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Background: Patients with advanced-stage iNHL, including follicular lymphoma (FL) and marginal zone lymphoma (MZL), frequently relapse with standard treatment, underscoring a need for novel therapies. Axi-cel autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy is approved for the treatment of R/R large B cell lymphoma after ≥ 2 lines of systemic therapy. Here, we present the primary analysis of ZUMA-5, a Phase 2, multicenter, single-arm study of axi-cel in patients with R/R iNHL. Methods: Adults with FL (Grades 1-3a) or MZL (nodal or extranodal) had R/R disease after ≥ 2 lines of therapy (must include an anti-CD20 mAb plus an alkylating agent), and ECOG 0 - 1. Patients underwent leukapheresis followed by conditioning therapy (cyclophosphamide/fludarabine) and a single infusion of axi-cel at 2 × 106 CAR T cells/kg. The primary endpoint was objective response rate (ORR) by central review (per Lugano classification; Cheson, et al. J Clin Oncol. 2014). Secondary endpoints included complete response (CR) rate, duration of response (DOR), progression-free survival (PFS), overall survival (OS), incidence of adverse events (AEs), and levels of CAR T cells in blood and cytokines in serum. The primary efficacy analysis occurred when ≥ 80 treated patients with FL had ≥ 12-months follow-up. Results: As of 3/12/2020, 146 patients with iNHL (124 FL; 22 MZL) received axi-cel; 84 patients with FL had ≥ 12-months follow-up. The median age was 61 years (range, 34 - 79); 57% of patients were male. Thirty-eight percent of patients had ECOG 1, 86% had stage III/IV disease, 47% had ≥ 3 FLIPI, and 49% had high tumor bulk (GELF). Patients had a median 3 prior lines of therapy (range, 1 - 10); 64% had ≥ 3 prior lines. Progression < 2 years after initial chemoimmunotherapy (POD24) occurred in 55% of patients, and 68% were refractory to last prior treatment. Axi-cel was successfully manufactured for all enrolled patients. With a median follow-up of 17.5 months (range, 1.4 - 31.6), the ORR was 92% among efficacy-evaluable patients with iNHL (n = 104), with a 76% CR rate. In patients with FL (n = 84), the ORR was 94% (80% CR rate); in those with MZL (n = 20), the ORR was 85% (60% CR rate). ORR was comparable across key risk groups analyzed by FLIPI, POD24, GELF, refractory status, and prior lines of therapy. As of the data cutoff, 62% of all treated patients had ongoing responses (64% for FL). The medians for DOR, PFS, and OS were not reached; 12-month estimated rates were 72% (95% CI, 61 - 80), 74% (95% CI, 63 - 82), and 93% (95% CI, 86 - 97), respectively. AEs of any grade occurred in 99% of all treated patients. Grade ≥ 3 AEs occurred in 86% of patients with iNHL (85% in FL; 95% in MZL), most commonly neutropenia (33%), decreased neutrophil count (27%), and anemia (23%). Grade ≥ 3 cytokine release syndrome (CRS; per Lee, et al, Blood. 2014) occurred in 7% of patients with iNHL (6% in FL; 9% in MZL). Grade ≥ 3 neurologic events (NEs; per CTCAE v4.03) occurred in 19% of patients with iNHL (15% in FL; 41% in MZL). Most CRS (118/119) and NEs (81/87) of any grade resolved by data cutoff. Grade 5 AEs occurred in 3 patients: multisystem organ failure in the context of CRS (Day 7; related to axi-cel; n = 1 FL), aortic dissection (Day 399; unrelated to axi-cel; n = 1 FL), and coccidioidomycosis infection (Day 327; unrelated to axi-cel; n = 1 MZL). The median peak CAR T cell level was 38 cells/µL (range, 0 - 1415) in all treated patients with iNHL, with 36 cells/µL (range, 0 - 1415) in those with FL and 53 cells/µL (range, 2 - 453) in those with MZL. The AUC0 - 28 was 448 cells/µL × days (range, 6 - 19,900) in all treated patients with iNHL, with 422 cells/µL × days (range, 6 - 19,900) and 552 cells/µL × days (range, 13 - 6468) in those with FL and MZL, respectively. The median time to peak was 9 days (range, 8 - 371) in all patients, 8 days (range, 8 - 371) in patients with FL, and 15 days (range, 8 - 29) in patients with MZL. In efficacy-evaluable patients with FL, median peak CAR T cell levels were numerically greater in those with ongoing response at 12 months than in those who relapsed (P = .057). In all treated patients with FL, CAR T cell peak was associated with Grade ≥ 3 CRS (P = .031) and NEs (P = .005). Conclusions: Axi-cel had considerable and durable clinical benefit in patients with iNHL, with high ORR and CR rates. Axi-cel had a manageable safety profile, with lower rates of Grade ≥ 3 NEs observed in patients with FL vs those in patients with MZL and those previously reported in aggressive NHL (Locke, et al. Lancet Oncol. 2019). Disclosures Chavez: Genentech: Speakers Bureau; Epizyme: Speakers Bureau; Gilead: Consultancy; Verastem: Consultancy; Novartis: Consultancy; Kite, a Gilead Company: Consultancy, Speakers Bureau; Pfizer: Consultancy; Celgene: Consultancy; AstraZeneca: Speakers Bureau; Morphosys: Consultancy, Speakers Bureau; Merck: Research Funding; Bayer: Consultancy; BeiGene: Speakers Bureau; Karyopharm: Consultancy; AbbVie: Consultancy. Sehgal:Juno Therapeutics: Research Funding; TP Therapeutics: Research Funding; Prothena: Research Funding; Gilead Sciences: Research Funding; Bristol-Myers Squibb: Research Funding; Merck: Research Funding. William:Celgene: Consultancy, Honoraria; Incyte: Research Funding; Seattle Genetics: Research Funding; Merck: Research Funding; Dova: Research Funding; Guidepoint Global: Consultancy; Kyowa Kirin: Consultancy, Honoraria. Munoz:Incyte: Research Funding; Fosunkite: Consultancy; Innovent: Consultancy; Acrotech/Aurobindo: Speakers Bureau; Verastem: Speakers Bureau; AstraZeneca: Speakers Bureau; Genentech/Roche: Research Funding, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; Kyowa: Consultancy, Honoraria, Speakers Bureau; Seattle Genetics: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Bayer: Consultancy, Research Funding, Speakers Bureau; Kite, a Gilead Company: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy; Janssen: Consultancy, Research Funding, Speakers Bureau; Juno/Celgene/BMS: Consultancy, Research Funding, Speakers Bureau; Alexion: Consultancy; Beigene: Consultancy, Speakers Bureau; Merck: Research Funding; Portola: Research Funding; Millenium: Research Funding. Salles:BMS/Celgene: Honoraria, Other: consultancy or advisory role; Kite, a Gilead Company: Honoraria, Other: consultancy or advisory role ; Epizyme: Honoraria, Other: consultancy or advisory role; Janssen: Honoraria, Other: consultancy or advisory role; MorphoSys: Honoraria, Other: consultancy or advisory role; Novartis: Honoraria, Other: consultancy or advisory role; Roche: Honoraria, Other: consultancy or advisory role; Abbvie: Other: consultancy or advisory role; Autolos: Other: consultancy or advisory role; Debiopharm: Consultancy, Honoraria, Other: consultancy or advisory role; Genmab: Honoraria, Other; Karyopharm: Honoraria; Takeda: Honoraria. Munshi:Kite, a Gilead Company: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau. Maloney:Celgene: Consultancy, Honoraria, Research Funding; Bioline Rx: Consultancy, Honoraria; Juno Therapeutics: Consultancy, Honoraria, Patents & Royalties, Research Funding; Gilead Science: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; A2 Biotherapeutics: Consultancy, Current equity holder in publicly-traded company, Honoraria; Genentech: Consultancy, Honoraria. de Vos:Bayer: Consultancy; Verastem: Consultancy. Reshef:Kiadis: Research Funding; Monsanto: Consultancy; Novartis: Honoraria; Magenta: Consultancy; Atara: Consultancy, Research Funding; Celgene: Consultancy; Bristol-Myers Squibb: Research Funding; Takeda: Research Funding; Incyte: Research Funding; Pharmacyclics: Research Funding; Immatics: Research Funding; Shire: Research Funding; Bluebird: Research Funding; Gilead: Consultancy, Honoraria, Other: Travel support, Research Funding. Leslie:Epizyme: Honoraria, Speakers Bureau; Karyopharm: Honoraria, Speakers Bureau; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Speakers Bureau; BMS: Speakers Bureau; KitePharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BeiGene: Honoraria, Speakers Bureau; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Yakoub-Agha:Celgene: Honoraria; Novartis: Honoraria; Gilead/Kite: Honoraria, Other: travel support; Janssen: Honoraria; Jazz Pharmaceuticals: Honoraria. Oluwole:Bayer: Consultancy; Spectrum Pharmaceuticals: Consultancy; Pfizer: Consultancy; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding. Fung:AstraZeneca: Honoraria, Other: speakers' bureau, travel support; Genentech: Honoraria, Other: speakers' bureau, travel support; AbbVie: Honoraria, Other: speakers' bureau, travel support; Kite, a Gilead Company: Honoraria, Other: speakers' bureau, travel support; Sanotif: Honoraria, Other: speakers' bureau, travel support; Takeda: Honoraria, Other: speakers' bureau, travel support; Janssen Oncology: Honoraria, Other: speakers' bureau, travel support. Rosenblatt:Merck: Other: consultancy or advisory role ; Biograph55: Other: consultancy or advisory role, Research Funding; Synergy: Patents & Royalties; University of Miami: Other: Leadership. Rossi:Kite, a Gilead Company: Current Employment; Gilead Sciences: Current equity holder in publicly-traded company. Goyal:Kite, a Gilead Company: Current Employment. Plaks:Kite, a Gilead Company: Current Employment, Other: travel support; Gilead Sciences: Other: stock or other ownership . Yang:Kite, a Gilead Company: Current Employment. Lee:Kite, a Gilead Company: Current Employment; Gilead Sciences: Current equity holder in publicly-traded company. Godfrey:IGM Biosciences: Current Employment, Current equity holder in publicly-traded company. Vezan:Kite, a Gilead Company: Current Employment, Honoraria, Other: Travel support; Abbvie: Current equity holder in publicly-traded company; Merck: Current equity holder in publicly-traded company. Avanzi:Kite, a Gilead Company: Current Employment, Other: travel support; Gilead Sciences: Other: stock or other ownership ; MSKCC: Patents & Royalties. Neelapu:N/A: Other; Calibr: Other; Poseida: Research Funding; Cellectis: Research Funding; Merck: Other: personal fees, Research Funding; Kite, a Gilead Company: Other: personal fees, Research Funding; Adicet Bio: Other; Legend Biotech: Other; Precision Biosciences: Other: personal fees, Research Funding; Incyte: Other: personal fees; Cell Medica/Kuur: Other: personal fees; Allogene Therapeutics: Other: personal fees, Research Funding; Pfizer: Other: personal fees; Celgene: Other: personal fees, Research Funding; Novartis: Other: personal fees; Bristol-Myers Squibb: Other: personal fees, Research Funding; Unum Therapeutics: Other, Research Funding; Karus Therapeutics: Research Funding; Acerta: Research Funding; Takeda Pharmaceuticals: Patents & Royalties.

Introduction : Bispecific antibodies that bridge lymphoma cells to T-cells have shown promise in treating B-cell malignancies. However, existing T cell engaging antibodies are associated with toxicity, especially cytokine release syndrome (CRS), which may limit dose intensity and efficacy. CRS results from overstimulation of T cells, which can also lead to subsequent downregulation of T cell function. IGM-2323 is a novel type of bispecific antibody, based on an engineered pentameric IgM framework, with a recombinant J-chain that is fused to an anti-CD3 scfv. The resulting construct has 10 high-affinity binding domains for CD20 and one binding domain for CD3. In preclinical studies, IGM-2323 has been shown to bind irreversibly to CD20-expressing cells and deplete them in human PBMC cell cultures with limited cytokine secretion. This first-in-human Phase 1, open-label, multicenter, dose escalation study was designed to evaluate the safety, tolerability, biomarkers and preliminary anti-tumor activity of IGM-2323 in adults with B- cell malignancies. Methods : Patients with relapsed or refractory CD20-positive B-cell NHL who had received >= 2 prior systemic therapies, acceptable organ function and ECOG 0-1 are eligible for enrollment. IGM-2323 is administered as an IV infusion on Days 1, 8, and 15 of a 21-day cycle, for up to 8 cycles, or longer if there is evidence of clinical benefit. The study utilized an accelerated titration design, with single patient cohorts for the first two dose levels, followed by traditional 3+3 design with further dose levels. The primary objectives are to evaluate the safety and tolerability of IGM-2323, and to determine a recommended phase 2 dose and schedule. Results : As of June 12, 2020, 8 patients have been treated at 4 dose levels (0.5, 2.5, 10, and 30 mg). NHL subtypes include 3 follicular lymphoma (FL), 2 mantle cell lymphoma, 2 marginal zone lymphoma, and 1 diffuse large B-cell lymphoma. Median age is 65 (range 47-81), median number of prior therapies is 4 (range 2-6). 2 patients had prior autologous transplant (ASCT). All 8 patients have completed at least one cycle and are evaluable for safety and dose limiting toxicities (DLT). 6/8 patients remain on active treatment at the time of the database cutoff. Two patients have discontinued at 25 and 7 weeks, due to patient and investigator decision. Six patients remain on study at 37, 27, 18, 9, 6, and 5 weeks ongoing. Median number of doses is 14. There were no DLT observed and no drug-related SAE. Two patients experienced Grade 3-4 treatment-emergent adverse events of transient neutropenia (0.5 mg cohort G4 Two patients in the 30 mg cohort experienced transient low-grade fever (one grade 1, one grade 2), with neither requiring treatment with steroids. Analysis of pharmacodynamic biomarkers shows measurable levels of circulating B cells at baseline in 1/8 patients, where B cell depletion was observed with treatment. Furthermore, there is evidence of cytokine secretion consistent with repeatable T cell responses over successive doses in both 10 mg and 30 mg cohorts. When cytokines were detectable following dosing, they were transient and were mostly gone at less than 6-12 hours. Interferon-gamma (IFNg) was the primary cytokine observed, with significant levels of IL-6 detected in only one patient. Conclusions : IGM-2323 is the first engineered high-affinity, high-avidity bispecific IgM monoclonal antibody to be tested in the clinic, and is designed to bind irreversibly to CD20-expressing cells while providing a more physiologic stimulation to T cells. Preliminary results from this first in human T-cell engaging antibody study show an improved safety and tolerability profile at higher doses. There is also evidence of a novel mechanism of action based on repeatable T cell activation and preservation of T cell function compared with other T cell engaging antibodies. The study continues to enroll patients in dose escalation. Updated safety, pharmacokinetic, biomarker, and efficacy data will be presented at the meeting. This study is registered with clinicaltrials.gov identifier NCT04082936. Disclosures Budde: F. Hoffmann-La Roche, Kite Pharma: Consultancy; Merck, Amgen, AstraZeneca, Mustang Therapeutics: Research Funding; AstraZeneca: Speakers Bureau. Gopal:Seattle Genetics; Janssen; Takeda; IgM Bio; IMab Bio; BMS; Astra Zeneca; Merck; Gilead: Research Funding; imab bio, takeda,astrazeneca,gilead: Research Funding; IgM bio, BMS, merck: Research Funding; Seattle Genetics; Janssen; IMab Bio; TG Therapeutics; Astra Zeneca; Merck; Gilead; ADC Therapeutics; Nurix; TG therapeutics, Cellectar; Actinium: Consultancy. Flinn:BeiGene: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Celgene: Research Funding; Kite Pharma: Consultancy, Research Funding; Karyopharm Therapeutics: Research Funding; IGM Biosciences: Research Funding; Calithera Biosciences: Research Funding; Loxo: Research Funding; ArQule: Research Funding; Agios: Research Funding; Teva: Research Funding; Seattle Genetics: Consultancy, Research Funding; Portola Pharmaceuticals: Research Funding; Pfizer: Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Novartis: Research Funding; Nurix Therapeutics: Consultancy; Curis: Research Funding; MorphoSys: Consultancy, Research Funding; Curio Science: Consultancy; Constellation Pharmaceuticals: Research Funding; Merck: Research Funding; Forty Seven: Research Funding; Iksuda Therapeutics: Consultancy; Genentech, Inc.: Research Funding; Great Point Partners: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Unum Therapeutics: Consultancy, Research Funding; Infinity Pharmaceuticals: Research Funding; TG Therapeutics: Consultancy, Research Funding; Trillium Therapeutics: Research Funding; Triphase Research & Development Corp.: Research Funding; Verastem: Consultancy, Research Funding; Yingli Pharmaceuticals ≠: Consultancy, Research Funding; Rhizen Pharmaceuticals: Research Funding; Johnson & Johnson: Other; Roche: Consultancy, Research Funding; Vincera Pharma: Consultancy; Gilead Sciences: Consultancy, Research Funding; Forma Therapeutics: Research Funding; F. Hoffmann-La Roche: Research Funding; Incyte: Research Funding; Takeda: Consultancy, Research Funding; Acerta Pharma: Research Funding; AbbVie: Consultancy, Research Funding. Nastoupil:Gilead/KITE: Honoraria; Bayer: Honoraria; Pfizer: Honoraria, Research Funding; Genentech, Inc.: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Karus Therapeutics: Research Funding; Novartis: Honoraria, Research Funding; LAM Therapeutics: Research Funding; TG Therapeutics: Honoraria, Research Funding; Merck: Research Funding; Janssen: Honoraria, Research Funding; Gamida Cell: Honoraria. Keyt:IGM Biosciences: Current Employment, Current equity holder in publicly-traded company. Carroll:IGM Biosciences: Current Employment, Current equity holder in publicly-traded company. Leabman:IGM Biosciences: Current Employment. Hernandez:IGM Biosciences: Current Employment. Sison:IGM Biosciences: Current Employment. Chen:IGM Biosciences: Current Employment, Current equity holder in publicly-traded company. Godfrey:IGM Biosciences: Current Employment, Current equity holder in publicly-traded company. Armand:Adaptive: Consultancy, Research Funding; IGM: Research Funding; Otsuka: Research Funding; Roche: Research Funding; Celgene: Consultancy; Affimed: Consultancy, Research Funding; Genentech: Research Funding; ADC Therapeutics: Consultancy; Sigma Tau: Research Funding; Infinity: Consultancy; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Merck & Co., Inc.: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy; Tensha: Research Funding.

e15007 Background: Bispecific antibodies which tether T-cells to cancer cells, and additionally activate T-cell mediated killing, have shown promise in treating B-cell malignancies. Dose intensity, however, is limited by cytokine release from the activated T-cells. Side effects include fever, hypotension, and hypoxia. Methods: A novel type of bispecific antibody (IGM-2323) was constructed using the pentameric IgM framework. Recombinant IgM heavy chains and kappa light chains with high affinity variable domains derived from an anti-CD20 IgG, were co-expressed with a J-chain fused to anti-CD3 single chain Fv domain. IGM-2323 has 10 binding domains for CD20, and one binding domain for CD3. Results: IGM-2323 binds to the CD20 antigen with > 1000-fold increased avidity relative to an IgG antibody format. IGM-2323 in human cell cultures shows highly potent T cell dependent cytotoxicity (EC50 20 pM), even on cells with very low cell surface expression of CD20, rituximab-resistant lymphoma cell lines, and patient derived CLL cells. Significantly, IGM-2323 exhibits vastly reduced cytokine release, with equivalent T cell dependent killing of CD20-expressing cancer cells, as compared to corresponding IgG-based T cell bispecifics made with the same binding domains. IGM-2323 mediated killing can occur at low T-cell/cancer cell ratios, as low as (1:5). In addition, IGM-2323 mediates complement dependent cytotoxicity (CDC) of CD20-expressing cells with > 100-fold higher potency than IgG. IgM-based bispecific (anti-CD20 x anti-CD3) completely depletes circulating B cells in cynomolgus monkeys at doses of > 300 µg per kg. Furthermore, durable depletion of B cells in spleen and lymphoid tissues was noted with multiple doses of > 1 mg/kg. Importantly, in these studies no observable adverse effects were seen, despite repeated doses at up to 25 mg/kg (the highest dose tested). Low levels of transiently increased circulating IL-6 and no increased levels of circulating TNF-α and IFN-γ were observed in these primate studies. Conclusions: These data indicate that IgM-based bispecifics can induce T cell engagement and very potent cytotoxicity that can be dissociated from cytokine release syndrome in the primate model. These preclinical data demonstrate the potential for broad application of this novel modular IgM-based bispecific antibody format. A Phase I dose-escalation study of IGM-2323 in patients with relapsed/refractory Non-Hodgkin’s lymphoma has been initiated (NCT04082936) and is currently enrolling patients.

Idelalisib, an oral inhibitor of phosphatidylinositol-3-kinase δ (PI3Kδ), was evaluated in a 48-week phase 1 study (50-350 mg daily or twice daily) enrolling 40 patients with relapsed or refractory mantle cell lymphoma (MCL). Primary outcome was safety and dose-limiting toxicity (DLT). Secondary outcomes were pharmacokinetic parameters, pharmacodynamic effects, overall response rate (ORR), progression-free survival (PFS), and duration of response (DOR). Patients without DLT and no evidence of disease progression after 48 weeks enrolled in the extension study. Patients had median age of 69 years (range, 52-83) and received median of 4 prior therapies (1-14); 17 of 40 patients (43%) were refractory to their most recent treatment. Median duration of idelalisib treatment was 3.5 months (range, 0.7-30.7), with 6 (15%) continuing extension treatment. Common grade ≥3 adverse events (AEs) included (total%/grade ≥3%) diarrhea (40/18), nausea (33/5), pyrexia (28/0), fatigue (25/3), rash (23/3), decreased appetite (20/15), upper respiratory infection (20/0), pneumonia (13/10), and alanine transaminase or aspartate transaminase elevations (60/20). ORR was 16 of 40 patients (40%), with CR in 2 of 40 patients (5%). Median DOR was 2.7 months, median PFS was 3.7 months, and 1-year PFS was 22%. These data provide proof of concept that targeting PI3Kδ is a viable strategy and worthy of additional study in MCL. This trial was registered at www.clinicaltrials.gov as #NCT00710528.

Activation of T cells induces the production of T cell growth and survival factor interleukin (IL) 2. Regulatory T cells intrinsically fail to induce IL-2 expression upon activation and can suppress IL-2 production in conventional T cells. Thus, the control of IL-2 expression is critically important to T cell immune responses, yet the mechanisms remain incompletely understood. Nuclear factor (NF) 90 is a zinc-finger DNA- and double-stranded RNA-binding protein subunit that binds specifically to the antigen receptor response element (ARRE)/NF of activated T cells target sequence in the IL-2 proximal promoter. Inducible binding of NF90 to the IL-2 promoter in vivo is shown by chromatin immunoprecipitation. NF90 gene-targeted mice exhibit perinatal lethality. Compared with newborn NF90(+/+) mice, newborn NF90(-/-) mice demonstrate severe impairment of IL-2 expression. Compared with wild-type cells, T cells deficient in NF90 are impaired in ARRE and IL-2 transcriptional activation and IL-2 mRNA stabilization. Fetal liver cells from NF90 gene-targeted mice were transplanted into irradiated adult recombination activating gene (RAG)-2(-/-) and IL-2Rgamma(-/-) mice deficient in T cells, B cells, and natural killer cells. NF90(+/+)- and NF90(-/-)-RAG chimeric mice showed grossly normal repopulation of the thymus and spleen, but only NF90(-/-) T cells were severely impaired in IL-2 gene expression. Compared with littermates, NF90(-/-) RAG chimeric mice exhibited profound T cell lymphocytopenia in the peripheral circulation. Thus, NF90 regulates inducible IL-2 transcription, mRNA stability, and gene expression in T cells and represents a novel therapeutic target for the modulation of T cell immune responses.

NF90 and splice variant NF110/ILF3/NFAR are double-stranded RNA-binding proteins that regulate gene expression. Mice with targeted disruption of NF90 were engineered. NF90(-/-) mice were born small and weak and succumbed to perinatal death within 12 h because of neuromuscular respiratory failure. Lung inflation and morphology were normal in NF90(-/-) mice. The diaphragm and other skeletal muscles in NF90(-/-) mice demonstrated disorganized arrangement and paucity of myofibers, evidence of myocyte degeneration and increased apoptosis. The expression of myogenic regulators, MyoD, myogenin, and p21WAF1/CIP1, was severely decreased in NF90(-/-) mice. These myogenic transcription factors and cell cycle inhibitors are regulated in part through post-transcriptional mRNA stabilization. Northwestern blotting revealed that NF90 is the principal and specific p21WAF1/CIP1 and MyoD 3'-untranslated region RNA-binding protein in developing skeletal muscles. NF90 regulates transcription factors and a cell cycle inhibitor essential for skeletal muscle differentiation and for survival.

CD4+CD25+ T-regulatory (Treg) cells have been shown to critically regulate self- and allograft tolerance in several model systems. Studies of human Treg cells have been restricted by the small number present in peripheral blood and their naturally hypoproliferative state. To better characterize Treg suppressor cell function, we determined methods for the isolation and expansion of these cells. Stringent magnetic microbead-based purification was required for potent suppressor cell line generation. Culture stimulation with cell-sized Dynabeads coated with anti-CD3 and anti-CD28 monoclonal antibodies, CD4+ feeder cells, and interleukin 2, provided for marked expansion in cell number (100-fold), with retention and enhancement of suppressor function. The potent Treg cell lines suppressed proliferation in dendritic cell-driven allo-mixed lymphocyte reaction (MLR) cultures by more than 90%. The Treg-derived suppressor cells functioned early in allo-MLR because expression of activation antigens and accumulation of cytokines was nearly completely prevented. Importantly, cultured Treg cells also suppressed activated and matured dendritic cell-driven responses. These results demonstrate that short-term suppressor cell lines can be generated, and they can express a very potent suppressive activity. This approach will enable more detailed biologic studies of Treg cells and facilitate the evaluation of cultured Treg cells as a novel form of immunosuppressive therapy. (Blood. 2004;104:453-461)

Idelalisib (GS-1101, CAL-101), an oral inhibitor of phosphatidylinositol 3-kinase-δ, was evaluated in a phase I study in 64 patients with relapsed indolent non-Hodgkin lymphoma (iNHL). Patients had a median (range) age of 64 (32-91) years, 34 (53%) had bulky disease (≥1 lymph nodes ≥5 cm), and 37 (58%) had refractory disease. Patients had received a median (range) of 4 (1-10) prior therapies. Eight dose regimens of idelalisib were evaluated; idelalisib was taken once or twice daily continuously at doses ranging from 50 to 350 mg. After 48 weeks, patients still benefitting (n = 19; 30%) enrolled into an extension study. Adverse events (AEs) occurring in 20% or more patients (total%/grade ≥3%) included diarrhea (36/8), fatigue (36/3), nausea (25/3), rash (25/3), pyrexia (20/3), and chills (20/0). Laboratory abnormalities included neutropenia (44/23), anemia (31/5), thrombocytopenia (25/11), and serum transaminase elevations (48/25). Twelve (19%) patients discontinued therapy due to AEs. Idelalisib induced disease regression in 46/54 (85%) of evaluable patients achieving an overall response rate of 30/64 (47%), with 1 patient having a complete response (1.6%). Median duration of response was 18.4 months, median progression-free survival was 7.6 months. Idelalisib is well tolerated and active in heavily pretreated, relapsed/refractory patients with iNHL. These trials were registered at clinicaltrials.gov as NCT00710528 and NCT01090414.

CD134 (OX40) is a member of the TNF receptor family that is expressed on activated T lymphocytes. T cells from mice that lack expression of CD134 made strong responses to a range of challenges, but they showed impaired proliferation in response to direct stimulation through the TCR with monoclonal anti-CD3ε Ab. CD134-deficient mice controlled infection with Leishmania major, Nippostrongylus brasiliensis, and Theiler’s murine encephalomyelitis virus, and they made overtly normal Ab responses to a variety of antigens. Thus, CD134 is not essential for many T cell responses in vivo, nor is it required for the provision of help to B cells. Nonetheless, a subtle role in the regulation of T cell reactivity is suggested by the effect of CD134 deficiency on in vitro T cell responses.

Background Phosphatidylinositol-3-kinase delta (PI3Kδ) mediates B-cell receptor signaling and microenvironmental support signals that promote the growth and survival of malignant B lymphocytes. In a phase 1 study, idelalisib, an orally active selective PI3Kδ inhibitor, showed antitumor activity in patients with previously treated indolent non-Hodgkin’s lymphomas. Methods In this single-group, open-label, phase 2 study, 125 patients with indolent non-Hodgkin’s lymphomas who had not had a response to rituximab and an alkylating agent or had had a relapse within 6 months after receipt of those therapies were administered idelalisib, 150 mg twice daily, until the disease progressed or the patient withdrew from the study. The primary end point was the overall rate of response; secondary end points included the duration of response, progression-free survival, and safety. Results The median age of the patients was 64 years (range, 33 to 87); patients had received a median of four prior therapies (range, 2 to 12). Subtypes of indolent non-Hodgkin’s lymphoma included follicular lymphoma (72 patients), small lymphocytic lymphoma (28), marginal-zone lymphoma (15), and lymphoplasmacytic lymphoma with or without Waldenstrom’s macroglobulinemia (10). The response rate was 57% (71 of 125 patients), with 6% meeting the criteria for a complete response. The median time to a response was 1.9 months, the median duration of response was 12.5 months, and the median progression-free survival was 11 months. Similar response rates were observed across all subtypes of indolent non-Hodgkin’s lymphoma, though the numbers were small for some categories. The most common adverse events of grade 3 or higher were neutropenia (in 27% of the patients), elevations in aminotransferase levels (in 13%), diarrhea (in 13%), and pneumonia (in 7%). Conclusions In this single-group study, idelalisib showed antitumor activity with an acceptable safety profile in patients with indolent non-Hodgkin’s lymphoma who had received extensive prior treatment. (Funded by Gilead Sciences and others; ClinicalTrials.gov number, NCT01282424.)

Phenotypic analysis of hematopoietic stem and progenitor cells (HSCs) has been an invaluable tool in defining the biology of stem cell populations. We have recently described the production of AC133, a monoclonal antibody (MoAb) that binds to a novel cell surface antigen present on a CD34bright subset of human HSCs. This antigen is a glycosylated protein with a molecular weight of 120 kD. Here, we report the molecular cloning of a cDNA encoding this antigen and show that it does not share homology with any previously described hematopoietic or other cell surface antigen(s). The AC133 polypeptide has a predicted size of 97 kD and contains five-transmembrane (5-TM) domains with an extracellular N-terminus and a cytoplasmic C-terminus. Whereas the expression of tetraspan (4-TM) and 7-TM molecules is well documented on mature and immature hematopoietic cells and leukocytes, this 5-TM type of structure containing two large (255–amino acid [aa] and 290-aa) extracellular loops is unique and does not share sequence homology with any known multi-TM family members. Expression of this protein appears limited to bone marrow in normal tissue by immunohistochemical staining; however, Northern analysis suggests that the mRNA transcript is present in a variety of tissues such as the kidney, pancreas, placenta, and fetal liver. The AC133 antigen is also expressed on subsets of CD34+ leukemias, suggesting that it may be an important early marker for HSCs, as well as the first described member of a new class of TM receptors.

Purpose Therapeutic prostate-specific antigen (PSA) –targeted poxviral vaccines for prostate cancer have been well tolerated. PROSTVAC-VF treatment was evaluated for safety and for prolongation of progression-free survival (PFS) and overall survival (OS) in a randomized, controlled, and blinded phase II study. Patients and Methods In total, 125 patients were randomly assigned in a multicenter trial of vaccination series. Eligible patients had minimally symptomatic castration-resistant metastatic prostate cancer (mCRPC). PROSTVAC-VF comprises two recombinant viral vectors, each encoding transgenes for PSA, and three immune costimulatory molecules (B7.1, ICAM-1, and LFA-3). Vaccinia-based vector was used for priming followed by six planned fowlpox-based vector boosts. Patients were allocated (2:1) to PROSTVAC-VF plus granulocyte-macrophage colony-stimulating factor or to control empty vectors plus saline injections. Results Eighty-two patients received PROSTVAC-VF and 40 received control vectors. Patient characteristics were similar in both groups. The primary end point was PFS, which was similar in the two groups (P = .6). However, at 3 years post study, PROSTVAC-VF patients had a better OS with 25 (30%) of 82 alive versus 7 (17%) of 40 controls, longer median survival by 8.5 months (25.1 v 16.6 months for controls), an estimated hazard ratio of 0.56 (95% CI, 0.37 to 0.85), and stratified log-rank P = .0061. Conclusion PROSTVAC-VF immunotherapy was well tolerated and associated with a 44% reduction in the death rate and an 8.5-month improvement in median OS in men with mCRPC. These provocative data provide preliminary evidence of clinically meaningful benefit but need to be confirmed in a larger phase III study.

CD4+CD25+ regulatory T cells (Tregs) are essential negative regulators of immune responses. Here, we examined the signaling properties of human Tregs, using CD4+CD25+ Treg and CD4+CD25– control (Tcont) cell lines generated from cord blood. Treg cell lines were markedly hyporesponsive to stimulation with dendritic cells and with anti-CD3/CD28–coated beads. Hyporesponsiveness was reversed by exogenous interleukin-2 (IL-2). T-cell receptor (TCR)–CD3/CD28–mediated activation of Rap1 and Akt was retained in Tregs, but activation of Ras, mitogenactivated protein kinase 1/2 (MEK1/2), and extracellular signal-regulated kinase 1/2 (Erk1/2) was impaired. Tregs were blocked from cell cycle progression due to decrease of cyclin E and cyclin A and increase of p27kip1 (p27kip cyclin dependent kinase inhibitor). IL-2 induced sustained increase of cyclin E and cyclin A and prevented up-regulation of p27kip1. Tregs had high susceptibility to apoptosis that was reversed by IL-2, which correlated with activation of Erk1/2, up-regulation of Bcl-xL (B-cell CLL/lymphoma 2-like nuclear gene encoding mitochondrial protein, transcript variant 2), and phosphorylation of Bad (Bcl2 antagonist of cell death) at Ser112. Thus, Tregs share biochemical characteristics of anergy, including abortive activation of Ras-MEK-Erk, increased activation of Rap1, and increased expression of p27kip1. In addition, our results indicate that TCR–CD3/CD28–mediated and IL-2 receptor–mediated signals converge at the level of MEK-Erk kinases to regulate Treg survival and expansion and suggest that manipulation of the MEK-Erk axis may represent a novel strategy for Treg expansion for immunotherapy.

Eliminating alloreactive cells from T-cell populations would enable the transfer of immune function to patients who receive stem cell transplants. However, high-efficiency depletion has proved difficult to achieve. We sought to develop ex vivo approaches for the maximal depletion of alloreactive CD4+ T cells. Using a flow cytometric cell sorting approach after mixed lymphocyte reaction (MLR) culture, we have found that sorted CFSEbright (5-(and-6)-carboxyfluorescein diacetate succinmidyl ester) (nondivided) and activation antigen-negative cells are markedly depleted of alloreactivity. With HLA-mismatched peripheral blood mononuclear cell (PBMC) stimulators we have consistently attained (90%-95%) depletion of alloreactivity. Importantly, when purified matured monocyte-derived dendritic cells (DCs) are used as stimulators, a 100-fold (99%) reduction in alloreactivity was attained, resulting in abrogation of the secondary MLR. Significantly, the CFSEbright CD25- cells recovered from these cultures retained general immunoreactivity, including responses to Candida and cytomegalovirus (CMV) antigens. In addition, a CFSE-based approach was tested and found to be sufficient for graft-versus-host disease (GVHD) prevention in vivo, in a major histocompatibility complex (MHC) class II disparate murine model. This efficient approach to selectively deplete mature alloantigen-specific T cells may permit enhanced immune reconstitution without GVHD. (Blood. 2004;103:1158-1165)

Introduction: PI3K-delta signaling is critical for activation, proliferation and survival of B cells, and is hyperactive in many B-cell malignancies. Idelalisib, a selective oral inhibitor of PI3Kd, demonstrated considerable clinical activity as monotherapy in recurrent (Flinn, Blood 2014) or refractory iNHL subjects (Gopal, NEJM 2014). FDA granted accelerated approval for Idelalisib (ZYDELIG®) in patients who have received at least two prior systemic therapies with relapsed FL or SLL. This study evaluated Idelalisib in combination with rituximab, bendamustine, or both. We now present mature safety and response data with up to 4 years of follow up. Methods: Eligible patients had relapsed/refractory indolent NHL. Idelalisib (Z) was administered continuously with rituximab (R) (375 mg/m2 given weekly for 8 doses) (R/Z regimen), with bendamustine (B) (90 mg/m2 given on Days 1 and 2, for 6 cycles) (B/Z regimen), or in combination with R (375 mg/m2, on Day 1) and B (90 mg/m2 given on Days 1 and 2 of each cycle, for 6 cycles (BR/Z regimen). Initial subjects in the R/Z and B/Z groups (n=8 each), received Idelalisib 100 mg/dose BID. Thereafter, all patients received an Idelalisib dose of 150 mg/dose BID. Tumor response was evaluated according to standard criteria (Cheson 2007). The cutoff date for this analysis was June 2014, 26 months after the last patient enrolled. Results: Between April 2010 and May 2012, 79 subjects with iNHL were enrolled (including 59 with FL, 15 with SLL, and 5 with MZL). Median [range] age was 61 [37-84] years. At baseline patients had elevated beta-2 microglobulin (59%), stage IV disease (58%), bulky adenopathy (> 5cm) (48%), anemia (Hgb Frequent adverse events (all grade %/grade 3-4 %) included pyrexia (54/3), nausea (44/0), fatigue (43/4), diarrhea (39/15), rash (38/9), cough (35/0), pneumonia (22/19), pneumonitis (4/3), and febrile neutropenia (3/3). Laboratory abnormalities included lymphopenia (75/62), neutropenia (56/41), anemia (47/10), thrombocytopenia (42/8), and serum transaminase elevations (56/17). Drug was temporarily held for Grade 3/4 ALT/AST elevations, and 8/13 pts (62%) were re-treated without recurrence of ALT/AST elevation. 27% of pts have discontinued therapy due to adverse events. Of the 79 subjects enrolled, 64 had an objective response with an ORR of 81% (95% CI: 70.6-89.0). Complete responses were demonstrated in 26 patients (33%), and partial responses in 38 patients (48%). In addition, 7 patients had stable disease (9%), and 4 patients had progressive disease (5%) as best response on-study. Four patients were non-evaluable, as they did not have follow up CT scans. By treatment subgroup, the ORR were (n=24/32) 75% (95% CI: 57-89) for R/Z, (n=29/33) 88% (95% CI: 72-97) for B/Z, and (n=11/14) 79% (95% CI: 49-95) BR/Z. The CR rates were 25% (n=8/32), 36% (n=12/33), and 43% (n=6/14) respectively; stable disease was noted in 4/32 patients (13%), 3/33 patients (9%), and 0/14 patients in the three groups respectively. ORR/CR by iNHL subtype is: FL (81%/39%), SLL (73%/13%), and MZL (100%/20%). The median progression-free survival is 32.8 months. Median PFS for R/Z group is 29.7 months, B/Z group 32.8 months, and BR/Z group 37.1 months. The PFS at 24 months was 55%, 64%, and 71% for the R/Z, B/Z, and BR/Z groups respectively. The median duration of response has not yet been reached. Median DOR for the R/Z group is 28.6 months, for the B/Z, and BR/Z groups it is not yet reached. The DOR at 24 months was 65%, 67%, and 64% for the R/Z, B/Z, and BR/Z groups respectively. Figure 1: Median overall survival is not yet reached. Conclusions: Idelalisib in combination therapy was well tolerated, had an acceptable safety profile, and was highly effective in this recurrent iNHL population with an ORR of 81%, and CR rate of 33%. Responses are durable beyond 2 years, supporting further evaluation of these combination regimens. Phase 3 trials evaluating the efficacy of Idelalisib in combination with R or BR in iNHL are ongoing (NCT01732913, NCT01732929). Figure 1 Figure 1. Disclosures de Vos: Gilead Sciences: Research Funding. Off Label Use: Zydelig is a kinase inhibitor indicated for the treatment of patients with: 1) Relapsed chronic lymphocytic leukemia (CLL), in combination with rituximab, in patients for whom rituximab alone would be considered appropriate therapy due to other co-morbidities; 2) Relapsed follicular B-cell non-Hodgkin lymphoma (FL) in patients who have received at least two prior systemic therapies; and 3) Relapsed small lymphocytic lymphoma (SLL) in patients who have received at least two prior systemic therapies.. Wagner-Johnston:Gilead Sciences: Research Funding. Coutre:Gilead Sciences: Research Funding. Flinn:Gilead Sciences: Research Funding. Schreeder:Gilead Sciences: Research Funding. Fowler:Gilead Sciences: Research Funding. Sharman:Gilead Sciences: Research Funding. Boccia:Gilead Sciences: Research Funding. Barrientos:Gilead Sciences: Research Funding. Rai:Gilead Sciences: Research Funding. Boyd:Gilead Sciences: Research Funding. Furman:Gilead Sciences: Research Funding. Holes:Gilead Sciences: Employment, Equity Ownership. Kim:Gilead Sciences: Employment, Equity Ownership. Godfrey:Gilead Sciences: Employment, Equity Ownership. Leonard:Gilead Sciences: Research Funding.

Introduction: Rituximab-alkylator combinations are the standard therapies for patients (pts) with iNHL, however, refractory disease nearly uniformly develops. Once iNHL becomes “double-refractory” to both rituximab + alkylating agents, there are limited options to induce durable remissions. PI3K-delta signaling is critical for activation, proliferation and survival of B cells, and is hyperactive in many B-cell malignancies. Idelalisib, a selective oral inhibitor of PI3Kd, demonstrated considerable clinical activity in double-refractory iNHL (Gopal NEJM 2014). FDA granted accelerated approval for Idelalisib (Zydelig®) in patients who have received at least two prior systemic therapies with relapsed FL or SLL. Based on these encouraging initial results, we now describe long-term follow up, safety, and remission durations of this double-refractory iNHL population treated with idelalisib. Methods: Eligible iNHL pts included those with measurable disease refractory to both rituximab and an alkylating agent. Refractory was defined as lack of response to, or progression of lymphoma within 6 months of completion of index therapy, confirmed by imaging. Idelalisib 150 mg PO BID was administered continuously until disease progression or intolerance. Responses were evaluated by an independent review committee, using standard criteria (Cheson, 2007, and Owen 2013). The new data cutoff date for this analysis was June 2014, 20 months after the last patient enrolled. Results: Enrolled pts (N = 125) had a median age of 64 years and included follicular lymphoma (FL) n=72 (58%), small lymphocytic lymphoma (SLL) n=28 (22%), marginal zone lymphoma (MZL) n=15 (12%) and lymphoplasmacytic lymphoma (LPL)/Waldenstrom's macroglobulinemia (WM) n=10 (8%). The median number of prior therapies was 4 [range 2-12], including bendamustine/rituximab (BR) (n=60) and rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP) (n=56) and autologous transplant (n=14). 81 pts (65%) had prior bendamustine, of which 61/81 (75%) were refractory. 112 pts (90%) were refractory to their last regimen, and 99 pts (79%) were refractory to ≥2 regimens. 38 pts (30%) had elevated LDH, and 33 pts (26%) had bulky disease >7 cm. The median time to progression from last prior therapy was 3.9 months. With a median exposure of 11.1 months (range 0.7 to 35.4), the overall response rate (ORR) is 56% (95% CI = 46.8-64.9) with 70 responders, comprising 12 CRs (9.6%), 58 PRs (46.4%). The median time to response was 1.9 months (time of first evaluation) and time to CR was 4.5 months. There were 43 pts with stable disease (SD) (34.4%). 90% of pts experienced some decrease in tumor burden. ORR for iNHL subtypes is: FL (54%), SLL (61%), MZL (47%), and LPL/WM (70%). CR rate for iNHL subtypes is: FL (14%), SLL (4%), MZL (7%), and LPL/WM (0%). Among responders, median DOR is 13.9 (0.03-31.3) months. DOR for iNHL subtypes in months (Figure 1) is: FL 11.8, SLL 13.9, MZL 18.4, and LPL/WM (not yet reached). Median PFS for all pts is 11.0 months, in comparison to a median PFS of the last prior regimen of 3.9 months (p The adverse events include (total%/≥ grade 3%) diarrhea/colitis (50/18), fatigue (30/2), nausea (31/2), cough (32/0), pyrexia (30/2), dyspnea (18/5), rash (14/2), pneumonia (14/11), and pneumonitis (4/3). Based on central laboratory measurements, Grade ≥3 ALT/AST elevations occurred in 18 pts (14%). Drug was temporarily held in these pts, and 11/15 pts (73%) were re-treated without recurrence of ALT/AST elevation. Overall, 30 pts (24%) have discontinued therapy due to adverse events. Conclusions: The prolonged administration of idelalisib was well tolerated, had an acceptable safety profile, and was highly effective in inducing and maintaining remissions in double-refractory iNHL population with an ORR of 56%, PFS of 11 months, and DOR of 13.9 months. The response rate and long term duration of responses in the small number of subjects with LPL/WM is very promising, and will be evaluated in larger trials of this disease. The observed disease control compared to prior regimens suggests the potential for prolonged clinical benefit in this challenging patient population with unmet medical need. Figure 1: Duration of Response by Disease Group. Figure 1:. Duration of Response by Disease Group. Disclosures Gopal: Gilead Sciences: Research Funding. Off Label Use: Zydelig is a kinase inhibitor indicated for the treatment of patients with: 1) Relapsed chronic lymphocytic leukemia (CLL), in combination with rituximab, in patients for whom rituximab alone would be considered appropriate therapy due to other co-morbidities; 2) Relapsed follicular B-cell non-Hodgkin lymphoma (FL) in patients who have received at least two prior systemic therapies; and 3) Relapsed small lymphocytic lymphoma (SLL) in patients who have received at least two prior systemic therapies.. Kahl:Gilead Sciences: Research Funding. de Vos:Gilead Sciences: Research Funding. Wagner-Johnston:Gilead Sciences: Research Funding. Schuster:Gilead Sciences: Research Funding. Jurczak:Gilead Sciences: Research Funding. Flinn:Gilead Sciences: Research Funding. Flowers:Gilead Sciences: Research Funding. Martin:Gilead Sciences: Research Funding. Viardot:Gilead Sciences: Research Funding. Blum:Gilead Sciences: Research Funding. Goy:Gilead Sciences: Research Funding. Davies:Gilead Sciences: Research Funding. Zinzani:Gilead Sciences: Research Funding. Dreyling:Gilead Sciences: Research Funding. Holes:Gilead Sciences: Employment, Equity Ownership. Sorensen:Gilead Sciences: Employment, Equity Ownership. Godfrey:Gilead Sciences: Employment, Equity Ownership. Salles:Gilead Sciences: Research Funding.

Introduction Alkylating agent-rituximab combinations are a current standard of care for patients with iNHL. Most iNHL will eventually become refractory to current therapies. Particularly, once iNHL becomes “double-refractory” to rituximab + alkylating agents, there are few data available on beneficial therapeutic options and development of novel therapies is essential for this patient population. PI3K-delta signaling is critical for activation, proliferation and survival of B cells, and is hyperactive in many B-cell malignancies. Idelalisib, a selective oral inhibitor of PI3Kδ, demonstrated considerable activity in recurrent iNHL in a phase 1 trial (Kahl, ICML 2011). We thus evaluated idelalisib in a phase 2 trial for patients with double-refractory iNHL, an area of unmet medical need. We present mature response data and extended follow-up of this study. Methods Eligible iNHL patients (pts) included those with measurable disease who were refractory to both rituximab and an alkylating agent. Refractory status was defined as lack of response to, or progression of lymphoma within 6 months of completion of therapy, documented by imaging. Idelalisib 150 mg PO BID was administered continuously until disease progression or intolerance. Responses were evaluated by an independent review committee, using standard criteria (Cheson, 2007). The new data cutoff date for this analysis was June 2013, 8 months after the last patient enrolled. Results Enrolled pts (N=125) had a median age of 64 years [range 33-87] and were 64% male. Indolent NHL subtypes included follicular lymphoma (FL) n=72 (58%), small lymphocytic lymphoma (SLL) n=28 (22%), marginal zone lymphoma (MZL) n=15 (12%) and lymphoplasmacytic lymphoma (LPL)/Waldenstrom's macroglobulinemia (WM) n=10 (8%). The median number of prior therapies was 4 [range 2-12], most common regimens included bendamustine/rituximab (BR) (n=60) and rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP) (n=56) and 14 pts (11%) had a prior autologous transplant. 81 pts (65%) had prior bendamustine, of which 61/81 (75%) were refractory. 112 pts (90%) were refractory to their last regimen, and 99 pts (79%) were refractory to ≥2 regimens. Median time since completion of last regimen was 3.9 months. At baseline, pts had elevated LDH (30%), bulky disease >7 cm (26%), anemia ≥grade 1 (51%), neutropenia ≥ grade 1 (24%), and thrombocytopenia ≥grade 1 (34%). With a median follow up 9.4 months, the overall response rate (ORR) was 57% (95% CI = 47.6, 65.6) with 71 responders, comprising 7 CRs (6%), 63 PRs (50%), and 1 minor response (MR) in a WM pt. There were 42 pts with stable disease (SD) (33%). 90% of pts experienced some decrease in tumor burden (Figure 1). Median time to response was 1.9 months (range 1.6-8.3), median time to CR was 3.7 months (range 1.9-12). ORR for iNHL subtypes was: FL (54%), SLL (61%), LPL/WM (80%), and MZL (47%). ORR for bendamustine refractory pts was 59%. ORR for pts with The most common adverse events were (total%/≥ grade 3%) diarrhea (43/13), fatigue (30/2), nausea (30/2), cough (29/0), pyrexia (28/2), dyspnea (18/3), rash (13/2), and pneumonia (11/7). Based on central laboratory measurements, Grade ≥3 ALT/AST elevations occurred in 16 pts (13%). Drug was held for these pts, and 11/14 pts (79%) were re-treated without recurrence of ALT/AST elevation. Grade ≥3 neutropenia occurred in 27%, thrombocytopenia in 6%, and anemia in 2%. 20% of pts have discontinued therapy due to adverse events. Conclusions Idelalisib was well tolerated, had an acceptable safety profile, and was highly effective in this double-refractory iNHL population with an ORR of 57%. The ORR was consistent across all subgroups, regardless of disease histology, number of prior regimens, refractoriness to bendamustine, or tumor bulk. With continued administration of idelalisib, responses were durable beyond one year, substantially exceeding the median DOR for the last prior therapy. Mature response data demonstrate that idelalisib can provide clinical benefit to patients with the unmet medical need of double-refractory iNHL. Disclosures: Gopal: Gilead Sciences: Research Funding. Off Label Use: Idelalisib is a PI3K-delta inhibitor currently in phase III trials for multiple hematologic malignancies. Kahl:Gilead Sciences: Advisory Board Other, Research Funding. De Vos:Gilead Sciences: Advisory Board Other, Research Funding. Wagner-Johnston:Gilead Sciences: Research Funding. Schuster:Gilead Sciences: Research Funding. Blum:Gilead Sciences: Research Funding. Jurczak:Gilead Sciences: Research Funding. Flinn:Gilead Sciences: Research Funding. Flowers:Gilead Sciences: Research Funding. Martin:Gilead Sciences: Research Funding. Viardot:Gilead Sciences: Research Funding. Goy:Gilead Sciences: Research Funding. Davies:Gilead Sciences: Research Funding. Zinzani:Gilead Sciences: Research Funding. Dreyling:Gilead Sciences: Research Funding. Holes:Gilead Sciences: Employment. Li:Gilead Sciences: Employment. Dansey:Gilead Sciences: Employment. Godfrey:Gilead Sciences: Employment. Salles:Gilead Sciences: Research Funding.

8526 Background: PI3K-delta signaling is critical for activation, proliferation and survival of B cells, plays a role in homing and retention in lymphoid tissues, and is hyperactive in many B-cell malignancies. Idelalisib (GS-1101) is a first-in-class, selective, oral inhibitor of PI3Kδ. Initial response rate of 38% was reported previously in iNHL (Kahl, ICML 2011). Long-term follow-up is now presented. Methods: This phase 1 study evaluated the activity of continuous idelalisib monotherapy in pts with relapsed hematologic malignancies. Doses ranged from 50 to 350 mg QD or BID in 8 cohorts. Response was evaluated based on investigator assessments using standard criteria (Cheson, 2007). Pts who continued to benefit were able to enroll in an ongoing extension study. Results: Study enrolled 64 pts with indolent iNHL. iNHL subtypes included 38 FL, 11 SLL, 9 LPL/WM, and 6 MZL. Pts were 69% male, median age [range] of 64 [32E91] years, 58% with refractory disease and 53% with bulky disease (LN diameter ≥5 cm). The median [range] number of prior therapies was 4 [1E10]. The median [range] duration of treatment was 3.8 [0-41] months, with 19 (30%) pts continuing on treatment extension protocol. ORR across all cohorts were 31/64 (48%), with 1 CR (1.6%). The median duration of response (mDOR) was 18.4 months, and median PFS (mPFS) was 7.6 months. For pts dosed with ≥100 mg BID (N=36); the ORR was 24/36 (67%), the mDOR was 15.4 months, and the mPFS was 16.6 months. The ORR for iNHL subtypes was: FL (45%), SLL (64%), LPL/WM (56%), and MZL (33%). Adverse events included (total%/≥G3%) diarrhea (36/8), fatigue (36/3), rash (27/3), nausea (25/2), pyrexia (20/3), chills (20/0), cough (19/2), pneumonia (17/16), and URI (17/0). Lab abnormalities included (total%/≥G3%) ALT/AST elevations (56/25). 8/64 (12.5%) pts discontinued therapy due to potentially treatment-related adverse events. Conclusions: The oral PI3Kδ inhibitor idelalisib is active in heavily pretreated pts with iNHL, can produce durable responses, and has a favorable safety profile. These data support further clinical development; phase 2 and 3 trials in iNHL are ongoing (NCT01732913, NCT01732926). Clinical trial information: NCT00710528.

8501 Background: PI3K-delta is critical for activation, proliferation and survival of B cells and plays a role in homing and retention in lymphoid tissues. PI3Kδ signaling is hyperactive in many B-cell malignancies. Idelalisib is a first-in-class, selective, oral inhibitor of PI3Kδ that has shown monotherapy activity in recurrent MCL (Kahl, ICML 2011). Methods: This phase 1 study is evaluating the activity of continuous idelalisib (Id), 150 mg BID, in combination with everolimus (E) (10 mg PO qD) (Id+E regimen), with bortezomib (V) (1.3 mg/m2 SC day 1, 8, 15 per 28 day cycle) (Id+V regimen), or with rituximab (R) (375 mg/m2, on Day 1) and bendamustine (B) (90 mg/m2x 2), for 6 cycles (Id+BR regimen). Investigators assessed response according to standard criteria (Cheson 2007). Results: Study enrolled 22 patients with relapsed/refractory MCL. Results are from 14 Jan 2013 data cutoff. The 3 cohorts included Id+E (N=12), Id+V (N=6), and Id+BR (N=4). Patients were 73% male, median age [range] of 68 [47E79] years, 32% with refractory disease and 73% stage III/IV. The median [range] number of prior therapies was 3 [1E7]. The median [range] duration of treatment was 2.5 [0.5-8.3+] months. Overall response rate (ORR) was 10/22 (46%), with 2 CR (9%). The ORR/CR for Id+E, was 25%/0%, Id+V was 50%/0%, and Id+BR was 100%/50%. The median duration of response (mDOR) and median PFS (mPFS) were not reached. Most common adverse events included (total%/≥G3%) diarrhea (41/9), fatigue (41/0), rash (27/14), cough (27/0), decreased appetite (23/0), and epistaxis (23/0). Lab abnormalities included (total%/≥G3%) thrombocytopenia (82/27), neutropenia (32/14), and ALT/AST elevations (50/5). Conclusions: Preliminary data indicates idelalisib-based combination therapy is active in patients with relapsed/refractory MCL. All combinations were tolerable. These data support further clinical development in larger trials to further characterize safety and response duration. Clinical trial information: NCT01088048.

8500 Background: PI3K-delta signaling is critical for activation, proliferation and survival of B cells, and is hyperactive in many B-cell malignancies. Idelalisib is a first-in-class, selective, oral inhibitor of PI3Kδ that has shown considerable monotherapy activity in recurrent iNHL (Kahl, ICML 2011), as well as combination therapy (Fowler, ASCO 2012). Methods: This phase I study evaluated the activity of continuous (48 weeks) idelalisib (Id), 100/150 mg BID, in combination with rituximab (R) (375 mg/m2 weekly x 8 doses) (Id+R), with bendamustine (B) (90 mg/m2 x 2, for 6 cycles) (Id+B), or in combination with R (375 mg/m2 monthly x 6) and B (90 mg/m2 x 2), for 6 cycles (Id+BR). Investigators assessed response according to standard criteria (Cheson 2007). Patients who continued to benefit were able to enroll on an extension study. Results: Study enrolled 78 pts with relapsed/refractory iNHL, with 34 (44%) pts continuing on treatment in the ongoing extension protocol. The 3 cohorts included Id+R (N=30), Id+B (N=34), and Id+BR (N=14). Pts were 67% male, median age [range] of 62 [37E84] years, 41% with refractory disease, 88% stage III/IV, and 36% of FL with high FLIPI scores. The median [range] number of prior therapies was 3 [1E10]. The median [range] duration of treatment was 10.6 [0.5-29.2] months. Overall response rate (ORR) was 63/78 (81%), with 22/78 (28%) CR. The ORR/CR for Id+R was 77%/20%, Id+B was 85%/29%, and Id+BR was 79%/43%. At 20 months, the PFS was 66%. For responders, 73% were progression-free at 20 months. Most common adverse events included (total%/≥G3%) pyrexia (56/4), fatigue (45/4), nausea (41/0), rash (40/8), cough (37/0), diarrhea (36/8), chills (18/0), URI (18/1), and pneumonia (17/15). Lab abnormalities included (total%/≥G3%) ALT/AST elevations (56/17). Conclusions: Idelalisib-based combination therapy is highly active and well tolerated in patients with relapsed/refractory iNHL. These data support further clinical development. Phase III trials evaluating the efficacy of idelalisib in combination with R, or BR in iNHL are ongoing (NCT01732913, NCT01732926). Clinical trial information: NCT01732913, NCT01732929.

TPS8618 Background: PI3K-delta is critical for activation, proliferation and survival of B cells and plays a role in homing and retention in lymphoid tissues. PI3Kδ signaling is hyperactive in many B-cell malignancies. Idelalisib is a first-in-class, selective, oral inhibitor of PI3Kδ that reduces proliferation, enhances apoptosis, and inhibits homing and retention of malignant B cells in lymphoid tissues (Lannutti et al, 2011). Phase I trials demonstrated that idelalisib is highly active in pts with heavily pretreated iNHL: pts experienced reductions in disease-associated chemokines, profound and rapid reductions in lymphadenopathy, and durable clinical benefit with an acceptable safety profile (de Vos et al, 2011). Methods: 450 pts with previously treated iNHL, who have measurable lymphadenopathy, require therapy for iNHL, have received prior anti-CD20-antibody-containing therapy and chemotherapy, and who have iNHL that is not refractory to bendamustine (B) are randomized in a 2:1 ratio into Arm A or B. In Arm A, pts receive idelalisib at 150 mg BID continuously + rituximab (R) at 375 mg/m2 every 28 days for 6 cycles + B at 90 mg/m2 on days 1 and 2 of each 28-d cycle. In Arm B, pts receive placebo BID instead of idelalisib. Stratification factors include tumor type (follicular lymphoma vs others), tumor burden (high vs low), and time since completion of last prior therapy for iNHL (

TPS8617 Background: PI3K-delta is critical for activation, proliferation and survival of B cells and plays a role in homing and retention in lymphoid tissues. PI3Kδ signaling is hyperactive in many B-cell malignancies. Idelalisib is a first-in-class, targeted, highly selective, oral inhibitor of PI3Kδ that reduces proliferation, enhances apoptosis, and inhibits homing and retention of malignant B cells in lymphoid tissues (Lannutti et al, 2011). Phase 1 trials demonstrated that idelalisib is highly active in pts with heavily pretreated iNHL: pts experienced reductions in disease-associated chemokines, profound and rapid reductions in lymphadenopathy, and durable clinical benefit with acceptable safety profile (de Vos et al, 2011). Methods: 375 pts with previously treated iNHL, who have measurable lymphadenopathy, have received prior anti-CD20-antibody-containing therapy, and who have iNHL that is not refractory to rituximab (R) are randomized in a 2:1 ratio into Arm A or Arm B. In Arm A, pts receive idelalisib at 150 mg BID continuously + R at 375 mg/m2 (weekly x 4 then every 8 weeks x 4). In Arm B, pts receive placebo BID instead of idelalisib. Stratification factors include tumor type (follicular lymphoma vs others), tumor burden (high vs low), and time since completion of last prior therapy for iNHL (

8519 Background: PI3K-delta signaling is critical for activation, proliferation and survival of B cells and plays a role in homing and retention in lymphoid tissues. PI3Kδ signaling is hyperactive in many B-cell malignancies. Idelalisib is a first-in-class, selective, oral inhibitor of PI3Kδ. Initial response rate of 42% was previously reported in MCL (Kahl, ICML 2011). Long-term follow-up is now presented. Methods: This phase 1 study evaluated the activity of continuous (48 weeks) idelalisib monotherapy in pts with relapsed or refractory hematologic malignancies. Doses ranged from 50 mg BID to 350 mg BID in 8 cohorts. Response was based on investigator assessments using standard criteria (Cheson et al, 2007). Patients who continued to benefit were able to enroll in an extension study. Results: 40 patients with recurrent MCL enrolled. Patients were 88% male, median age [range] of 69 [52-83] years, 43% with refractory disease. The median [range] number of prior therapies was 4 [1E14]. The median [range] duration of idelalisib treatment was 3.5 [1-26+] months, with 6 (15%) patients continuing on treatment in the extension protocol. Overall response rate (ORR) was 16/40 (40%), with 2/40 CR (5%). The median duration of response (mDOR) was 2.7 months, and median PFS (mPFS) was 3.7 months. The 1-year PFS was 22%. For patients dosed with ≥100 mg BID, ORR was 12/23 (52%), for patients dosed with ≥150 mg BID, ORR was 11/16 (69%) including both CR (12.5%). Most common adverse events included (total%/≥G3%); diarrhea (40/18), nausea (33/5), pyrexia (28/0), fatigue (25/3), rash (25/3), decreased appetite (20/15), URI (20/0), and pneumonia (13/13). Abnormal lab values included (total%/≥G3%) ALT/AST elevations (65/20). 6/40 (15%) patients discontinued therapy due to AEs, potentially treatment related. Conclusions: The oral PI3Kδ inhibitor idelalisib (GSE1101) is active and well tolerated in heavily pre-treated pts with MCL. A proportion of patients have long-term (>1 year) clinical benefit. These data support further clinical evaluation of idelalisib in MCL. Clinical trials with idelalisib in combination with other agents are in progress. Clinical trial information: NCT00710528.

The OX–40 protein was selectively upregulated on encephalitogenic myelin basic protein (MBP)–specific T cells at the site of inflammation during the onset of experimental autoimmune encephalomyelitis (EAE). An OX–40 immunotoxin was used to target and eliminate MBP–specific T cells within the central nervous system without affecting peripheral T cells. When injected in vivo, the OX–40 immunotoxin bound exclusively to myelin–reactive T cells isolated from the CNS, which resulted in amelioration of EAE. Expression of the human OX–40 antigen was also found in peripheral blood of patients with acute graft–versus–host disease and the synovia of patients with rheumatoid arthritis during active disease. The unique expression of the OX–40 molecule may provide a novel therapeutic strategy for eliminating autoreactive CD4+T cells that does not require prior knowledge of the pathogenic autoantigen.

193 Background: MVA-BN-PRO is an investigational prostate cancer immunotherapy comprising of a highly attenuated non-replicating vaccinia virus engineered to encode prostate specific antigen (PSA) and prostate acid phosphatase (PAP) proteins. Preclinical studies in mouse tumor models demonstrated vaccine-mediated induction of anti-PSA and PAP specific immune responses and anti-tumor activity. Results of the open-label multi-center evaluation in subjects with non-metastatic castration resistant prostate cancer are presented. Methods: Eligible subjects had documented prostate cancer with a rising PSA while on androgen suppression therapy and were chemotherapy naïve. Three cohorts of subjects were immunized subcutaneously receiving either 1, 2 or 4 injections of study drug (1 injection = 1×108 tissue culture infectious dose, TCID50) at monthly intervals for three months (treatment). If the vaccinations were tolerated, re-treatment with an additional 3 monthly vaccinations was administered at the same dose. Responders, defined as subjects with stable or declining PSA at week 29 compared with baseline, were offered a 1-year extended treatment of up to 12 monthly vaccinations. Subjects were followed for one year after their last vaccination. The primary endpoint was safety. Immune responses (humoral and cellular) to MVA-BN-PRO including the transgenes PSA and PAP were assessed by the ELISA and ELISPOT assays, respectively. Exploratory analyses included anti-tumor activity as evaluated by serum PSA levels and progression by bone scan, per investigator determined, or death. Results: Twenty-four subjects were dosed. All subjects completed the initial 3 vaccinations (treatment) and 21 subjects received 6 vaccinations (re-treatment). Seven responders received additional vaccinations during the extended treatment. No dose-limiting toxicities were reported. There were no reported ≥ Grade 3 treatment-related adverse events (AE). The most common related AEs were Grade 1 or 2 general disorders and administration site reactions. Conclusions: MVA-BN-PRO was well tolerated. Results from immune analysis and clinical activity measured by PSA levels or radiological progression will be presented. Clinical trial information: NCT00629057.

Although reliable antibodies are available that distinguish human suppressor T (Ts) cells from CTL and other T cells, few are available for murine Ts cells. We have developed a mAb (984D4.6.5) that, in the presence of complement, depletes alloantigen-specific Ts cells but not CTL. This antibody recognizes activated Ts cells but not their precursors. In these studies, flow cytometric analysis demonstrates that 984D4.6.5 reacts with several Ts cell hybridomas, cloned Ts cell lines and WEHI-3 (a myelomonocytic tumor cell line). Reactivity was not detected with BW5147, Th cell hybridomas, cloned Th cells, CTL lines and hybridomas, B cell lines, thymocytes, splenocytes, bone marrow cells nor a variety of tumor cells. Among 984D4.6.5 positive lines, expression is heterogeneous and the number of cells expressing high levels of the epitope is increased when the hybridomas are maintained at a relatively high cell density. Neuriminidase and pronase deplete the epitope recognized by mAb 984D4.6.5. Protein synthesis and glycosylation inhibitors also reduce expression of this epitope. These observations suggest that the epitope recognized by 984D4.6.5 is a carbohydrate linked to a polypeptide. This antibody was tested by ELISA for binding to a large panel of carbohydrates and glycolipids coupled to BSA. The only one that bound 984D4.6.5 was LS tetrasaccharide c (NeuNAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc), an O-linked carbohydrate. Comparative analysis shows that both the sequence and the linkage of these sugars are essential to the reactivity with the 984D4.6.5 antibody. This epitope is expressed by a glycoprotein of approximately 200 kDa, as shown by Western blots. The identity of this glycoprotein remains to be determined, but indirect evidence suggests that it is not CD45.

Abstract 3645 Introduction: PI3Kδ drives proliferation and survival in malignant B-cells. GS-1101 is an orally bioavailable, small-molecule inhibitor of PI3Kδ that has shown considerable monotherapy activity when given at dose levels of ≥100 mg BID in patients with heavily pretreated indolent non-Hodgkins lymphoma (iNHL). Methods and Patients: This Phase 1 combination study has evaluated repeated 28-day cycles of GS-1101 with rituximab and/or bendamustine in patients with previously treated iNHL. GS-1101 was administered starting on Day 1 of Cycle 1 with rituximab (R) (375 mg/m2 given weekly for 8 doses) (GS-1101/R regimen), with bendamustine (B) (90 mg/m2 given on Days 1 and 2 of each cycle for 6 cycles) (GS-1101/B regimen), or in combination with R (375 mg/m2, on Day 1 of each cycle for 6 cycles) and B (90 mg/m2 given on Days 1 and 2 of each cycle for 6 cycles (GS-1101/BR regimen). Initial cohorts received a GS-1101 dose of 100 mg/dose BID. Thereafter, all patients received a GS-1101 dose of 150 mg/dose BID. Tumor response was evaluated according to standard criteria (Cheson 2007). Chemokine/cytokine plasma levels were assessed at baseline and on Day 28 of therapy using multiplexed bead suspension arrays. Results: The study enrolled 76 patients with iNHL. Patient characteristics, histological sub-typing, safety, and efficacy results are depicted in the table. The majority of patients were >60 years of age and had undergone extensive prior therapy. Grade ≥3 adverse events and lab abnormalities were generally consistent with those expected with each of the single agents. Lymph node shrinkage was rapid and all evaluable patients had reductions in lymphadenopathy, resulting in overall response rates (ORR) of 77%, 85%, and 77% for the GS-1101/R, GS-1101/B, and GS-1101/BR regimens, respectively. Complete responses (with bone marrow confirmation) were observed in 13%, 16%, and 30% of patients. With median follow-up duration ranging from 28 to 48 weeks, 1-year progression- free survival (PFS) rates were >75% in all treatment groups. Disease-associated chemokines/cytokines were commonly elevated at baseline and were significantly reduced by GS-1101-based combination treatment. Conclusions: A lack of overlapping toxicities allows the oral PI3Kδ inhibitor, GS-1101, to be delivered at the full single-agent starting dose when coadministered with chemoimmunotherapies in heavily pretreated patients with iNHL. GS-1101-based combination therapy with rituximab and/or bendamustine offers major and rapid reductions in lymphadenopathy. All 3 regimens provide durable tumor control. The data from this trial support the development of Phase 3 combination trials of GS-1101 with rituximab- and/or bendamustine-containing regimens in patients with iNHL. Disclosures: Fowler: Gilead Sciences: Membership on an entity's Board of Directors or advisory committees. Off Label Use: Phase I trial of GS-1101, a PI3K delta inhibitor, in B cell malignancies. de Vos:Gilead Sciences: Membership on an entity's Board of Directors or advisory committees. Schreeder:Gilead Sciences: Research Funding. Leonard:Gilead: Consultancy. Coutre:Gilead Sciences: Membership on an entity's Board of Directors or advisory committees. Sharman:Gilead: Honoraria, Research Funding. Boccia:Gilead Sciences: Research Funding. Barrientos:Gilead Sciences: Research Funding. Holes:Gilead: Employment. Lannutti:Gilead Sciences Inc: Employment. Johnson:Gilead Sciences: Employment. Jahn:Gilead: Employment. Miller:Gilead: Employment. Godfrey:Gilead Sciences: Employment.

Three recently published works represent the first descriptions of a potential new family of 5-trans-membrane cell surface glycoproteins.[1][1] [2][2] [3][3] The human AC133 molecule[2][2] [3][3]may indeed be the homologue of mouse prominin,[1][1] [4][4] and Drs Weigmann and colleagues have very

Membrane-type serine protease 1 (MT-SP1)/Matriptase is a type II transmembrane serine protease that is primarily expressed in epithelial cells and is over-expressed in several cancers. MT-SP1 has been implicated in the processes of tumor cell invasion and metastasis, primarily due to its ability to degrade extracellular matrix proteins and to activate the latent forms of hepatocyte growth factor (HGF), urokinase type plasminogen activator and protease-activated receptor 2. To further explore the role of this protein in human cancer, we developed a fully human IgG1, high affinity, neutralizing antibody against MT-SP1 (95/96) using XenoMax® technology. Using this antibody, we confirmed the expression of MT-SP1 on epithelial cells. In addition, we observed MT-SP1 expression on human peripheral blood B lymphocytes and monocytes. Furthermore, expression was also observed on several B cell lymphomas including Ramos, Raji and Daudi cell lines where, in contrast to epithelial cells, MT-SP1 was found in the absence of its inhibitor and cognate receptor, HGF activator inhibitor -1 (HAI-1). As 95/96 is a potent inhibitor of MT-SP1 enzymatic activity, we used the antibody to examine the functional role of MT-SP1 in B cell lymphoma. Using a tripeptide fluorogenic substrate of MT-SP1, we detected cell surface enzymatic activity on Ramos cells that was completely inhibited by 95/96, implying that the MT-SP1 expressed on Ramos cells is catalytically active. The ability of 95/96 to modulate the invasive properties of Ramos lymphoma cells was evaluated using a rodent hind-limb paralysis model. The median survival of SCID mice systemically inoculated with Ramos lymphoma cells, and receiving 95/96, was significantly extended compared to the animals receiving an isotype-matched control antibody (35 days vs. 26 days, p < 0.01). 95/96 had no effect on the in vitro proliferation or invasion of Ramos cells and was unable to induce cell death in a whole blood assay or by complement-dependent cytolysis. Collectively, these data suggest a role for MT-SP1 catalytic activity in the invasion or metastasis of Ramos B cell lymphoma.

CD4+CD25+ regulatory T cells (Tr) are negative regulators of immune responses. Studies of human Tr are restricted by their small numbers in peripheral blood and their hypoproliferative state. A recently established method achieved in vitro expansion and generation of Tr cell lines (Godfrey et al; Blood 2004,104:453-61). This approach facilitates the evaluation of cultured Tr cells as a novel form of immunosuppressive therapy and provides a system for molecular analysis of Tr. Activation of Ras and MAP kinases is mandatory for IL-2 production, viability and cell cycle progression of T cells. In anergic T cells activation of these signaling events is impaired, whereas activation of Rap1 is retained. Subsequently, anergic cells have defective IL-2 production, impaired cell cycle progression, and increased susceptibility to apoptosis. In the current study, we sought to determine the signaling and biochemical properties of Tr. Human CD4+CD25+ (Tr) and control CD4+CD25− (Tc) cell lines were generated from human cord blood cells. We examined activation of Ras, Rap1 and MAP kinases as well as cell cycle progression and cell viability, in response to TCR/CD3-plus-CD28 mediated stimulation. Stimulation was done for 15 min, 2 and 16 hrs for assessment of signaling events or for 24, 48 and 72 hrs for assessment of cell cycle progression and viability. Although activation of Rap1 was not affected, activation of Ras was reduced in Tr as compared to Tc. Activation of JNK and Erk1/2 MAP kinases was also significantly impaired. Both Tr and Tc entered the cell cycle and expressed cyclin E and cyclin A at 24 and 48 hrs of culture. However, p27 was downregulated only in Tc and not in Tr and hyperphosphorylation of Rb, which is the hallmark of cell cycle progression, was detected only in the Tc and not in the Tr population. At 72 hrs of culture, expression of cyclin E and cyclin A was dramatically diminished in Tr whereas it remained unchanged in Tc. More strikingly, expression of p27 in Tr was increased to levels higher than background. Since Tr do not produce IL-2, we examined whether addition of exogenous IL-2 would downregulate p27 and rescue Tr from defective cell cycle progression, similarly to its effect on anergic cells. Addition of exogenous IL-2 resulted in decrease of p27, sustained increase of cyclin E and cyclin A and cell cycle progression. Besides inhibiting cell cycle progression, p27 also promotes apoptosis. Therefore, we examined whether Tr had a higher susceptibility to apoptosis. As determined by Annexin V staining, Tr had a high degree of apoptosis only at 72 hrs of culture, when p27 expression was highly upregulated. Exogenous IL-2 reversed both p27 upregulation and apoptosis. Addition of IL-2 to Tr, also resulted in sustained IL-2-receptor-mediated activation of Erk1/2 at levels equivalent to those of Tc. Thus Tr cells share many biochemical and molecular characteristics of anergy, including defective TCR/CD3-plus-CD28-mediated activation of Ras and MAP kinases, increased expression of p27, defective cell cycle progression and high susceptibility to apoptosis. Moreover, these results suggest that TCR/CD3-mediated and IL-2 receptor-mediated signals converge at the level of MAP kinases to determine the fate of Tr cells towards expansion or cell cycle arrest and subsequent apoptosis.

CD4+CD25+ T regulatory (Treg) cells have been shown to critically regulate self and more recently allograft tolerance in mice. Studies of human Treg have been hindered by the presence of CD25-dim conventional T cells that copurify during Treg isolation. We compared adult and cord blood sources for Treg, with the hypothesis that cord blood should lack significant numbers of memory cells or environmentally reactive T cells. We found that cord blood was a superior source for Treg isolation compared to adult blood. CD4+CD25+ cells were readily purified, and generated cell lines that consistently exhibited potent suppressor activity, with >95% suppression of allogeneic MLR (29/30 donors). The cells could markedly suppress an allo-MLR at a 1/16-1/32 suppressor/responder cell ratio. The cultured Treg cells blocked cytokine accumulation in MLR, with a less robust inhibition of chemokine production. Cultured Treg uniformly expressed CD25, CD62L, CCR7, CD27, and intracellular CTLA4. Upon re-stimulation with anti-CD3/CD28 beads, the cultured Treg produced minimal cytokines (IL2, IFN-gamma, and IL10), and preferentially expressed TGF-beta latency associated protein on the cell surface, while conventional CD4+CD25− derived cell lines did not. Cytokine production however, could be largely restored by stimulation with PMA/ionomycin. Abundant FoxP3 mRNA was detected in fresh, cultured, and anti-CD3/CD28 bead restimulated CD4+CD25+ cells (approximately 2–4 fold less than cyclophillin A). Low amounts of FoxP3 mRNA were present in fresh CD25− cells, and they increased 32 fold on culture. However, FoxP3 protein, as assessed by western blot, was specifically expressed in the CD25+ derived cell lines, and was not detected in the CD25− derived cell lines. Restimulation with anti-CD3/CD28 beads led to increased expression of FoxP3 protein in CD25+ derived cell lines, but not in CD25− derived cell lines. Cord blood derived cultured suppressor cells were not cytolytic in chromium release assays, and mediated suppression of alloreactivity that was cell contact dependent and predominantly independent of IL10 and TGF-beta. Antibodies to GITR, GITR-L, OX40, OX40-L, CTLA4, and PD1 did not significantly affect suppression of the culture activated Treg cells. These results demonstrate virtually pure populations of potent suppressor cells can be cultured from cord blood, and these cell lines form an ideal model system for the evaluation of suppressor cell biology and mechanisms of action.