Wang, Victoria

Abstract The targeted therapy ibrutinib inhibits B cell receptor signaling (BTK inhibitor) and has yielded high response rates and durable remissions in patients with chronic lymphocytic leukemia (CLL). However, it is widely believed that the addition of immune therapies to targeted drugs will be required to activate anti-tumor immunity and work towards curative therapy. Identifying effective combinations of targeted drugs and/or standard chemotherapy with immunotherapy is a priority research area and particularly relevant for CLL, as patients' T cells have been shown to exhibit profound tolerance/exhaustion and notably, no activity was reported in a recent trial of anti-PD-1 immunotherapy for relapsed disease. Ibrutinib has shown beneficial immunomodulatory activity in CLL by inhibiting IL-2-inducible T cell kinase (ITK) as well BTK that is associated with increased effector CD4+and CD8+ T cell numbers and decreased expression of inhibitory checkpoint receptors such as PD-1 on patient T cells. Here we have performed comparative immune bioassays from a randomized phase III trial comparing ibrutinib-based therapy to traditional FCR chemoimmunotherapy to assess the effects of treatments on anti-tumor T cell function. Viable peripheral blood mononuclear cell samples were collected serially (baseline, 6 months and 12 months) from CLL patients on the randomized phase III E1912 trial of ibrutinib and rituximab versus FCR for previously untreated disease to allow longitudinal batched immune analysis. Cytotoxicity assays revealed that highly purified CD3+ T cells from the FCR treated patients at 6 and 12-month time-points did not change their activated killing function against autologous baseline CD19+ CLL tumor B cells (acting as target antigen-presenting cells pulsed with superantigen, sAg) compared to pre-treatment/baseline exhausted T cells (n=22). In contrast, patients treated with ibrutinib-based therapy had a significant increase in activated anti-tumor T cell killing function (P<.01, n=22) at both 6-month (66% increase) and 12-month (89% increase) time-points. Flow cytometric analysis of circulating immune subsets revealed that the percentage of PD-1 and PD-L1 positive cells among CD8+ and CD4+ T cells (particularly effector compartments) were reduced with ibrutinib-based therapy, whereas only a partial reduction was detected following FCR treatment. However, patients' T cells from both treatment arms responded normally to T cell receptor engagement by upregulating these checkpoint molecules. This led us to explore ex vivo treatment of highly purified CD3+ T cells and CD19+ CLL B cells from both treatment arms with anti-PD-L1 or anti-PD-1 immunotherapy prior to cytotoxicity assays. Our functional data revealed that the T cells from both FCR time-points (6 and 12-months) were not sensitive to either anti-PD-L1 (n=14) or anti-PD-1 (n=14) treatment. In contrast, ibrutinib-based treatment sensitized anti-tumor T function (23% increase in killing) following anti-PD-L1 treatment (n=14) at the 6-month time-point only (P<.01) but not with anti-PD-1. To investigate the mechanism underlying these effector function differences, we compared the ability of highly purified CD3+ T cells from each treatment arm (n=45) time-point to form F-actin immunological synapses with baseline autologous CLL tumor B cells. Quantitative confocal image analysis revealed that ibrutinib-based therapy significantly (P<.01) enhanced polarization of F-actin, tyrosine-phosphorylated proteins and granzyme B at immune synapses with tumor cells at both 6 and 12-month time-points, whereas FCR treated patient T cells failed to mobilize these lytic synapse molecules. Importantly, our assays have revealed that T cells from both FCR treatment time-points formed "non-polarized" immune synapses with tumor cells, in keeping with cytotoxic dysfunction and insensitivity to additional checkpoint immunotherapy. In contrast, our functional correlative bioassays have revealed that ibrutinib-based therapy can reactivate exhausted cytolytic T cell function and suggest to us, a potential therapeutic window for anti-PD-L1 immunotherapy at the earlier 6-month time-point. We believe this data supports the concept of incorporating functional bioassays to immune-monitoring assays associated to clinical trials that should aid knowledge-led design of future combination immunotherapy. Disclosures Kay: Janssen: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Shanafelt:Pharmacyclics: Research Funding; Genentech: Research Funding; GlaxoSmithKline: Research Funding; Jansen: Research Funding. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding; MedImmune: Research Funding.

Abstract BACKGROUND: Chronic lymphocytic leukemia (CLL) is one of the most common lymphoid malignancies, accounting for ~11% of all hematologic neoplasms. Over the last 15 years, a series of phase 3 trials have established that chemoimmunotherapy (CIT) with fludarabine, cyclophosphamide, and rituximab (FCR) improves both progression free survival (PFS) and overall survival (OS) compared with chemotherapy alone. FCR is the gold standard for young fit patients with treatment naïve CLL. In parallel with the advances in CIT, a profound increase in the understanding of CLL B-cell biology led to new therapeutic approaches.1 Among these, ibrutinib (an irreversible inhibitor of Bruton's Tyrosine Kinase [BTK]) has had the largest impact on clinical practice to date. Initial trials of ibrutinib demonstrated robust and durable efficacy in patients with relapsed/refractory disease. Subsequent phase 3 trials showed improved PFS and OS with ibrutinib relative to chlorambucil in previously untreated, older CLL patients. Despite these advances, the efficacy of ibrutinib as a first-line treatment for younger CLL patients (i.e. <70) relative to the most efficacious CIT regimens, such as FCR, is unknown. METHODS: Eligible patients were treatment-naive individuals with CLL who were age <70 and required therapy. Patients with deletion 17p- were excluded from participating given the poor response of these patients to FCR therapy. Participants were randomly assigned in a 2:1 ratio to receive ibrutinib (420 mg/day until disease progression) and rituximab (50 mg/m2 on day 1 of cycle 2; 325 mg/m2 on day 2 of cycle 2; 500 mg/m2 on day 1 of cycles 3-7) or six courses of intravenous fludarabine (25 mg/m2 ) and cyclophosphamide (250 mg/m2) days 1-3 with rituximab (50 mg/m2 on day 1 of cycle 1; 325 mg/m2 on day 2 of cycle 1; 500 mg/m2 on day 1 of cycles 2-6) every 28-days. The planned accrual was 519 patients. Hematologic toxicity was graded according to the 2008 IWCLL Working Group scale. All other adverse events were graded according to the NCI Common Toxicity Criteria (version 4). The primary endpoint was PFS with a secondary endpoint of overall survival (OS). Analysis was by intention to treat. The first planed interim analysis for PFS was scheduled to occur 24-27 months after full accrual with the first interim analysis for OS scheduled to occur if the boundary for PFS was crossed. The primary analysis was a stratified logrank test applied to all patients as randomized. Treatment effect p-values are one-sided. The study was approved by the Central Institutional Review Board for the National Cancer Institute, conducted in accordance with the principles of the Declaration of Helsinki, and registered with ClinicalTrials.gov (NCT02048813). RESULTS: A total of 529 patients were accrued between January 31, 2014 and June 9, 2016. 354 patients were assigned to ibrutinib and rituximab (IR) and 175 to FCR. Nineteen patients did not start protocol therapy. The first interim analysis was performed September 2018. With median follow-up of 33.4 months, we observed 77 PFS events and 14 deaths. The hazard ratio (HR) for PFS favored IR over FCR (HR=0.352; 95% CI 0.223-0.558; p<0.0001) which crossed the pre specified boundary. The HR for OS also favored the IR arm (HR=0.168, 95% CI 0.053-0.538; p=0.0003, pre-specified boundary for superiority p=0.0005). Kaplan-Meier estimates for PFS and OS are shown in Figure 1A and 1B. In subgroup analysis for PFS, IR was superior to FCR independent of age, sex, performance status, disease stage or the presence/absence of del11q23. With current follow-up, IR was also superior to FCR for IGHV unmutated patients (HR=0.262; 95% CI 0.137-0.498; p<0.0001) but not IGHV mutated patients (HR=0.435; 95% CI 0.140-0.1350; p=0.07). Grade 3 and 4 treatment-related adverse events were observed in 58% of IR and 72% of FCR treated patients (p=0.0042). Specifically, FCR was more frequently associated with grade 3 and 4 neutropenia (FCR: 69 [44%] of 158 vs. IR: 80 [23%] of 352; p<0·0001) and infectious complications (FCR: 28/158 [17.7%] vs. IR: 25/352 [7.1%]; p<0.0001). CONCLUSIONS: The combination of ibrutinib and rituximab provides superior PFS and OS relative to FCR for patients with previously untreated CLL age <70. These findings have immediate practice changing implications and establish ibrutinib-based therapy as the most efficacious first-line therapy for patients with CLL. Disclosures Shanafelt: Mayo Clinic: Patents & Royalties: Physician Well-being Index, Medical Student Well-being Index, Well-being index; Celgene: Research Funding; GlaxoSmithKline: Research Funding; Genentech: Research Funding; Abbvie: Research Funding; Pharmacyclics: Research Funding; Janssen: Research Funding. Kay:Janssen: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees. O'Brien:Amgen: Consultancy; Astellas: Consultancy; Celgene: Consultancy; GlaxoSmithKline: Consultancy; Janssen: Consultancy; Aptose Biosciences Inc.: Consultancy; Vaniam Group LLC: Consultancy; Abbvie: Consultancy; Alexion: Consultancy; Kite Pharma: Research Funding; Regeneron: Research Funding; Acerta: Research Funding; Gilead: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding. Barrientos:Pharmacyclics/AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; astra Zeneca: Membership on an entity's Board of Directors or advisory committees. Erba:Seattle Genetics: Consultancy, Research Funding; Pfizer: Consultancy, Other: grant; Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy, Other: grant; Pfizer: Consultancy, Other: grant; Incyte: Consultancy, Speakers Bureau; Agios: Consultancy, Speakers Bureau; Agios: Consultancy, Speakers Bureau; Astellas: Research Funding; Janssen: Research Funding; Janssen: Research Funding; Amgen: Research Funding; Amgen: Research Funding; Novartis: Consultancy, Speakers Bureau; Astellas: Research Funding; Incyte: Consultancy, Speakers Bureau; Astellas: Research Funding; Pfizer: Consultancy, Other: grant; Novartis: Consultancy, Speakers Bureau; Juno: Research Funding; Juno: Research Funding; Novartis: Consultancy, Speakers Bureau; MacroGenics: Consultancy; MacroGenics: Consultancy; Amgen: Research Funding; Takeda/Millenium: Research Funding; Amgen: Research Funding; Takeda/Millenium: Research Funding; Seattle Genetics: Consultancy, Research Funding; Jazz: Consultancy, Speakers Bureau; Immunogen: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Jazz: Consultancy, Speakers Bureau; Seattle Genetics: Consultancy, Research Funding; Celgene: Consultancy, Speakers Bureau; Takeda/Millenium: Research Funding; Immunogen: Consultancy, Research Funding; Takeda/Millenium: Research Funding; Glycomimetics: Consultancy, Other: Chair, Data and Safety Monitoring Committee; Celgene: Consultancy, Speakers Bureau; MacroGenics: Consultancy; Daiichi Sankyo: Consultancy, Research Funding; MacroGenics: Consultancy; Glycomimetics: Consultancy, Other: Chair, Data and Safety Monitoring Committee; Daiichi Sankyo: Consultancy, Research Funding; Juno: Research Funding; Juno: Research Funding; Agios: Consultancy, Speakers Bureau; Agios: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; Immunogen: Consultancy, Research Funding; Jazz: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Immunogen: Consultancy, Research Funding; Glycomimetics: Consultancy, Other: Chair, Data and Safety Monitoring Committee; Celgene: Consultancy, Speakers Bureau; Daiichi Sankyo: Consultancy, Research Funding; Glycomimetics: Consultancy, Other: Chair, Data and Safety Monitoring Committee; Daiichi Sankyo: Consultancy, Research Funding. Stone:Juno: Consultancy; Macrogenics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding; FujiFilm: Consultancy; Sumitomo: Consultancy; Ono/Theradex: Consultancy; Otzuka/Astex: Consultancy; Pfizer: Consultancy; Roche: Consultancy; AbbVie: Consultancy, Research Funding; Actinium: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Argenx: Membership on an entity's Board of Directors or advisory committees; Arog: Consultancy, Research Funding; Astellas: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; Celator / Jazz: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cornerstone: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Tallman:ADC Therapeutics: Research Funding; Daiichi-Sankyo: Other: Advisory board; BioSight: Other: Advisory board; AbbVie: Research Funding; Cellerant: Research Funding; AROG: Research Funding; Orsenix: Other: Advisory board.