Introduction: The BTK inhibitor, ibrutinib is FDA-approved in MCL and CLL, with activity in the majority of CD20+ B-cell malignancies. As rituximab-combination chemotherapy is today's standard of care in CD20+ B-cell malignancies, we previously investigated and determined ibrutinib antagonizes rituximab-dependent NK-cell mediated cytotoxicity (ADCC) due to ibrutinib's secondary irreversible binding to interleukin-2 inducible tyrosine kinase (ITK) which is required for FcR-stimulated NK cell function including calcium mobilization, granule release, and overall ADCC. We hypothesized that BTK inhibitors, BTK-InhA (ACP-196), BTK-InhB (BGB-3111) and BTK-InhC (undisclosed), each with lower ITK binding, may preserve NK cell function and therefore synergize rather than antagonize rituximab. Methods: Rituximab and trastuzumab-dependent NK-cell mediated cytotoxicity was assessed using lymphoma and HER2+ breast cancer cell lines as well as autologous CLL tumor cells. In vitro NK cell cytokine secretion, degranulation and cytotoxicity were assessed by IFN-g release, CD107a mobilization and chromium release. Results: FcR-stimulated NK cells following exposure to rituximab-coated lymphoma cells or trastuzumab-coated HER2+ breast cancer cells express high and moderate levels of ITK and BTK, respectively. Ibrutinib, in a dose-dependent manner (0.1 and 1uM), and not BTK-InhA, BTK-InhB or BTK-InhC (each at 1uM), inhibited both rituximab- and trastuzumab-induced NK cell cytokine secretion in vitro (Fig A *p=.018, **p=.002, ***p<.001). Similarly, BTK-InhA, BTK-InhB, and BTK-InhC had no inhibitory effect while ibrutinib prevented rituximab- and trastuzumab-stimulated NK cell degranulation by ~50% and ~85% at 1uM, respectively (Fig B *p=.034, **p=.024, ***p=.004, ****p=.002). Interestingly, BTK-InhA, BTK-InhB, and BTK-InhC each resulted in greater in vitro cytotoxicity of rituximab-coated, chromium-labeled lymphoma cells compared to ibrutinib at high NK:target cell ratios (Fig C *p=.048, **p=.009). Similarly, only ibrutinib abrogated in vitro cytotoxicity of trastuzumab-coated, chromium-labeled breast cancer cells with no impact due to BTK-InhA, BTK-InhB, or BTK-InhC (Fig C ***p<.001). At a constant NK:target cell ratio (25:1) and at all concentrations of rituximab tested (range 0.1 to 10ug/mL), 1uM of ibrutinib but not CGI-1746, BTK-InhA, BTK-InhB or BTK-InhC, reduced lysis of chromium-labeled Raji and autologous CLL tumor cells by ~4-fold (Fig D *p<.001). Conclusions: Ibrutinib is clinically effective as monotherapy and in combination with rituximab, despite inhibition of ADCC in vitro and in vivo murine models due to ibrutinib's secondary irreversible binding to ITK. Preclinically, the efficacy of therapeutics which do not inhibit NK cell function, including three novel BTK inhibitors, is superior to ibrutinib. Clinical investigation is needed to determine the impact of this finding on patients with lymphoma receiving rituximab. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures Wang: BeiGene: Employment. Lannutti:Acerta Pharma: Employment. Johnson:Acerta Pharma: Employment.

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Abscopal effects, systemic tumor regression following localized therapy, are induced by radiation therapy and augmented with intratumoral immunostimulation. Based on a preclinical lymphoma model, we previously investigated low-dose immunostimulation with a Toll-like receptor 9 (TLR9) agonist in combination with fractionated, low-dose radiation therapy in relapsed/refractory NHL (NCT00185965) and Mycosis Fungoides (NCT00226993). In an attempt to improve the potency of the immune responses and the rate of clinical responses, the dose of CpG was increased 3-fold and enrollment broadened to include treatment-naive as well as relapsed/refractory low-grade lymphoma (NCT00880581). We treated 15 treatment-naive patients and 15 relapsed/refractory patients with follicular lymphoma using low-dose radiotherapy to a single tumor site followed by 18mg of the C-G enriched, synthetic oligodeoxynucleotide (CpG) TLR9 agonist, PF-3512676 injected at the same site, with injections repeated 10 times weekly. Clinical responses were assessed at distant, untreated tumor sites. Immune responses were evaluated by measuring T-cell activation after in vitro re-stimulation with autologous tumor cells. The in situ vaccination with escalated-dose CpG was well tolerated with 16 cases of grade 1 to 2 local or systemic reactions including 2 cases of possibly-related autoimmune disease and no treatment-limiting adverse events. Among treatment-naive and relapsed/refractory patients, four and three patients, respectively, had partial responses with median duration of response of 29 and 12 weeks, respectively. Two and four patients, respectively, had stable disease of duration greater than one year with median time to best clinical benefit among patients with a response or stable disease of 31 and 12 weeks. The range of time to best response was broad, from 10 to 184 weeks (see Figure). Median progression-free survival was similar among treatment-naive and relapsed/refractory patients, at 41 and 35 weeks, respectively, and median overall survival was not reached in either cohort with median follow-up of 2.6 and 3.5 years. Importantly, in response to in situ vaccination, all patients made tumor-specific immune responses within 2 to 4 weeks post-vaccination with the most informative markers being the activation marker CD278 (ICOS) for CD4 T cell response among the CD45RO+ memory subset, and perforin and granzyme B for CD8 T cell responses. Based on the anti-lymphoma activity observed we have recently initiated two Phase I/II dose-escalation trials of a second-generation TLR9 agonist and radiation therapy in relapsed/refractory low-grade NHL and relapsed NHL post-allogeneic transplant (NCT01745354). Figure 1 Figure 1. Disclosures Advani: Seattle Genetics, Inc.: Other, Research Funding; Genentech: Research Funding; Janssen Pharmaceuticals: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; Takeda International Pharmaceuticals Co.: Research Funding.

Introduction: Kohrt et al., Blood, 2014 demonstrated that ibrutinib antagonizes ADCC function of rituximab in vitro in ADCC assays and in vivo in the DHL-4 xenograft model through inhibition of FcgammaR signaling in immune effector cells, possibly mediated by inhibition of ITK. Obinutuzumab (GA101) is a glycoengineered type II CD20 antibody that mediates higher direct cell death induction than rituximab, and by being glycoengineered mediates enhanced induction of ADCC and ADCP. Here we aimed to investigate the impact of ibrutinib on the immune effector function of obinutuzumab as compared to rituximab. Experimental methods: The impact of ibrutinib (dose range 30, 100, 300 ng/ml to cover Cmax and Ctrough in patients) on NK cell mediated ADCC induction by obinutuzumab and rituximab was investigated using SU-DHL4 and Z138 cells as targets in LDH and chromium release assays or measuring CD16 downmodulation and the degranulation marker CD107a. IFNg release as a surrogate for NK cell activation was investigated using DHL-4 target cells or an autologous in vitro system using leukemic cells derived from CLL/NHL patients. Depletion of CD19 positive B-cells was determined in whole blood from healthy volunteers in flow cytometry-based whole blood assay. In vivo the combination of obinutuzumab or rituximab (10 mg/kg once weekly for 3 weeks) with ibrutinib (25mg/kg BID days 14-28) was investigated in the DHL-4 xenograft model. Results: In ADCC assays, ibrutinib (dose range 30, 100, 300 ng/ml) resulted in a reduction of the ADCC potency of obinutuzumab and rituximab. However, at saturating antibody concentrations of 10 ug/ml, ADCC mediated by obinutuzumab was retained while ADCC mediated by rituximab was strongly reduced as measured by chromium release (Figure 1A). Interestingly, in the whole blood B cell depletion assay only little impact of ibrutinib on obinutuzumab-mediated B cell depletion in terms of EC50 and maximal killing was observed at clinically meaningful concentrations of ibrutinib (30, 100, 300 ng/ml), while the activity of rituximab could be completely abolished with 300 ng/ml ibrutinib (Figure 1B). Notably, control experiments using an effector dead version of obinutuzmab that cannot any longer mediate ADCC or ADCP demonstrate that the retained B cell depletion by obinutuzumab in presence of ibrutinib is not due to direct cell death induction, but also due to immune effector cell mediated function (ADCC and ADCP). In the DHL-4 xenograft model where ibrutinib as a single agent has no anti-tumoral efficacy, the combination resulted in a reduced anti-tumoral efficacy of rituximab, whereas efficacy of obinutuzumab was not affected (Figure 1C). Conclusions: Surprisingly, we found that the inhibitory effect of ibrutinib on the immune effector mediated activity of obinutuzumab is not observed when compared to rituximab. Most notably, ADCC at saturating antibody doses, whole blood B cell depletion and in vivo efficacy of obinutuzumab were retained in presence of clinically relevant concentrations of ibrutinib covering Cmax and Ctrough levels, whereas the activity of rituximab was almost completely abolished under these conditions. We hypothesize that the differential behavior of obinutuzumab and rituximab may be related to the enhanced FcgRIII affinity and stronger FcgRIII signaling activation mediated by obinutuzumab as a consequence of glycoengineering that may subsequently overwrite inhibitory effects of ibrutinib. While the clinical relevance of the observed preclinical antagonism for the combination of rituximab with ibrutinib still needs further clinical investigation, these preclinical data strongly support the clinical investigation of ibrutinib in combination with the glycoengineered Type II CD20 antibody obinutuzumab for the treatment of chronic lymphocytic leukemia and other B-cell malignancies. Figure 1 Figure 1. Disclosures Herter: Roche: Employment. Bacac:Roche: Employment. Umana:Roche: Employment. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

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