ALBERT

Description: Monoclonal antibodies can block cellular interactions that negatively regulate T-cell immune responses, such as CD80/CTLA-4 and PD-1/PD1-L, amplifying preexisting immunity and thereby evoking antitumor immune responses. Ibrutinib, an approved therapy for B-cell malignancies, is a covalent inhibitor of BTK, a member of the B-cell receptor (BCR) signaling pathway, which is critical to the survival of malignant B cells. Interestingly this drug also inhibits ITK, an essential enzyme in Th2 T cells and by doing so it can shift the balance between Th1 and Th2 T cells and potentially enhance antitumor immune responses. Here we report that the combination of anti–PD-L1 antibody and ibrutinib suppresses tumor growth in mouse models of lymphoma that are intrinsically insensitive to ibrutinib. The combined effect of these two agents was also documented for models of solid tumors, such as triple negative breast cancer and colon cancer. The enhanced therapeutic activity of PD-L1 blockade by ibrutinib was accompanied by enhanced antitumor T-cell immune responses. These preclinical results suggest that the combination of PD1/PD1-L blockade and ibrutinib should be tested in the clinic for the therapy not only of lymphoma but also in other hematologic malignancies and solid tumors that do not even express BTK.

Description: Small-molecule drugs that target the B-cell antigen receptor (BCR) signalosome show clinical efficacy in the treatment of B-cell non-Hodgkin lymphoma. These agents, including the Bruton tyrosine kinase (BTK) inhibitor PCI-32765, display an unexpected response in patients with chronic lymphocytic leukemia (CLL): a rapid and sustained reduction of lymphadenopathy accompanied by transient lymphocytosis, which is reversible upon temporary drug deprivation. We hypothesized that this clinical response reflects impaired integrin-mediated adhesion and/or migration. Here, we show that PCI-32765 strongly inhibits BCR-controlled signaling and integrin α4β1-mediated adhesion to fibronectin and VCAM-1 of lymphoma cell lines and primary CLL cells. Furthermore, PCI-32765 also inhibits CXCL12-, CXCL13-, and CCL19-induced signaling, adhesion, and migration of primary CLL cells. Our data indicate that inhibition of BTK by PCI-32765 overcomes BCR- and chemokine-controlled integrin-mediated retention and homing of malignant B cells in their growth- and survival-supporting lymph node and bone marrow microenvironment, which results in clinically evident CLL regression.

Description: Key Points In B-ALL, cells that express a functional pre-BCR ibrutinib abrogate leukemia cell growth in vitro and in vivo. Effects of ibrutinib in B-ALL not only are mediated through inhibition of BTK but also involve BLK inhibition.

Description: Abstract 883 Specific expression of Bruton's tyrosine kinase (Btk) in osteoclasts (OC), but not osteoblasts (OB), suggests its role in regulating osteoclastogenesis. Although Btk is critical in B cell maturation and myeloid function, it has not been characterized in plasma cell malignancies including multiple myeloma (MM) and Waldenström Macroglobulinemia (WM). We here investigate effects of PCI-32765, an oral, potent, and selective Btk inhibitor with promising clinical activity in B-cell malignancies, on OC differentiation and function within MM bone marrow (BM) microenvironment, as well as on MM and WM cancer cells. We further define molecular targets of Btk signaling cascade in OCs and MM in the BM milieu. In CD14+ OC precursor cells, RANKL and M-CSF stimulate phosphorylation of Btk in a time-dependent fashion; conversely, PCI-32765 abrogates RANKL/M-CSF-induced activation of Btk and downstream PLCγ2. Importantly, PCI-32765 decreased number of multinucleated OC (〉3 nuclei) by tartrate-resistant acid phosphatase (TRAP) staining and the secretion of TRAP5b (ED50 = 17 nM), a specific mature OC marker. It increased size of OCs and number of nuclei per OC, with significantly defective bone resorption activity as evidenced by diminished pit formation on dentine slices. Moreover, lack of effect of Dexamethasone on OC activity was overcome by combination of Dexamethasone with PCI-32765. PCI-32765 significantly reduced cytokine and chemokine secretion from OC cultures, including MIP1α, MIP1β, IL-8, TGFβ1, RANTES, APRIL, SDF-1, and activin A (ED50 = 0.1–0.48 nM). It potently decreased IL-6, SDF-1, MIP1α, MIP1β, and M-CSF in CD138-negative cell cultures from active MM patients, associated with decreased TRAP staining in a dose-dependent manner. In MM and WM cells, immunoblotting analysis confirmed a higher Btk expression in CD138+ cells from majority of MM patients (4 out of 5 samples) than MM cell lines (5 out of 9 cell lines), whereas microarray analysis demonstrated a higher expression of Btk and its downstream signaling components in WM cells than in CD19+ normal bone marrow cells. PCI-32765 significantly inhibits SDF-1-induced adhesion and migration of MM cells. It further blocked cytokine expression (MIP1a, MIP-1β) at mRNA level in MM and WM tumor cells, correlated with inhibition of Btk-mediated pPLCγ2, pERK and NF-kB activation. Importantly, PCI-32765 inhibited growth and survival triggered by IL-6 and coculture with BM stromal cells (BMSCs) or OCs in IL-6-dependent INA6 and ANBL6 MM cells. Furthermore, myeloma stem-like cells express Btk and PCI-32765 (10–100 nM) blocks their abilities to form colonies from MM patients (n=5). In contrast, PCI-32765 has no adverse effects on Btk-negative BMSCs and OBs, as well as Btk-expressing dendritic cells. Finally, oral administration of PCI-32765 (12 mg/kg) in mice significantly suppresses MM cell growth (p〈 0.03) and MM cell-induced osteolysis on implanted human bone chips in a humanized myeloma (SCID-hu) model. Together, these results provide compelling evidence to target Btk in the BM microenvironment against MM and WM., strongly supporting clinical trials of PCI-32765 to improve patient outcome in MM and WM. Disclosures: Chang: Pharmacyclics Inc: Employment. Buggy:Pharmacyclics, Inc.: Employment, Equity Ownership. Elias:Pharmacyclics Inc: Consultancy. Treon:Millennium: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Actelion: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 982 Bruton's tyrosine kinase (Btk) is critical for B-lymphocyte function, due to its involvement in key functions in B-cells, e.g., maturation, activation, and trafficking. These functions are mediated by several receptors, including the B-cell antigen receptor (BCR) and the chemokine receptor CXCR4. Both BCR and CXCR4 play important roles in chronic lymphocytic leukemia (CLL), providing leukemic cells with survival and proliferation advantages. Initial studies in CLL suggest that inhibiting Btk with PCI-32765 (Pharmacyclics, Inc) is an effective therapeutic, reducing the size of solid lymphoid tissues leading initially to lymphocytosis and eventually to decreased blood absolute lymphocyte counts (ALCs). To understand the effect of blocking Btk-mediated signaling in CLL, we utilized an accelerated adoptive transfer CLL mouse model, injecting 5×106 TCL1 leukemia cells into SCID mice that succumb 5–6 weeks after cell transfer. Total 45 mice were injected, separated into 3 groups, and then treated with PCI-32765, at either 2, 3 or 4 weeks after cell transfer, with 5 mice from each group receiving either vehicle control or PCI-32765 (5 or 25 mg/kg/day) in daily drinking water. Mice were bled to track changes in ALCs. Animals treated at 2-weeks post cell transfer with the suboptimal (5mg/kg/day) and optimal (25mg/kg/day) doses exhibited a transient lymphocytosis at day 4, with a 7- and 10-fold increase in circulating TCL1 leukemia cells, respectively (p=0.002). By day 7, these levels had fallen to those of untreated mice. Until week 6, mice receiving the optimal dose of PCI-32765 at week 2 and 3 but not week 4, appeared healthy and had significantly reduced ALCs (p

Description: Abstract 183 CLL is a common leukemia which demonstrates variable reactivity of the B cell receptor (BCR) to antigen ligation, but constitutive activation of this pathway. Bruton's Tyrosine Kinase (BTK) is a member of this pathway which shows transcriptional upregulation and constitutive activity in CLL. Ongoing trials of ibrutinib, an orally bioavailable inhibitor of BTK, have shown outstanding preliminary activity in CLL. However results with other known reversible and more selective irreversible BTK inhibitors have had variable results. As ibrutinib potentially has other targets in addition to BTK, this raises the question of whether BTK is an important target in CLL. To answer this question and determine the role of BTK in the development and expansion of CLL, we have used Eμ-TCL1 (TCL1) transgenic mouse model of CLL, where the TCL1 oncogene is under the control of the VH promoter-IgH-Eμ enhancer and adult transgenic mice spontaneously develop CLL. To determine whether BTK is an important target in CLL as opposed to an alternative target of ibrutinib, we crossed the B6/TCL1 mouse with the XID mouse, which harbors a mutation in the PH domain of BTK, rendering it kinase-inactive. In B-cells derived from these mice we observed continued TCL1 expression and diminished BCR signaling with BCR ligation. XID/TCL1 mice (n=65) had a significantly prolonged overall survival (OS) compared to those with wild-type (WT) BTK (n=79) (Median not reached versus (vs) 13.4 months respectively, p

Description: The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib has been approved by the FDA for the treatment of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma. Patients receiving this treatment often develop lymphocytosis and concomitant reduced organomegaly. These actions of ibrutinib are believed due to egress of CLL cells from lymphoid compartments, although the mechanism(s) responsible for this are not clear. Overexpressed surface membrane chemokine receptor CXCR4 (smCXCR4) is a hallmark of CLL cells and is involved in CLL cell migration and interaction with protective niches. Surface expression of CXCR4 is regulated by phosphorylation at serine/theronine residues in the receptor’s cytoplasmic tail. Kinases including GRK and PIM are known to regulate phosphorylation and surface expression of CXCR4. The transcript and protein levels of BTK, GRK and PIM also correlate with smCXCR4 expression. Here we studied the effect of ibrutinib on CLL cell distribution using TCL1-192 cells, a clonal murine cell line that mimics aggressive CLL and involves active B-cell antigen receptor (BCR) signaling. Similar to patients, TCL1-192-bearing mice receiving ibrutinib developed almost immediately (≤ 1 hour) lymphocytosis that consisted of both non-divided and recently-divided cells. While TCL1-192 cells overexpress smCXCR4, ibrutinib promoted lymphocytosis was associated with a fall in smCXCR4 to a level similar to that on normal B cells from wild type C3H/B6 mice. Reduced levels of smCXCR4 were also observed at the later timepoints when mice still had the continued release of cells into circulation even though lymphocytosis was no longer evident. Despite this change in smCXCR4 expression, total CXCR4 was not changed after ibrutinib treatment. Importantly, cells obtained from ibrutinib treated animals that had reduced levels of smCXCR4 also failed to respond to their ligand CXCL12 in vitro, as measured by absence of smCXCR4 internalization or calcium mobilization. Furthermore, re-expression of smCXCR4 after withdrawal of CXCL12 was not observed in treated cells. We therefore next studied in vitro the mechanism whereby ibrutinib lowered smCXCR4 expression. Western blot analysis using cells treated with ibrutinib at the dose ≥0.1μM at 37⁰C for 2 hours in the presence of CXCL12 showed significantly reduced levels of phosphorylated CXCR4 at Ser339. Phosphorylation of Ser339 in the CXCR4 intracellular domain is known to be essential for normal receptor recycling, hyper-phosphorylated CXCR4 on Ser339 is associated with overexpressed smCXCR4 in CLL patient cells. Correlating with this, TCL1-192 cells treated with ibrutinib at 0.1μM not only de-phosphorylated CXCR4 on Ser339, but also significantly enhanced internalization of smCXCR4 after CXCL12 stimulation. We then examined the kinases responsible for the de-phosphorylation of CXCR4. Western blot analysis of CLL cells from animals treated with ibrutinib for 4 weeks showed minimal levels of total BTK protein. There was a direct positive correlation in between BTK protein expression and smCXCR4 expression. In addition to BTK, reduced levels of kinases known to regulate CXCR4 phosphorylation on Ser339 were also observed in cells collected from ibrutinib treated mice. Together, these results suggest blocking BTK, directly or indirectly causes de-phosphorylation of CXCR4, resulting in impaired smCXCR4 recycling and blocked CXCL12 signaling. The function of CXCR4 is dependent on its location on the cell membrane. Impaired CXCR4 recycling after ibrutinib treatment would lead to defective cell trafficking. Indeed, cell transfer studies involving TCL1-192 cells previously treated with ibrutinib indicated that persistent decrease in smCXCR4, which was ibrutinib-dependent, blocked the return of CLL cells to solid tissue niches. Finally, ibrutinib-fed mice eventually developed lower lymphocyte counts, reduced organomegaly, and prolonged survival. Ibrutinib-fed mice also had defective response to BCR stimulation, including abolished calcium mobilization, blocked cell proliferation and survival after anti-IgM antibody stimulation. Together, the data provide direct in vivo evidence of ibrutinib impairing CLL cell homing and retention and suggest a mechanism by which BTK targeted therapy affects not only BCR signaling but also CXCR4 phosphorylation and signaling, with the latter contributing to defective smCXCR4 expression. Disclosures Chang: Pharmacyclics, Inc: Employment, Equity Ownership. Chang:Pharmacyclics: Employment. Buggy:Pharmacyclics: Employment. Burger:Pharmacyclics: Consultancy, Honoraria, Research Funding.

Description: Introduction: Ibrutinib, a covalent inhibitor of Bruton's tyrosine kinase (BTK), is clinically established as an effective treatment for chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Further, recent trials reported that ibrutinib combined with anti-CD20 monoclonal antibodies (CD20 mAbs) led to overall response rates (ORRs) of 95% and 100% in relapsed CLL patients (Burger et al Lancet Oncol 2014, Jaglowski et al Blood 2015), and an ORR of 87% in relapsed MCL patients (Wang et al ASH 2014), indicating complementary therapeutic effects from the 2 drugs. However, some preclinical studies showed that ibrutinib inhibited antibody-dependent cell-mediated cytotoxicity and phagocytosis (ADCC and ADCP). This discrepancy may be due to differences between experimental conditions (eg, drugs used at micromolar concentrations for sustained periods of time) relative to the pharmacokinetics (PK) of ibrutinib, which has a typical peak plasma concentration (Cmax) of ~200 nM 2 hours (tmax) after oral administration of 420-560 mg, and is cleared rapidly from circulation (Davis et al ASH 2014; Advani et al J Clin Oncol 2013). Using clinical PK as a basis, we designed in vitro and in vivo studies to re-evaluate the impact of ibrutinib on ADCC and ADCP. Methods: To model the effect of covalent inhibition by ibrutinib long after tmax, peripheral blood mononuclear cells (PBMC) from multiple healthy donors were incubated for 2 hrs with 200 nM ibrutinib, washed, and rested for 12 hours to represent a time halfway to the next daily dose. Natural killer (NK) cells purified from these PBMC were cultured with JeKo-1 (MCL line) or primary CLL cells opsonized with the CD20 mAbs rituximab (RTX), ofatumumab (OFA), or obinutuzumab (OBZ; washout protocol). To assess the effect of ibrutinib at tmax, treated PBMC, without being washed or rested, were used to purify NK cells for an ADCC assay in the presence of 200 nM ibrutinib (no wash protocol). Dead target tumor cells were counted by flow cytometry and presented as % maximum killing by NK cells from PBMC treated with vehicle. In ADCP assays, % PBMC-derived macrophages that had phagocytosed target cells are shown. In a xenograft model of MCL, SCID mice (10/group) bearing subcutaneous Mino tumors were treated with CD20 mAbs with or without daily oral dosing with ibrutinib at 12 mg/kg. Tumor size was recorded twice weekly. Plasma concentration of ibrutinib was measured using LC-MS. BTK occupancy in tumor cells by ibrutinib was determined as described previously (Dubovsky et al Blood 2013). Results: The ADCC activity of NK cells treated with ibrutinib rebounded significantly after the washout procedure. Killing of RTX-opsonized JeKo-1 and primary CLL cells rose from 36% and 21% (no wash) to 77% and 71% (wash), respectively. Similar results were obtained using OFA. The killing of OBZ-opsonized JeKo-1 cells was not substantially affected with or without washouts (Fig 1A). Phagocytosis was not affected in the presence of 200 nM ibrutinib (Fig 1B). In tumor-bearing mice, ibrutinib administered at 12 mg/kg by oral gavage resulted in a Cmax of 338 ng/mL (767 nM), exceeding the Cmax observed in patients receiving up to 560 mg a day. However, total drug exposure over time (AUC0-last = 391 ng·h/mL) was within range (Advani et al J Clin Oncol 2013, de Jong et al Cancer Chem Pharmacol 2015). In contrast to previous preclinical study results, ibrutinib significantly enhanced the therapeutic effect of CD20 mAbs (Fig 2). At study termination, we confirmed 98.4 ± 0.3% BTK occupancy in tumor cells. Conclusions: We recommend ibrutinib concentrations used in cellular assays and in vivo models reflect pharmacologic levels of ibrutinib in patients administered 420-560 mg once daily. The optimal dose in animal studies can be determined by PK/PD measurements. Our data indicate that NK-mediated ADCC recovers significantly 12 hours after ibrutinib has been removed, which may be attributed to turnover of ITK, or other signaling components of ADCC that are targets of ibrutinib during the rest period. ADCP activity of macrophages was not affected by 200 nM ibrutinib. These data suggest that the 2 major effector cell types for tumor-targeting antibodies are not as inhibited by clinically relevant concentrations of ibrutinib as have been described previously. This is supported by the present in vivo study data showing that ibrutinib did not inhibit, but augmented the therapeutic effect of CD20 mAbs. Disclosures Ng: Pharmacyclics LLC, an AbbVie Company: Employment. Lu:Pharmacyclics LLC, an AbbVie Company: Employment. Sukbuntherng:Pharmacyclics LLC, an AbbVie Company: Employment. Neuenburg:Pharmacyclics LLC, an AbbVie Company: Employment. James:Pharmacyclics LLC, an AbbVie Company: Employment. Chang:Pharmacyclics LLC, an AbbVie Company: Employment.

Description: Introduction Proliferation of chronic lymphocytic leukemia (CLL) cells is highly dependent on the microenvironment. B-cell receptor (BCR) signaling and interactions of the tumor cells with elements of the tissue microenvironment including T cells and macrophages appear to be of particular importance (Burger et al, Blood 2009; Herishanu at al, Blood 2011; Bagnara at al, Blood 2011). The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib is highly effective in blocking BCR signaling and leads to impressive clinical responses in CLL (Byrd et al, NEJM 2013). BTK is a member of the TEC kinase family that also includes TEC, IL2-inducible T cell kinase (ITK), and BMX/ETK. BTK is not expressed in T cells; however ITK, which is expressed in T cells, is directly inhibited by ibrutinib, and the drug reduces cytokine secretion from activated T cells without inducing apoptosis (Herman et al, Blood, 2011). Here, we sought to determine the in vivo effect of ibrutinib on T cells and cytokine levels in CLL patients treated with single agent ibrutinib. Methods The effect of ibrutinib on T-cell subsets, T-cell activation, and cytokine profiles was assessed in 10 CLL patients treated with 420mg ibrutinib daily in an ongoing phase II trial (NCT01500733). Matched samples of viably frozen peripheral blood mononuclear cells obtained from patients pre-treatment and after 6 months on ibrutinib were analyzed by flow cytometry. Cytokine levels pre-treatment and on days 1, 28, months 2, and 6 on ibrutinib were measured in the same patients using the Milliplex human cytokine assay. Results Consistent with inhibition of BCR signaling in CLL cells, CCL3 and CCL4 serum levels were rapidly and significantly decreased by ibrutinib as described previously (Ponader et al, Blood, 2012). In addition, serum levels of a number of inflammatory cytokines including IL6, IL8, IFNg, and TNFα were decreased by 〉 50% by day 28 of ibrutinib treatment and remained so by 6 months. This is of specific interest as “pseudoexhausted” T cells from CLL patients were recently shown to secrete high amounts of IFNg, and TNFα (Riches et al, Blood 2013). Thus, the decreased levels of inflammatory cytokines may reflect a reversal of T cell “pseduoexhaustion”. Furthermore, the immunosuppressive cytokine IL10, a Th1-type cytokine that is secreted by CLL cells and activated T cells, was also rapidly and significantly reduced. These in vivo data are consistent with previous in vitro data showing decreased secretion of IL6 and IL10 from T cells upon exposure to ibrutinib (Herman et al, Blood, 2011). Thus, ibrutinib appears to reduce cytokine and chemokine secretion from both CLL and T cells resulting in an overall decrease in inflammatory cytokines. While absolute T-cell numbers showed little change on treatment, we found that ibrutinib reduced the frequency of activated CD4+ T cells (Table). Furthermore, for 3 out of 4 patients, the percentage of Ki67 positive T cells in the peripheral blood decreased on ibrutinib therapy (mean decrease 63%). The frequency of the Th17 T-cell subset was also diminished. Consistently, a decrease in serum levels of IL17 was seen in the two patients having detectable IL17 levels pre-treatment. While changes in the cytokine pattern (decrease in IFNg and IL10) might suggest inhibition of a Th1-type response, there was no change in the ratio of Th1 to Th2 T-cell subsets by immunophenotyping. Conclusions We here demonstrate a decrease in the levels of inflammatory cytokines and in T-cell activation in CLL patients treated with ibrutinib. Whether this is a direct consequence of BTK inhibition in B-cells or, at least in part, results from inhibition of T-cell signaling remains to be determined. Nevertheless, our data indicate that ibrutinib significantly alters the composition of the tumor microenvironment in CLL, affecting soluble as well as cellular elements. These effects may be important for clinical response and the development of combination therapies and therefore deserve further study. Supported by the Intramural Research Program of NHLBI. We thank our patients for participating and acknowledge Pharmacyclics for providing study drug. Disclosures: Off Label Use: Ibrutinib in chronic lymphocytic leukemia. Chang:Pharmacyclics: Employment, Equity Ownership. Buggy:Pharmacyclics: Employment, Equity Ownership.