNFα, IFNγ, GM-CSF, and MIP1b. B, CD107a degranulation assay. CAR19 T cells showed specific CD107a degranulation when cocultured with JEKO-1 and MCL-RL MCL cell lines, similar to the positive control PMA and ionomycin stimulation (PI). C, CTL019 cytotoxicity and proliferation assays. For the cytotoxicity assay (left), CTL019 were cocultured at different effector-to-target ratio (E:T) with luciferase-positive MCL cell lines or control (K562). At 24 hours, cell killing was assessed by luminescence relative to controls. CTL019 cells are able to induce cell death in both MCL cell lines (one-way ANOVA significant at all ratios > 0:1 compared with control cell line K562) with a dose correlation effect; no cytotoxicity is observed against the CD19-negative control cell line (K-562). For the proliferation assay (right), CFSE-labeled CTL019 cells were cocultured with the MCL cell lines (JEKO-1, MCL-RL) or control (K562) for 5 days. CTL019 show specific proliferation (CFSE dilution) when cocultured with MCL cell lines but not with control. D, in vivo potent antilymphoma activity of CTL019 against both ibrutinib-resistant (JEKO-1) and ibrutinib-sensitive cell line (MCL-RL). NSG mice were engrafted with either luciferase-positive JEKO-1 cells (top) or MCL-RL (bottom). At day 6, mice were randomized to receive 3 different doses of CTL019 (0.5 × 106, 1 × 106, 2 × 106/mouse, CAR+ 70%) or control T cells (UTD, 1 × 106/mouse). A dramatic antilymphoma activity was observed in all doses, with the highest dose (2 × 106), leading to long-term complete remission in JEKO-1 (ANOVA at day 28, P < 0.0001 for all CTL019 doses). In MCL-RL, luminescence values are shown at 1 week after T-cell infusion; a significant dose-dependent CTL019 antilymphoma activity is observed (one-way ANOVA, P < 0.05 for the doses of 1 × 106 and 2 × 106 CATL019/mouse). In the MCL-RL model, late relapses are observed, also at the dose of 2 × 106 CART19/mouse (data not shown). Each graph is representative of two independent experiments, each with 4–5 mice per group. BLI, bioluminescence.

Figure 2.

CTL019 cells exhibit potent in vitro and in vivo effector functions against diverse MCL cell lines. A, feasibility of CTL019 production in MCL patients and antilymphoma effector activity. Peripheral blood (PB; #001) or bone marrow (BM; #002) samples were obtained from patients with active MCL (infiltration 68% and 4%, respectively). CAR19 T cells were expanded according to the standard protocol used at our institution (see Materials and Methods). CTL019 expansion was feasible for both peripheral blood and bone marrow T cells, with a range of population doublings from 3.5 (BM#002) to 6.5 (PB#001). Expanded T cells included both CD8 and CD4 cells with a variable CAR19 expression (from 10% to 49%) similar to what is currently obtained in other CTL019 trials at our institution (right; ref. 28) As shown in the bottom panel, CTL019 and MCL cells from the same patient were cocultured for 6 hours and then harvested and analyzed by flow cytometry for CD107a degranulation or cytokine production. Autologous CTL019 but not control T cells (UTD) showed significant activation with CD107a degranulation and intracytoplasmic production of cytokines, including IL2, TNFα, IFNγ, GM-CSF, and MIP1b. B, CD107a degranulation assay. CAR19 T cells showed specific CD107a degranulation when cocultured with JEKO-1 and MCL-RL MCL cell lines, similar to the positive control PMA and ionomycin stimulation (PI). C, CTL019 cytotoxicity and proliferation assays. For the cytotoxicity assay (left), CTL019 were cocultured at different effector-to-target ratio (E:T) with luciferase-positive MCL cell lines or control (K562). At 24 hours, cell killing was assessed by luminescence relative to controls. CTL019 cells are able to induce cell death in both MCL cell lines (one-way ANOVA significant at all ratios > 0:1 compared with control cell line K562) with a dose correlation effect; no cytotoxicity is observed against the CD19-negative control cell line (K-562). For the proliferation assay (right), CFSE-labeled CTL019 cells were cocultured with the MCL cell lines (JEKO-1, MCL-RL) or control (K562) for 5 days. CTL019 show specific proliferation (CFSE dilution) when cocultured with MCL cell lines but not with control. D, in vivo potent antilymphoma activity of CTL019 against both ibrutinib-resistant (JEKO-1) and ibrutinib-sensitive cell line (MCL-RL). NSG mice were engrafted with either luciferase-positive JEKO-1 cells (top) or MCL-RL (bottom). At day 6, mice were randomized to receive 3 different doses of CTL019 (0.5 × 106, 1 × 106, 2 × 106/mouse, CAR+ 70%) or control T cells (UTD, 1 × 106/mouse). A dramatic antilymphoma activity was observed in all doses, with the highest dose (2 × 106), leading to long-term complete remission in JEKO-1 (ANOVA at day 28, P < 0.0001 for all CTL019 doses). In MCL-RL, luminescence values are shown at 1 week after T-cell infusion; a significant dose-dependent CTL019 antilymphoma activity is observed (one-way ANOVA, P < 0.05 for the doses of 1 × 106 and 2 × 106 CATL019/mouse). In the MCL-RL model, late relapses are observed, also at the dose of 2 × 106 CART19/mouse (data not shown). Each graph is representative of two independent experiments, each with 4–5 mice per group. BLI, bioluminescence.

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Impact of ibrutinib on CTL019 function in vitro

Ibrutinib was originally thought not to impact T cells based on short-term activity assays (33). However, a comprehensive analysis of the impact of ibrutinib on the T-cell kinase ITK subsequently supported an overall immunomodulatory role of ibrutinib in CD4 T cells as a suppressor of the Th2-type polarization (20). Cytokine expression pattern analysis of patients treated with anti-CD19 CAR T cells performed by several groups indicates that this therapy is associated with both Th1-type (IL2, IFNγ, TNF) and Th2-type (IL4, IL5, IL10), as well as other cytokine-secretion patterns. (13,28) Therefore, we evaluated the effect of ibrutinib on CTL019 function at, above, and below the concentrations that would be expected in patients (mean peak concentration in patient serum is 100–150 ng/mL; ref. 6). We found that CTL019 cells express ITK and that stimulation of CTL019 cells, whether through the TCR complex or through the CAR, led to the phosphorylation of ITK. The presence of ibrutinib resulted in a modest reduction in ITK phosphorylation that was only evident at the highest concentration of ibrutinib (Fig. 3A).

Figure 3.

Impact of ibrutinib on CTL019 functions in vitro. A, Western blot analysis of p-ITK inhibition by ibrutinib (IBRU) in CAR19 cells. CTL019 cells were stimulated with anti-CD3/CD28 Dynabeads or anti-CAR19 beads for 2 minutes in the presence of different concentrations of ibrutinib (10–1,000 nmol/L) or control (DMSO). Western blot analysis on protein lysates revealed modest inhibition of the phosphorylation of ITK (at Y180) particularly in CAR-stimulated CTL019 at the highest concentration (1 μmol/L). β-Actin and p-Erk were used as loading and activation controls, respectively. B, CTL019 degranulation assay and cytokine production in the presence of ibrutinib. CTL019 or control T cells (UTD) were cocultured with JEKO-1 or MCL-RL for 4 hours in the presence of increasing doses of ibrutinib (10–1,000 nmol/L). Positive (PMA/ionomycin) and negative controls (media alone, K562) were also included. Flow cytometric analysis revealed significant activation of CTL019 cells, but not UTD, in the presence of MCL cell lines as shown by CD107a degranulation and intracytoplasmic cytokine production (IL2, TNFα). C, CTL019 proliferation assay in the presence of ibrutinib. CFSE-labeled CTL019 cells were cocultured with lethally irradiated MCL-RL cells or JEKO-1 (or controls, K562) for 5 days in the presence of increasing doses of ibrutinib (10–1,000 nmol/L, added at every change of media). Cells were then analyzed for CFSE dilution, as a marker of cell proliferation. Profound CTL019 proliferation was observed; however, significant reduction in CFSE-positive T cells was observed with the highest (supraphysiologic) ibrutinib doses (1,000 nmol/L) in the JEKO-1 group. D, CTL019 cytokine production in the presence of ibrutinib. CTL019 or control T cells were cocultured with irradiated MCL-RL cells for 3 days and supernatants were analyzed for 30 human cytokines (Luminex, 30-plex). Intense production of both Th1 and Th2 cytokines was observed with significant reduction of all cytokines at the highest ibrutinib dose (1,000 nmol/L). E, effect of ibrutinib on CTL019 cytotoxic machinery. CTL019 were expanded with anti-CD3/CD28 beads in the presence of increasing doses of ibrutinib (10–1,000 nmol/L). RT-PCR analysis of Fas ligand, granzyme B, perforin, and TRAIL mRNA expression was performed at the end of expansion (day 10). No clear effect of increasing doses of ibrutinib was observed (one-way ANOVA = ns). A trend in increased perforin expression was not statistically significant. F, CTL019 cytotoxicity in the presence of ibrutinib. CTL019 or control T cells (UTD) were cocultured at different effector-to-target ratio (E:T) with luciferase-positive MCL cell lines (JEKO-1, MCL-RL) with increasing doses of ibrutinib. At 24 hours, cell killing was assessed by luminescence. CTL019 are able to induce cell death in both MCL cell lines. At a specific E:T ratio, increased MCL killing was significantly correlated to increased ibrutinib dose. The P values (one-way ANOVA) comparing CART19-DMSO versus CART19 +IBRU 100 nmol/L at the different E:T ratios are summarized in the figure.

Figure 3.

Impact of ibrutinib on CTL019 functions in vitro. A, Western blot analysis of p-ITK inhibition by ibrutinib (IBRU) in CAR19 cells. CTL019 cells were stimulated with anti-CD3/CD28 Dynabeads or anti-CAR19 beads for 2 minutes in the presence of different concentrations of ibrutinib (10–1,000 nmol/L) or control (DMSO). Western blot analysis on protein lysates revealed modest inhibition of the phosphorylation of ITK (at Y180) particularly in CAR-stimulated CTL019 at the highest concentration (1 μmol/L). β-Actin and p-Erk were used as loading and activation controls, respectively. B, CTL019 degranulation assay and cytokine production in the presence of ibrutinib. CTL019 or control T cells (UTD) were cocultured with JEKO-1 or MCL-RL for 4 hours in the presence of increasing doses of ibrutinib (10–1,000 nmol/L). Positive (PMA/ionomycin) and negative controls (media alone, K562) were also included. Flow cytometric analysis revealed significant activation of CTL019 cells, but not UTD, in the presence of MCL cell lines as shown by CD107a degranulation and intracytoplasmic cytokine production (IL2, TNFα). C, CTL019 proliferation assay in the presence of ibrutinib. CFSE-labeled CTL019 cells were cocultured with lethally irradiated MCL-RL cells or JEKO-1 (or controls, K562) for 5 days in the presence of increasing doses of ibrutinib (10–1,000 nmol/L, added at every change of media). Cells were then analyzed for CFSE dilution, as a marker of cell proliferation. Profound CTL019 proliferation was observed; however, significant reduction in CFSE-positive T cells was observed with the highest (supraphysiologic) ibrutinib doses (1,000 nmol/L) in the JEKO-1 group. D, CTL019 cytokine production in the presence of ibrutinib. CTL019 or control T cells were cocultured with irradiated MCL-RL cells for 3 days and supernatants were analyzed for 30 human cytokines (Luminex, 30-plex). Intense production of both Th1 and Th2 cytokines was observed with significant reduction of all cytokines at the highest ibrutinib dose (1,000 nmol/L). E, effect of ibrutinib on CTL019 cytotoxic machinery. CTL019 were expanded with anti-CD3/CD28 beads in the presence of increasing doses of ibrutinib (10–1,000 nmol/L). RT-PCR analysis of Fas ligand, granzyme B, perforin, and TRAIL mRNA expression was performed at the end of expansion (day 10). No clear effect of increasing doses of ibrutinib was observed (one-way ANOVA = ns). A trend in increased perforin expression was not statistically significant. F, CTL019 cytotoxicity in the presence of ibrutinib. CTL019 or control T cells (UTD) were cocultured at different effector-to-target ratio (E:T) with luciferase-positive MCL cell lines (JEKO-1, MCL-RL) with increasing doses of ibrutinib. At 24 hours, cell killing was assessed by luminescence. CTL019 are able to induce cell death in both MCL cell lines. At a specific E:T ratio, increased MCL killing was significantly correlated to increased ibrutinib dose. The P values (one-way ANOVA) comparing CART19-DMSO versus CART19 +IBRU 100 nmol/L at the different E:T ratios are summarized in the figure.

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We next probed the short- and long-term in vitro function of CTL019 cells in the presence of ibrutinib. Following 4 to 6 hours of incubation with MCL cell lines, clinically relevant concentrations of ibrutinib did not influence CTL019 degranulation and cytokine production (Fig. 3B). In a 5-day proliferation assay, we observed a dose-dependent reduction in T-cell proliferation and total T-cell numbers, but this reduction occurred predominantly at supraphysiologic concentrations of ibrutinib (1 μmol/L and above) and, more frequently, upon the CTL019 cell exposure to JEKO-1 as compared with MCL-RL cells. (Fig. 3C and Supplementary Fig. S5A). Similarly, the cell-culture supernatant analysis for 30 different cytokines demonstrated that ibrutinib did not impact cytokine production except in the presence of supraphysiologic drug concentrations (Fig. 3D). We did not find differences in Th1/Th2 polarization between ibrutinib exposed and nonexposed CTL019 using two different techniques (Fig. 3D and Supplementary Fig. S5B). The intrinsic cytotoxic machinery of CTL019 was not significantly impacted in the presence of ibrutinib (Fig. 3E and Supplementary Fig. S5C) and there was no apparent difference in the expression of CD19 or of inhibitory ligands on MCL exposed to ibrutinib (data not shown). Notably, killing of MCL cells by CTL019 cells was significantly augmented in the presence of ibrutinib, suggesting an additive cytotoxic effect of the combination in both ibrutinib-sensitive (MCL-RL) and -resistant (JEKO-1) MCL cells (Fig. 3F). Collectively, these results indicate that ibrutinib has no adverse effect on CART cell function at physiologically relevant concentrations, and that the combination of two agents active against MCL is additive in vitro.

Impact of ibrutinib on circulating CTL019 cells

In our in vitro models, combination with ibrutinib clearly enhanced the already potent antitumor effect of CTL019 and hence it was important to evaluate the nature of the interaction of CTL019 with ibrutinib also in vivo.

Inhibition of ITK has been reported to antagonize Th2 polarization and promote a Th1 phenotype (20). However, in mice treated with CTL019 and ibrutinib we did not find an increase in Th1 cells when compared with CTL019 monotherapy (Supplementary Fig. S6A). Of note, exposure of tumor-bearing mice to ibrutinib led to an increase in peripheral blood T cells, regardless of antigen specificity, as ibrutinib augmented circulating T-cell numbers of both CTL019 and control untransduced cells (Fig. 4A and data not shown). This increase was not due to increased proliferation, as there was no difference in the proliferation marker Ki67 between the treatment groups (Fig. 4B, left). Similarly, we did not find any difference in the antiapoptotic marker Bcl2, suggesting that the difference in the number of circulating CTL019 cells was not related to an impairment of apoptosis (Fig. 4B, right). To differentiate whether the increased number of circulating T cells in ibrutinib-treated mice were due to accumulation in, or mobilization into, the peripheral blood compartment, we engrafted NSG mice with unlabeled MCL-RL cells followed by injection with luciferase-expressing T cells, wherein the bioluminescent signal (BLI) from the whole animal would correlate with total T-cell load. Ibrutinib treatment did not enhance BLI in either CTL019 or control T-cell–treated animals, suggesting that ibrutinib did not increase the total T-cell number but rather triggered T-cell mobilization to the blood (Fig. 4C). We then investigated the frequency of different T-cell subsets among the circulating T cells and could not detect any difference in the T-cell subset distribution between the CTL019- and CTL019/ibrutinib- engrafted mice (Fig Supplementary Fig. S6B and S6C). Because CXCR4 is involved in ibrutinib-driven B-cell mobilization in humans, we measured the expression of CXCR4 in vivo in the circulating T cells of mice treated with CTL019 or CTL019 and ibrutinib and found similar CXCR4 levels in the two groups indicating that the increased mobilization was not due to decreased CXCR4 expression (Supplementary Fig. S7A). Finally, we analyzed the expression of inhibitory/costimulatory receptors in the peripheral blood T cells of mice treated with CTL019 and CTL019 plus ibrutinib. There was a trend to reduced PD-1 expression when ibrutinib was added to CTL019 or untransduced T cell controls, but no differences in expression of TIM3, LAG3, CD137, or CTLA4 were found. (Supplementary Fig. S7B and S7C).

Figure 4.

Increase in circulating CTL019 cells in the presence of ibrutinib (IBRU). A, higher number of circulating CAR19 T cells in the combination treatment. Peripheral blood (PB) circulating T cells were monitored weekly by retro-orbital bleeding and flow cytometry analysis was performed. Expansion of CTL019 in the periphery was detected in both CTL019 and CTL019–ibrutinib (ibrutinib 125 mg/kg/day in the drinking water) treated mice; however, a significantly higher number of T cells was observed in the combination group (Student t test). Peak expansion is usually observed at 1–2 weeks after T-cell infusion. B, in vivo T-cell proliferation and apoptosis after treatment with CTL019/ibrutinib combination. One week after CTL019 infusion in MCL-RL bearing mice, peripheral blood was collected and analyzed for Ki67 and bcl-2 by flow cytometry. No statistically (Student t test) significant difference in T-cell proliferation (Ki67) or apoptosis (bcl-2) was observed. C, in vivo tracking of T-cell expansion. NSG mice were engrafted with WT MCL-RL cells. After one month, luciferase-positive CTL019 or control T cells were infused. Five days after infusion, mice were analyzed by bioluminescence imaging. A significant increase in T cell number was observed in both CTL019 and CTL019–ibrutinib treated mice as compared with control T cells (UTD) and UTD–ibrutinib. No difference in T-cell proliferation was detected between CTL019 and CTL019-ibrutinib (Student t test). BLI, bioluminescence.

Figure 4.

Increase in circulating CTL019 cells in the presence of ibrutinib (IBRU). A, higher number of circulating CAR19 T cells in the combination treatment. Peripheral blood (PB) circulating T cells were monitored weekly by retro-orbital bleeding and flow cytometry analysis was performed. Expansion of CTL019 in the periphery was detected in both CTL019 and CTL019–ibrutinib (ibrutinib 125 mg/kg/day in the drinking water) treated mice; however, a significantly higher number of T cells was observed in the combination group (Student t test). Peak expansion is usually observed at 1–2 weeks after T-cell infusion. B, in vivo T-cell proliferation and apoptosis after treatment with CTL019/ibrutinib combination. One week after CTL019 infusion in MCL-RL bearing mice, peripheral blood was collected and analyzed for Ki67 and bcl-2 by flow cytometry. No statistically (Student t test) significant difference in T-cell proliferation (Ki67) or apoptosis (bcl-2) was observed. C, in vivo tracking of T-cell expansion. NSG mice were engrafted with WT MCL-RL cells. After one month, luciferase-positive CTL019 or control T cells were infused. Five days after infusion, mice were analyzed by bioluminescence imaging. A significant increase in T cell number was observed in both CTL019 and CTL019–ibrutinib treated mice as compared with control T cells (UTD) and UTD–ibrutinib. No difference in T-cell proliferation was detected between CTL019 and CTL019-ibrutinib (Student t test). BLI, bioluminescence.

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In vivo antitumor activity of ibrutinib, CTL019, and their combination

Our in vivo MCL model provided a unique opportunity to perform a direct comparison of two novel therapies that are currently used clinically as single agents. A schema of the treatment protocol is provided in Fig. 5A. Mice treated with CTL019 showed a statistically significant improvement in lymphoma control compared with ibrutinib-treated mice (Fig. 5B). As depicted in Fig. 5C, all mice treated with ibrutinib monotherapy died before day 100, whereas CTL019 fostered long-term survival of the recipient mice, suggesting that CTL019 is therapeutically more effective than ibrutinib in this model.

Figure 5.

Direct comparison of the anti-MCL activity of ibrutinib and CTL019 in MCL xenografts. A, protocol schema. NSG mice were engrafted with luciferase-positive MCL-RL cells (2 × 106 cells/mouse, i.v.). At day 7 mice were randomized according to tumor burden, to receive vehicle, ibrutinib 125 mg/kg/day or CTL019 2 × 106/mouse. Ibrutinib (Ibru) and vehicle were continued for all the duration of the experiment. B and C, CTL019 therapy is more effective than ibrutinib against MCL-RL. Mice treated with CTL019 had a significantly improved antitumor activity compared with ibrutinib (Student t test, P < 0.0001 from day 18). CTL019 treatment also ensured a statistically improved overall survival compared with ibrutinib (log-rank test, P < 0.005; C). Graphs are representative of two experiments, each with 5 mice per group; P values compared with ibrutinib alone. The dotted bar represents the limit of detection. BLI, bioluminescence

Figure 5.

Direct comparison of the anti-MCL activity of ibrutinib and CTL019 in MCL xenografts. A, protocol schema. NSG mice were engrafted with luciferase-positive MCL-RL cells (2 × 106 cells/mouse, i.v.). At day 7 mice were randomized according to tumor burden, to receive vehicle, ibrutinib 125 mg/kg/day or CTL019 2 × 106/mouse. Ibrutinib (Ibru) and vehicle were continued for all the duration of the experiment. B and C, CTL019 therapy is more effective than ibrutinib against MCL-RL. Mice treated with CTL019 had a significantly improved antitumor activity compared with ibrutinib (Student t test, P < 0.0001 from day 18). CTL019 treatment also ensured a statistically improved overall survival compared with ibrutinib (log-rank test, P < 0.005; C). Graphs are representative of two experiments, each with 5 mice per group; P values compared with ibrutinib alone. The dotted bar represents the limit of detection. BLI, bioluminescence

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We next tested the combination of CTL019 and ibrutinib in vivo (Fig. 6A). Because we found no difference in the antitumor effect when comparing untransduced T cells plus ibrutinib with ibrutinib alone (Supplementary Fig. S8A), in all subsequent experiments the control groups were vehicle and ibrutinib alone. Ibrutinib monotherapy led to modestly delayed disease growth at early time points, whereas CTL019 monotherapy led to a profound reduction in tumor burden that was followed by the disease progression beginning at 6–7 weeks. In striking contrast, 80% to 100% of mice treated with the combination of CTL019 and ibrutinib experienced complete, long-term disease control (Fig. 6B and C).

Figure 6.

Combination of ibrutinib and CTL019 in MCL xenografts. A, protocol schema. NSG mice were engrafted with luciferase-positive MCL-RL cells (2 × 106 cells/mouse, i.v.). At day 7, mice were randomized according to tumor burden, to receive vehicle, ibrutinib 125 mg/kg/day, CTL019 2 × 106/mouse, or CTL019 with ibrutinib (IBRU; same doses). Ibrutinib and vehicle were continued for all the duration of the experiment. B and C, increased antilymphoma activity of the CTL019–ibrutinib combination. Mice treated with CTL019 in combination with ibrutinib displayed a significantly better antilymphoma effect compared either with ibrutinib (Student t test, P < 0.0001 at day 60) or CTL019 alone (P = 0.007 at day 110). At long-term follow-up, 5 of 5 mice in the ibrutinib group and 4 of 5 mice in the CTL019 group are progressing while only 1 mouse in the CTL019–ibrutinib combination has progressed. Graphs are representative of two experiments, each one with 5 mice per group. The dotted bar represents the limit of detection. The bioluminescence images of two representative mice per group are shown in C. Please note that the range of radiance for visualization varies among different time points, as detailed in D. Mouse liver histopathology after treatment with CTL019/ibrutinib combination. Animals were euthanized at the end of the experiment (day 120) or when required according to the animal welfare regulations; organs (liver, spleen, bone marrow) were collected for histopathology (H&E, PAX5, CD2). Representative mice are shown in the figure. Variable amount of disease is observed in the liver of untreated mice, and ibrutinib mice and CTL019-treated mice; most of mice treated with CTL019–ibrutinib combination had no disease. CD2+ T cells were detected in CTL019 mice (at the time of progression, together with MCL-RL cells) while CTL019-ibrutinib treated mice showed disappearance of T cells. The livers of these mice were analyzed by flow cytometry (E) and residual T cells showed differential expression of PD-1: PD-1 was significantly upregulated in mice not receiving ibrutinib (F), possibly explaining the lack of antitumor activity. G, coculture of CTL019 with MCL-RL cell line leads to overexpression of inhibitory receptors and this overexpression is reduced in the presence of ibrutinib. CART19 cells were cocultured with MCL-RL cells in the presence or not of ibrutinib (100 nmol/L). At day 6, inhibitory receptor expression (PD-1, LAG-3, TIM-3, and CTLA-4) was analyzed by flow cytometry. Marked upregulation of inhibitory receptor In T cells is observed. However, a significant reduction in the surface expression of PD-1, LAG-3, TIM-3, and CTLA-4 was detected when CART19 cells were cultured with ibrutinib 100 nmol/L.

Figure 6.

Combination of ibrutinib and CTL019 in MCL xenografts. A, protocol schema. NSG mice were engrafted with luciferase-positive MCL-RL cells (2 × 106 cells/mouse, i.v.). At day 7, mice were randomized according to tumor burden, to receive vehicle, ibrutinib 125 mg/kg/day, CTL019 2 × 106/mouse, or CTL019 with ibrutinib (IBRU; same doses). Ibrutinib and vehicle were continued for all the duration of the experiment. B and C, increased antilymphoma activity of the CTL019–ibrutinib combination. Mice treated with CTL019 in combination with ibrutinib displayed a significantly better antilymphoma effect compared either with ibrutinib (Student t test, P < 0.0001 at day 60) or CTL019 alone (P = 0.007 at day 110). At long-term follow-up, 5 of 5 mice in the ibrutinib group and 4 of 5 mice in the CTL019 group are progressing while only 1 mouse in the CTL019–ibrutinib combination has progressed. Graphs are representative of two experiments, each one with 5 mice per group. The dotted bar represents the limit of detection. The bioluminescence images of two representative mice per group are shown in C. Please note that the range of radiance for visualization varies among different time points, as detailed in D. Mouse liver histopathology after treatment with CTL019/ibrutinib combination. Animals were euthanized at the end of the experiment (day 120) or when required according to the animal welfare regulations; organs (liver, spleen, bone marrow) were collected for histopathology (H&E, PAX5, CD2). Representative mice are shown in the figure. Variable amount of disease is observed in the liver of untreated mice, and ibrutinib mice and CTL019-treated mice; most of mice treated with CTL019–ibrutinib combination had no disease. CD2+ T cells were detected in CTL019 mice (at the time of progression, together with MCL-RL cells) while CTL019-ibrutinib treated mice showed disappearance of T cells. The livers of these mice were analyzed by flow cytometry (E) and residual T cells showed differential expression of PD-1: PD-1 was significantly upregulated in mice not receiving ibrutinib (F), possibly explaining the lack of antitumor activity. G, coculture of CTL019 with MCL-RL cell line leads to overexpression of inhibitory receptors and this overexpression is reduced in the presence of ibrutinib. CART19 cells were cocultured with MCL-RL cells in the presence or not of ibrutinib (100 nmol/L). At day 6, inhibitory receptor expression (PD-1, LAG-3, TIM-3, and CTLA-4) was analyzed by flow cytometry. Marked upregulation of inhibitory receptor In T cells is observed. However, a significant reduction in the surface expression of PD-1, LAG-3, TIM-3, and CTLA-4 was detected when CART19 cells were cultured with ibrutinib 100 nmol/L.

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Histopathology of organs harvested at the conclusion of the experiment revealed MCL infiltrates in all untreated and ibrutinib-treated mice with the extent of involvement being relatively diminished in the ibrutinib-treated group. Most of the mice treated with CTL019 alone displayed persistent MCL and some CTL019 cells, while mice treated with CTL019–ibrutinib showed clearance of the tumor and disappearance of CTL019 (Fig. 6D).

Having shown that ibrutinib treatment was associated with a nonsignificant trend to lower PD-1 expression on CTL019 in the blood compartment, we next analyzed the expression of PD-1 on CTL019 in tumor-involved organs. We confirmed the presence of T cells in the livers of mice treated with CART19 and, to a lesser extent, in CART19 + ibrutinib–treated mice (Fig. 6E). Interestingly T cells from mice receiving CTL019 monotherapy had significantly higher levels of PD-1 as compared with mice receiving CTL019 + ibrutinib (Fig. 6F). We then evaluated the expression of inhibitory receptors on CTL019 cells exposed to increasing doses of ibrutinib in vitro as a possible mechanism of improved antitumor activity. Interestingly, we found that CTL019 cells cocultured with MCL-RL for 6 days markedly upregulated inhibitory receptors such as PD-1, LAG-3, TIM-3, CTLA-4 (Fig. 6G). Notably, the addition of ibrutinib to the coculture led to a significant reduction in all inhibitory receptors (Fig. 6G). This mechanism may illuminate the observation of better antitumor activity of the combination in vitro and in vivo.

Novel therapies for B-cell malignancies include small-molecule inhibitors of BCR signaling and CD19-directed T-cell–based therapies. The BTK inhibitor ibrutinib was recently approved by the FDA for the treatment of therapy-resistant MCL and engenders responses in most (68%) patients. However, these responses are typically partial and relatively short-lived: the median progression-free survival is 17.5 months (8). Anti-CD19 CAR T cell therapy leads to durable responses in subsets of patients with high-risk B-ALL (12–14), DLBCL (16), and to a lesser degree, CLL (15). Combination of chemotherapeutic agents with non–cross-resistant mechanisms of action has a long history in the treatment of cancer (33) and provides the rationale for the current study. Here we evaluated the combined effect of signal transduction (kinase) inhibition and cellular immunotherapy; these two novel therapeutic approaches are poised to revolutionize treatment of patients with lymphoma and cancer in general. Specifically, we investigated the impact of adding the BTK inhibitor ibrutinib to CTL019 using MCL as a model of a currently incurable disease responsive to both these modalities. Although ibrutinib exerted in vitro a profound detrimental effect on the sensitive MCL cells, we found that at all but high supraphysiologic doses of the drug, CTL019 cell function remains unimpaired, with intact proliferative capacity, tumor recognition and cytotoxicity, and cytokine synthesis. This observation was not a foregone conclusion, given that at least a subset of CAR T cells expresses a tyrosine kinase that is inhibited by ibrutinib (ITK). We also demonstrated an additive effect of combining BTK signaling inhibition with the direct cytotoxicity delivered by CTL019. This finding indicates that the combined ibrutinib and CART19 anti-MCL cell activity stems from their direct effect on the malignant B lymphocytes.

The in vitro studies were followed by a clear demonstration of superiority of CTL019 over ibrutinib in the MCL xenotransplant mouse model when each was used as monotherapy at clinically relevant doses and schedules of administration (single dose for CTL019, continuous administration for ibrutinib) and despite the fact that we used a higher dose of ibrutinib than that employed by most groups (20). This approach is supported by our dose-titration experiments and by the fact that the dose of ibrutinib that is used in MCL therapy is higher than that the one to treat CLL.

When combining ibrutinib with CTL019 in vivo, we observed complete and long-lasting tumor responses. We also noted higher numbers of circulating CTL019 cells; ibrutinib is known to lead to a peripheral blood lymphocytosis, predominantly thought to be due to mobilization of malignant B lymphocytes from lymph nodes through inhibition of CXCR4 pathway (34–36). To our knowledge, T-cell lymphocytosis has not been formally demonstrated in patients treated with ibrutinib. Our results indicate that the T-cell lymphocytosis is not specific to antigen-specific cells, as untransduced control T cells were also shown to increase in the peripheral blood. The observed lymphocytosis does not appear to be related to increased proliferation or enhanced T-cell survival, and may be related to differential T-cell trafficking. Current data implicate CXCR4 in malignant lymphocyte trafficking in some models (35,37) and although we did not find CXCR4 to be differentially expressed in ibrutinib-treated mice, our data do not exclude functional involvement of the CXCR4–SDF1 pathway.

Most preclinical work showing the efficacy of CTL019 has been performed using B-ALL cell lines, which are not sensitive to ibrutinib (23). Furthermore, the strongest clinical responses to date have been obtained in patients with B-ALL, whereas patients with diffuse large B-cell lymphoma and indolent B-cell lymphomas have somewhat lower response rates (15). The reasons for this seemingly tumor type–specific heterogeneous responses to CTL019 remain to be elucidated.

The kinetics of the tumor response and subsequent progression suggest that ibrutinib either deepens the initial response achieved by CTL019 alone, or enhances the long-term immunosurveillance capacity of CTL019 cells. In an infectious model, Dubovsky and colleagues (20) showed that ibrutinib enhances the percentage of antigen-specific CD8 T cells and increases the percentage of both CD4 and CD8 T cells that bear CD62L, a marker of memory T-cell differentiation. However, we did not see changes in T-cell polarization, effector function, or memory subsets in the combination therapy in our model; if found, these would have pointed toward immunologic memory as a potential mechanism of action. The most stringent test for initiation of memory is by tumor rechallenge in animals that have cleared disease. However, in this model, the only animals that successfully cleared tumor long-term are those who received the combination therapy and therefore there is not a suitable control group with which to compare. Therefore, the exact mechanism(s) of the strong antilymphoma effect of the CTL019/ibrutinib combination remains to be elucidated but most likely reflects the advantage of simultaneous direct targeting of malignant cells with two therapeutic modalities with vastly different modes of action. The observation that T cells, including CTL019 cells, are mobilized into the peripheral blood may also help to explain the augmented antitumor effect that we observed.

Recently, ibrutinib has been found to enhance the antitumor effect of blockade of the PD1/PD-L1 system in mouse models (38), a phenomenon that was accompanied by enhanced antitumor immune responses. These authors did not show reduction of PD1 or PD-L1 molecules upon exposure to ibrutinib. In contrast, here we found that tumor-infiltrating CTL019 cells had lower PD-1 expression if the animals were also treated with ibrutinib and these results were further corroborated by in vitro studies showing that exposure to MCL cells led to a marked increase in inhibitory receptors (“immune checkpoint molecules”) on CTL019 that was partially abrogated by cotreatment with ibrutinib. These observations may suggest that this two-pronged antitumor approach derives additional synergy from ibrutinib-mediated T-cell mobilization and from ibrutinib-mediated reduction in inhibitory receptor expression on CAR T cells.

Regardless of the above uncertainties, this is the first preclinical study that combines signal transduction inhibition with adoptive T-cell immunotherapy by targeting BTK and CD19, respectively. Our findings document a potent additive therapeutic effect of this novel and highly promising combination acting by enhanced killing of the MCL cells. They also pave the way for clinical trials of this and similar non–cross-resistant combinations in patients with MCL and other types of B-cell lymphoma.

M.V. Maus and M. Kalos have ownership interest (including patents) in Novartis. S.F. Lacey, M. Milone, C.H. June, S. Gill and M.A. Wasik report receiving commercial research grants from Novartis. S.J. Schuster is a consultant/advisory board member for Pharmacyclics, and reports receiving commercial research support from Janssen, Novartis, and Pharmacyclics. M. Ruella, S.S. Kenderian, J.A. Fraietta, M.V. Maus, M. Milone, M. Kalos, C.H. June, S. Gill, and M.A. Wasik are listed as co-inventors on patents in the area of CAR T cells that are owned by the University of Pennsylvania and licensed to Novartis. All authors work under a research alliance involving the University of Pennsylvania and Novartis pharmaceuticals. No potential conflicts of interest were disclosed by the other authors.

Conception and design: M. Ruella, S.S. Kenderian, J.A. Fraietta, Q. Zhang, S.J. Schuster, M. Kalos, S. Gill, M.A. Wasik

Development of methodology: M. Ruella, S.S. Kenderian, J.A. Fraietta, Q. Zhang, M.V. Maus, M. Milone, S. Gill

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Ruella, S.S. Kenderian, O. Shestova, J.A. Fraietta, M.V. Maus, X. Liu, S. Nunez-Cruz, M. Klichinsky, O.U. Kawalekar, S.F. Lacey, M. Milone, A.R. Mato, S.J. Schuster, S. Gill

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Ruella, S.S. Kenderian, J.A. Fraietta, S. Qayyum, Q. Zhang, X. Liu, O.U. Kawalekar, S. Gill, M.A. Wasik

Writing, review, and/or revision of the manuscript: M. Ruella, S.S. Kenderian, S. Nunez-Cruz, A. Mato, S.J. Schuster, M. Kalos, C.H. June, S. Gill, M.A. Wasik

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Ruella, O. Shestova, Q. Zhang

Study supervision: M. Ruella, Q. Zhang, S.F. Lacey, C.H. June, S. Gill, M.A. Wasik

The authors thank Fang Chen and Natalka Kengle for performing Luminex assay.

This was supported by grants from the University of Pennsylvania-Novartis Research Alliance, Lymphoma Research Foundation (2013–2015 MCL E/D), and JB Cox Charitable Lead Trust. S. Gill is an American Society of Hematology Scholar. M.V. Maus is supported by NCI K08-166039. M. Klichinsky is funded by a NIH T32-GM008076. Imaging was performed at the University of Pennsylvania Small Animal Imaging Facility (SAIF) Optical/Bioluminescence Core, supported by NIH grant CA016520. S.J. Schuster received Philanthropic support from the Jim and Frannie Maguire Lymphoma Research Fund.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Perez-Galan
P
,
Dreyling
M
,
Wiestner
A.
Mantle cell lymphoma: biology, pathogenesis, and the molecular basis of treatment in the genomic era
.
Blood
2011
;
117
:
26
38
.
2.
Gladden
AB
,
Woolery
R
,
Aggarwal
P
,
Wasik
MA
,
Diehl
JA.
Expression of constitutively nuclear cyclin D1 in murine lymphocytes induces B-cell lymphoma
.
Oncogene
2006
;
25
:
998
1007
.
3.
Chandran
R
,
Gardiner
SK
,
Simon
M
,
Spurgeon
SE.
Survival trends in mantle cell lymphoma in the United States over 16 years 1992–2007
.
Leuk Lymphoma
2012
;
53
:
1488
93
.
4.
Petro
JB
,
Rahman
SMJ
,
Ballard
DW
,
Khan
WN
. 
Bruton's tyrosine kinase is required for activation of I kappa B kinase and nuclear factor kappa B in response to B cell receptor engagement
.
J Exp Med
2000
;
191
:
1745
53
.
5.
Krysov
S
,
Dias
S
,
Paterson
A
,
Mockridge
CI
,
Potter
KN
,
Smith
KA
, et al
Surface IgM stimulation induces MEK1/2-dependent MYC expression in chronic lymphocytic leukemia cells
.
Blood
2012
;
119
:
170
9
.
6.
Advani
RH
,
Buggy
JJ
,
Sharman
JP
,
Smith
SM
,
Boyd
TE
,
Grant
B
, et al
Bruton tyrosine kinase inhibitor ibrutinib (PCI-32765) has significant activity in patients with relapsed/refractory B-cell malignancies
.
J Clin Oncol
2013
;
31
:
88
94
.
7.
Treon
SP
,
Tripsas
CK
,
Meid
K
,
Warren
D
,
Varma
G
,
Green
R
, et al
Ibrutinib in previously treated Waldenstrom's macroglobulinemia
.
N Engl J Med
2015
;
372
:
1430
40
.
8.
Wang
ML
,
Rule
S
,
Martin
P
,
Goy
A
,
Auer
R
,
Kahl
BS
, et al
Targeting BTK with ibrutinib in relapsed or refractory mantle-cell lymphoma
.
N Engl J Med
2013
;
369
:
507
16
.
9.
Wang
ML
,
Blum
KA
,
Martin
P
,
Goy
A
,
Auer
R
,
Kahl
BS
, et al
Long-term follow-up of MCL patients treated with single-agent ibrutinib: updated safety and efficacy results
.
Blood
2015
;
126
:
739
45
.
10.
Woyach
JA
,
Furman
RR
,
Liu
TM
,
Ozer
HG
,
Zapatka
M
,
Ruppert
AS
, et al
Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib
.
N Engl J Med
2014
;
370
:
2286
94
.
11.
Chiron
D
,
Di Liberto
M
,
Martin
P
,
Huang
X
,
Sharman
J
,
Blecua
P
, et al
Cell-cycle reprogramming for PI3K inhibition overrides a relapse-specific C481S BTK mutation revealed by longitudinal functional genomics in mantle cell lymphoma
.
Cancer Discov
2014
;
4
:
1022
35
.
12.
Maude
SL
,
Frey
N
,
Shaw
PA
,
Aplenc
R
,
Barrett
DM
,
Bunin
NJ
, et al
Chimeric antigen receptor T cells for sustained remissions in leukemia
.
N Engl J Med
2014
;
371
:
1507
17
.
13.
Davila
ML
,
Riviere
I
,
Wang
X
,
Bartido
S
,
Park
J
,
Curran
K
, et al
Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia
.
Sci Transl Med
2014
;
6
:
224ra25
.
14.
Lee
DW
,
Kochenderfer
JN
,
Stetler-Stevenson
M
,
Cui
YK
,
Delbrook
C
,
Feldman
SA
, et al
T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial
.
Lancet
2015
;
385
:
517
28
.
15.
Kochenderfer
JN
,
Dudley
ME
,
Kassim
SH
,
Somerville
RP
,
Carpenter
RO
,
Stetler-Stevenson
M
, et al
Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor
.
J Clin Oncol
2015
;
33
:
540
9
.
16.
Porter
DL
,
Levine
BL
,
Kalos
M
,
Bagg
A
,
June
CH
. 
Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia
.
N Engl J Med
2011
;
365
:
725
33
.
17.
Ruella
M
,
Gill
S
. 
How to train your T cell: genetically engineered chimeric antigen receptor T cells versus bispecific T-cell engagers to target CD19 in B acute lymphoblastic leukemia
.
Expert Opin Bio Ther
2015
;
15
:
761
6
.
18.
Fisher
DT
,
Chen
Q
,
Appenheimer
MM
,
Skitzki
J
,
Wang
WC
,
Odunsi
K
, et al
Hurdles to lymphocyte trafficking in the tumor microenvironment: implications for effective immunotherapy
.
Immunol Invest
2006
;
35
:
251
77
.
19.
Berg
LJ
,
Finkelstein
LD
,
Lucas
JA
,
Schwartzberg
PL
. 
Tec family kinases in T lymphocyte development and function
.
Ann Rev Immunol
2005
;
23
:
549
600
.
20.
Dubovsky
JA
,
Beckwith
KA
,
Natarajan
G
,
Woyach
JA
,
Jaglowski
S
,
Zhong
Y
, et al
Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes
.
Blood
2013
;
122
:
2539
49
.
21.
Kohrt
HE
,
Sagiv-Barfi
I
,
Rafiq
S
,
Herman
SE
,
Butchar
JP
,
Cheney
C
, et al
Ibrutinib antagonizes rituximab-dependent NK cell-mediated cytotoxicity
Blood
2014
;
123
:
1957
60
.
22.
Belaud-Rotureau
MA
,
Parrens
M
,
Dubus
P
,
Garroste
JC
,
de Mascarel
A
,
Merlio
JP
. 
A comparative analysis of FISH, RT-PCR, PCR, and immunohistochemistry for the diagnosis of mantle cell lymphomas
.
Mod Pathol
2002
;
15
:
517
25
.
23.
Milone
MC
,
Fish
JD
,
Carpenito
C
,
Carroll
RG
,
Binder
GK
,
Teachey
D
, et al
Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo
.
Mol Ther
2009
;
17
:
1453
64
.
24.
Gill
S
,
Tasian
SK
,
Ruella
M
,
Shestova
O
,
Li
Y
,
Porter
DL
, et al
Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells
.
Blood
2014
;
123
:
2343
54
.
25.
Kenderian
SS
,
Ruella
M
,
Shestova
O
,
Klichinsky
M
,
Scholler
J
,
Song
D
, et al
CD33 directed chimeric antigen receptor T cell therapy as a novel preparative regimen prior to allogeneic stem cell transplantation in acute myeloid leukemia
.
Biol Blood Marrow Transplantation
2015
;
21
:
S25
S6
.
26.
Zhang
Q
,
Wang
H
,
Kantekure
K
,
Paterson
JC
,
Liu
X
,
Schaffer
A
, et al
Oncogenic tyrosine kinase NPM-ALK induces expression of the growth-promoting receptor ICOS
.
Blood
2011
;
118
:
3062
71
.
27.
Zhang
Q
,
Wei
F
,
Wang
HY
,
Liu
X
,
Roy
D
,
Xiong
QB
, et al
The potent oncogene NPM-ALK mediates malignant transformation of normal human CD4(+) T lymphocytes
.
Am J Pathol
2013
;
183
:
1971
80
.
28.
Kalos
M
,
Levine
BL
,
Porter
DL
,
Katz
S
,
Grupp
SA
,
Bagg
A
, et al
T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia
.
Sci Transl Med
2011
;
3
:
95ra73
.
29.
Kenderian
SS
,
Ruella
M
,
Shestova
O
,
Klichinsky
M
,
Aikawa
V
,
Morrissette
JJ
, et al
CD33 Specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia
.
Leukemia
2015
;
29
:
1637
47
.
30.
Ma
J
,
Lu
P
,
Guo
A
,
Cheng
S
,
Zong
H
,
Martin
P
, et al
Characterization of ibrutinib-sensitive and -resistant mantle lymphoma cells
.
Br J Haematol
2014
;
166
:
849
61
.
31.
Balasubramanian
S
,
Lan
V
,
Chen
J
,
Tamayo
AT
,
Wang
M
,
O'Brien
S
, et al
Activity of Bruton's tyrosine kinase (Btk) inhibitor PCI-32765 in mantle cell lymphoma (MCL) identifies Btk as a novel therapeutic target [abstract]
. In:
Proceedings of the 53rd ASH Annual Meeting and Exposition; 2011 Dec 10–13; San Diego, CA
.
Washington, DC
:
ASH
; 
2011
. Abstract nr 3688.
32.
Noonan
KA
,
Huff
CA
,
Davis
J
,
Lemas
MV
,
Fiorino
S
,
Bitzan
J
, et al
Adoptive transfer of activated marrow-infiltrating lymphocytes induces measurable antitumor immunity in the bone marrow in multiple myeloma
.
Sci Transl Med
2015
;
7
:
288ra78
.
33.
Honigberg
LA
,
Smith
AM
,
Sirisawad
M
,
Verner
E
,
Loury
D
,
Chang
B
, et al
The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy
.
Proc Natl Acad Sci U S A
2010
;
107
:
13075
80
.
34.
Chang
BY
,
Francesco
M
,
De Rooij
MF
,
Magadala
P
,
Steggerda
SM
,
Huang
MM
, et al
Egress of CD19(+)CD5(+) cells into peripheral blood following treatment with the Bruton tyrosine kinase inhibitor ibrutinib in mantle cell lymphoma patients
.
Blood
2013
;
122
:
2412
24
.
35.
de Rooij
MF
,
Kuil
A
,
Geest
CR
,
Eldering
E
,
Chang
BY
,
Buggy
JJ
, et al
The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia
.
Blood
2012
;
119
:
2590
4
.
36.
Byrd
JC
,
Furman
RR
,
Coutre
SE
,
Flinn
IW
,
Burger
JA
,
Blum
KA
, et al
Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia
.
N Engl J Med
2013
;
369
:
32
42
.
37.
Ngo
HT
,
Leleu
X
,
Lee
J
,
Jia
X
,
Melhem
M
,
Runnels
J
, et al
SDF-1/CXCR4 and VLA-4 interaction regulates homing in Waldenstrom macroglobulinemia
.
Blood
2008
;
112
:
150
8
.
38.
Sagiv-Barfi
I
,
Kohrt
HE
,
Czerwinski
DK
,
Ng
PP
,
Chang
BY
,
Levy
R
. 
Therapeutic antitumor immunity by checkpoint blockade is enhanced by ibrutinib, an inhibitor of both BTK and ITK
.
Proc Natl Acad Sci U S A
2015
;
112
:
E966
72
.