Abstract: Background: Umbralisib is an oral, once-daily, novel, dual inhibitor of phosphatidylinositol-3-kinase-delta (PI3Kδ) and casein kinase-1ε (CK1ε) that exhibits improved selectivity for the delta isoform of PI3K. Ublituximab is a novel anti-CD20 monoclonal antibody glycoengineered for enhanced antibody-dependent cellular cytotoxicity that targets a unique epitope on CD20. U2 has been well-tolerated and demonstrated promising activity in heavily pre-treated CLL patients. Herein, results are presented for the randomized, multicenter, Phase 3 UNITY-CLL trial (NCT02612311), which evaluated U2 vs O+Chl in patients with TN and R/R CLL. Methods: Patients ≥18 years of age with treatment-naïve (TN) or relapsed/refractory (R/R) CLL requiring treatment per iwCLL criteria with adequate organ function and ECOG PS ≤2 were eligible. Stratification factors included treatment status (TN vs R/R) and del(17p) status. Patients were initially randomized 1:1:1:1 to receive U2, O+Chl, umbralisib monotherapy, or ublituximab monotherapy. Following establishment of contribution comparing U2 to the single agents, patients were randomized 1:1 to U2 or O+Chl. Umbralisib was given orally at 800 mg once-daily until progression or removal from treatment for other reasons. Ublituximab was administered intravenously at 900 mg on Days 1/2 [split 150/750 mg], 8, and 15 of Cycle 1, Day 1 of Cycles 2 - 6, and on Day 1 every 3 cycles after Cycle 6. O was given intravenously at 1000 mg on Days 1/2 [split 100/900] , 8, and 15 of Cycle 1, and Day 1 of Cycles 2 - 6. Chl was given orally at 0.5 mg/kg on Day 1 and 15 of Cycles 1 - 6. Each cycle was 28 days. The primary endpoint was independent review committee (IRC)-assessed progression-free survival (PFS) of U2 vs O+Chl. Key secondary endpoints included IRC-assessed overall response rate (ORR), complete response (CR), undetectable minimal residual disease (uMRD), duration of response (DOR), and safety (assessed from the first dose until 30 days after the last dose of study medication in each arm) as well as contribution of umbralisib and ublituximab to the U2 combination. Results: From Feb 2016 - Oct 2017, 421 pts were randomized to the U2 (n=210) or O+Chl (n=211) arms. The median age was 67 y (range, 36-91); 57% of patients (n=240) were treatment-naïve; 43% (n=181) had R/R CLL (median number of prior treatments = 1); 10% had del(17p), 20% del(11q), and 56% were IgHV unmutated. 66% were male. Demographics were well-balanced between treatment arms. At a median follow-up of 36.2 mos, U2 significantly prolonged PFS vs O+Chl (median 31.9 mos vs 17.9 mos; HR 0.546, 95% CI 0.413-0.720, P & lt;0.0001; Figure 1). Estimated 24-mo PFS rates with U2 and O+Chl were 60.8% and 40.4%, respectively. PFS improvement with U2 vs O+Chl was consistent across all subgroups examined including treatment naïve patients (median 38.5 mos vs 26.1 mos; HR [95% CI]; 0.482 [0.316-0.736] ); and relapsed/refractory patients (median 19.5 mos vs 12.9 mos; HR [95% CI]; 0.601 [0.415-0.869] ). IRC-assessed ORR was higher with U2 (83.3%; 95% CI, 78.1%-88.6%) vs O+Chl (68.7%; 95% CI, 62.2%-75.2%; P & lt;0.001). Among previously treated subjects, twenty-six (6%) received prior ibrutinib (14 on U2, 12 on O+Chl), with an ORR of 57% observed for U2 compared to 25% for O+Chl in this population. MRD data is currently being analyzed. The median treatment duration was 23 mos for U2 (range, 0.1 - 49 mos) and 5 mos (range, 0.1 - 7 mos) for O+Chl. G3/4 AEs of interest regardless of causality (U2 vs O+Chl) included neutropenia (30.6% vs 34.7%), thrombocytopenia (3.4% vs 13.1%), diarrhea (12.1% vs 2.5%), infusion related reaction (1.9% vs 3.5%), elevated AST/ALTs (8.3% vs 2%), colitis (3.4% vs 0%) and pneumonitis (2.9% vs 0%). AEs led to treatment discontinuation in 34 patients (16.5%) on U2 and 16 patients (7.6%) on O+Chl. Conclusions: UNITY-CLL is the first randomized Phase 3 study in CLL of a PI3Ki vs. chemoimmunotherapy, and the first randomized study of a PI3Ki in treatment-naive CLL. U2 exhibited a well-tolerated safety profile, and significantly improved PFS vs. standard of care chemoimmunotherapy in patients with treatment-naive and relapsed/refractory CLL. Figure Disclosures Gribben: Janssen: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Celgene: Research Funding; Abbvie: Honoraria. Jurczak:Celgene: Research Funding; Afimed: Research Funding; Sandoz-Novartis: Consultancy; European Medicines Agency,: Consultancy; AstraZeneca: Consultancy; Takeda: Research Funding; Janssen China R & D: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Bayer: Research Funding; Acerta: Consultancy, Research Funding; Pharmacyclics: Research Funding; Maria Sklodowska-Curie National Research Institute of Oncology, Krakow, Poland: Current Employment; MEI Pharma: Research Funding; Nordic Nanovector: Research Funding; Servier: Research Funding; Merck: Research Funding; Gilead Sciences: Research Funding; Epizyme: Consultancy; Roche: Research Funding; MorphoSys: Research Funding; TG Therapeutics, Inc.: Research Funding; Jagiellonian University, Krakow, Poland: Ended employment in the past 24 months. Jacobs:Sanofi Genzyme: Speakers Bureau; Genentech: Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Verastem: Consultancy; Seattle Genetics: Consultancy; Astra Zeneca: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; Pharmacyclics: Research Funding, Speakers Bureau; TG Therapeutics, Inc.: Research Funding. Giannopoulos:BMS-Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Janssen: Honoraria. Wrobel:Janssen-Cilag: Honoraria, Research Funding, Speakers Bureau. Zafar:Bristol Meyers Squibb: Honoraria, Other: TRAVEL, ACCOMODATIONS, EXPENSES (paid by any for-profit health care company); Florida Cancer Specialists and Research Institute: Current Employment; Sarah Canon Research Institute: Research Funding; Karyopharm: Honoraria; AstraZeneca: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company). Danilov:Nurix: Consultancy; Celgene: Consultancy; Astra Zeneca: Consultancy, Research Funding; Aptose Biosciences: Research Funding; Gilead Sciences: Research Funding; Verastem Oncology: Consultancy, Research Funding; Pharmacyclics: Consultancy; Abbvie: Consultancy; BeiGene: Consultancy; Takeda Oncology: Research Funding; Karyopharm: Consultancy; TG Therapeutics: Consultancy; Genentech: Consultancy, Research Funding; Rigel Pharmaceuticals: Consultancy; Bristol-Myers Squibb: Research Funding; Bayer Oncology: Consultancy, Research Funding. Burke:Gilead: Consultancy; Epizyme: Consultancy; Adaptive: Consultancy; Kura: Consultancy; Astra Zeneca: Consultancy; Bayer: Consultancy; Bristol Myers Squibb: Consultancy; AbbVie: Consultancy; Adaptive Biotechnologies: Consultancy; Morphosys: Consultancy; Verastem: Consultancy; Roche: Consultancy; Seattle Genetics: Speakers Bureau; Celgene: Consultancy. Goldschmidt:Bristol-Myers Squibb: Speakers Bureau; Amgen: Consultancy; Blue Ridge Cancer Care: Current Employment. Huntington:Genentech: Consultancy; Astrazeneca: Honoraria; TG Therapeutics: Research Funding; Pharmacyclics: Honoraria; Novartis: Consultancy; Celgene: Consultancy, Research Funding; Bayer: Consultancy, Honoraria; AbbVie: Consultancy; DTRM: Research Funding. Pinilla Ibarz:AstraZeneca: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Novartis: Consultancy; Sunesis Pharmaceuticals: Consultancy; TG Therapeutics: Consultancy; Sanofi: Consultancy; Pharmacyclics: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau. Sharman:Bristol Meyers Squibb: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Acerta: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; BeiGene: Research Funding. Siddiqi:AstraZeneca: Consultancy, Research Funding, Speakers Bureau; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy, Research Funding; Juno: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Oncternal: Research Funding; TG Therapeutics: Research Funding; Janssen: Speakers Bureau; Seattle Genetics: Speakers Bureau. Brander:MEI Pharma: Other, Research Funding; Pfizer: Consultancy, Other; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Other, Research Funding; Novartis: Consultancy, Other; Teva: Consultancy, Honoraria; Tolero: Research Funding; ArQule: Consultancy, Other, Research Funding; Ascentage: Other, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Tolero: Research Funding; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; NCCN: Other; Verastem: Consultancy, Honoraria, Other, Research Funding; Teva: Consultancy, Honoraria; Novartis: Consultancy, Other; AstraZeneca: Consultancy, Honoraria, Other, Research Funding; BeiGene: Other, Research Funding; DTRM: Other, Research Funding; NCCN: Other; Genentech: Consultancy, Honoraria, Other, Research Funding; Juno/Celgene/BMS: Other, Research Funding. Kolibaba:TG Therapeutics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Research Funding; Sumitomo Dainippon Pharma Oncology: Consultancy, Other; Verastem: Honoraria; Genentech: Research Funding; Gilead: Research Funding; Janssen: Research Funding; McKesson Life Sciences: Consultancy; Novartis: Research Funding; Pharmacyclics: Research Funding; Acerta: Research Funding; AbbVie: Research Funding; Atara Biotech: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Cell Therapeutics: Research Funding; Compass Oncology: Ended employment in the past 24 months. Ghosh:Karyopharm: Consultancy; Genmab: Consultancy, Speakers Bureau; AbbVie: Speakers Bureau; AstraZeneca: Speakers Bureau; Celgene/Bristol-Myers Squibb: Speakers Bureau; Forty Seven Inc: Consultancy, Other: Research Bureau, Research Funding; Janssen: Consultancy, Research Funding, Speakers Bureau; Juno/Celgene/Bristol-Myers Squibb: Consultancy, Research Funding; Kite/Gilead: Consultancy, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding, Speakers Bureau; SGN: Consultancy, Research Funding, Speakers Bureau; TG Therapeutics: Consultancy, Research Funding; Roche/Genentech: Research Funding. Sportelli:TG Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Miskin:TG Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. O'Connor:Astex Pharmaceuticals: Honoraria, Research Funding; Celgene: Honoraria, Other: Data Safety Monitoring Committee, Research Funding; TG Therapeutics: Current Employment, Current equity holder in publicly-traded company; Kymera Therapeutics: Current equity holder in private company, Honoraria, Membership on an entity's Board of Directors or advisory committees; Nomocan: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; Mundipharma: Other: Consulting; Servier: Consultancy. Weiss:TG Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Flinn:Iksuda Therapeutics: Consultancy; Curis: Research Funding; Infinity Pharmaceuticals: Research Funding; F. Hoffmann-La Roche: Research Funding; Vincera Pharma: Consultancy; Karyopharm Therapeutics: Research Funding; Gilead Sciences: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Triphase Research & Development Corp.: Research Funding; Trillium Therapeutics: Research Funding; TG Therapeutics: Consultancy, Research Funding; Teva: Research Funding; Seattle Genetics: Consultancy, Research Funding; ArQule: Research Funding; Unum Therapeutics: Consultancy, Research Funding; Incyte: Research Funding; Forma Therapeutics: Research Funding; Loxo: Research Funding; BeiGene: Consultancy, Research Funding; Kite Pharma: Consultancy, Research Funding; MorphoSys: Consultancy, Research Funding; Forty Seven: Research Funding; Genentech, Inc.: Research Funding; Great Point Partners: Consultancy; IGM Biosciences: Research Funding; Juno Therapeutics: Consultancy, Research Funding; Acerta Pharma: Research Funding; AbbVie: Consultancy, Research Funding; Celgene: Research Funding; Johnson & Johnson: Other; Yingli Pharmaceuticals ≠: Consultancy, Research Funding; Rhizen Pharmaceuticals: Research Funding; Roche: Consultancy, Research Funding; Curio Science: Consultancy; Nurix Therapeutics: Consultancy; Takeda: Consultancy, Research Funding; Calithera Biosciences: Research Funding; Merck: Research Funding; Constellation Pharmaceuticals: Research Funding; Agios: Research Funding; Portola Pharmaceuticals: Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Pfizer: Research Funding; Novartis: Research Funding.

Abstract: Identification of CD8 + cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro . Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4 + SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)–mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4 + T cells into strategies designed to enhance T cell immunity. IMPORTANCE HIV-1 epitopes catalogued by the Los Alamos National Laboratory (LANL) have yielded limited success in vaccine trials. Because the HLA of infected cells have not previously been assessed for HIV-1 ligands, the objective here was to directly characterize the viral ligands that mark infected cells. Recovery of HLA-presented peptides from HIV-1-infected CD4 + T cells and interrogation of the peptide cargo by mass spectrometric DLS show that typical and atypical viral ligands are efficiently presented by HLA and targeted by human CTLs. Nef and Gag ligands dominate the infected cell's antigenic profile, largely due to extensive ligand sampling from select hot spots within these viral proteins. Also, HIV-1 ligands are often longer than expected, and these length variants are quite antigenic. These findings emphasize that an HLA-based view of HIV-1 ligand presentation to CTLs provides previously unrealized information that may enhance the development of immune therapies and vaccines.