SummaryThe specificity of T lymphocyte activation is determined by engagement of the T cell receptor (TCR) by Peptide/major histocompatibility complexes expressed on the antigen-presenting cell (APC). Lacking costimulation by accessory molecules on the APC, T cell proliferation does not occur and unresponsiveness to subsequent antigenic stimulus is induced. The B7/BB1 receptor on APCs binds CD28 and CTLA-4 on T cells, and provides a costimulus for T cell proliferation. Here, we show that prolonged, specific T cell hyporesponsiveness to antigenic restimulation is achieved by blocking the interaction between CD28 and B7/BB1 in human mixed leukocyte culture (MLC). Secondary T cell proliferative responses to specific alloantigen were inhibited by addition to the primary culture of monovalent Fab fragments of anti-CD28 monoclonal antibody (mAb) 9.3, which block interaction of CD28 with B7/BB1 without activating T cells. Hyporesponsiveness was also induced in MLC by CTLA4Ig, a chimeric immunoglobulin fusion protein incorporating the extracellular domain of CTLA-4 with high binding avidity for B7/BB1. Cells previously primed could also be made hyporesponsive, if exposed to alloantigen in the presence of CTLA4Ig. Maximal hyporesponsiveness was achieved in MLC after 2 d of incubation with CTLA4Ig, and was maintained for at least 27 d after removal of CTLA4Ig. Accumulation of interleukin 2 (11.2) and interferon "y but not I1.4 mRNA was blocked by CTLA4Ig in T cells stimulated by alloantigen. Antigen-specific responses could be restored by addition of exogenous I1.2 at the time of the secondary stimulation. Addition to primary cultures of the intact bivalent anti-CD28 mAb 9.3, or B7/BB1 + transfected CHO cells or exogenous I~2, abrogated induction of hyporesponsiveness by CTLA4Ig. These data indicate that interaction of CD28 with B7/BB1 during TCR engagement with antigen is required to maintain T cell competence and that blocking such interaction can result in a state of T cell hyporesponsiveness.

A conditional block to transcriptional elongation is an important mechanism for regulating c-myc gene expression. This elongation block within the first c-myc exon was defined originally in mammalian cells by nuclear run-on transcription analyses. Subsequent oocyte injection and in vitro transcription analyses suggested that sequences near the end of the first c-myc exon are sites of attenuation and/or premature termination. We report here that the mapping of single stranded DNA in vivo with potassium permanganate (KMnO4) and nuclear run-on transcription assays reveal that polymerase is paused near position +30 relative to the major c-myc transcription initiation site. Deletion of 350 bp, including the sites of 3'-end formation and intrinsic termination defined in oocyte injection and in vitro transcription assays does not affect the pausing of polymerase in the promoter-proximal region. In addition, sequences upstream of +47 are sufficient to confer the promoter-proximal pausing of polymerases and to generate the polarity of transcription farther downstream. Thus, the promoter-proximal pausing of RNA polymerase II complexes accounts for the block to elongation within the c-myc gene in mammalian cells. We speculate that modification of polymerase complexes at the promoter-proximal pause site may determine whether polymerases can read through intrinsic sites of termination farther downstream.

These data indicate that approximately 60% of patients transplanted early after failure of initial therapy for malignant lymphoma are projected to be disease-free more than 2 years after treatment with fractionated TBI, etoposide, and Cy and infusion of autologous hematopoietic stem cells. The transplant-related mortality rate is low and relapse is the main cause of treatment failure in patients with less advanced disease. For patients with more advanced disease, the K-M estimates of both transplant-related deaths (25%) and relapse (53%) remain major problems.

To our knowledge, this is the first study to prospectively compare multiple measures of innate and adaptive immune responses in hematopoietic stem cell transplant recipients with CMV viremia. The strongest immune correlates with protection against CMV viremia in hematopoietic stem cell transplant recipients are reconstitution of CMV-specific T cell memory responses (LPA) and recovery of natural killer cell function. In contrast, positive CMV-LPA before transplantation may be a marker of high risk of CMV reactivation after transplantation.

Purpose Pinatuzumab vedotin is an antibody–drug conjugate with the potent antimicrotubule agent monomethyl auristatin E (MMAE) conjugated to an anti-CD22 antibody via a protease-cleavable linker. This phase I study determined its recommended phase II dose (RP2D) and evaluated its safety, tolerability, and antitumor activity alone and with rituximab in relapsed/refractory (r/r) non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Experimental Design Patients received escalating doses of pinatuzumab vedotin every 21 days. Clinical activity at the RP2D alone or with rituximab was evaluated in r/r diffuse large B-cell lymphoma (DLBCL) and r/r indolent NHL (iNHL) patients. Results Seventy-five patients received single-agent pinatuzumab vedotin. The RP2D was 2.4 mg/kg, based on dose-limiting toxicities (DLT) of grade 4 neutropenia >7 days in 1 of 3 patients and grade 4 neutropenia <7 days in 2 of 3 patients treated at 3.2 mg/kg (maximum assessed dose). No DLTs occurred at 2.4 mg/kg. At the RP2D, neutropenia was the mostcommon grade ≥3 adverse event. Peripheral neuropathy–related grade ≥2 adverse events most frequently resulted in treatment discontinuation. Rituximab cotreatment did not impact safety, tolerability, or pharmacokinetics of pinatuzumab vedotin. Unconjugated MMAE exposure was much lower than antibody-conjugated MMAE exposure, without accumulation with repeat dosing. At the RP2D, objective responses were observed in DLBCL (9/25) and iNHL (7/14) patients; 2 of 8 patients treated with pinatuzumab vedotin (RP2D) and rituximab had complete responses. CLL patients showed no objective responses. Conclusions The RP2D of pinatuzumab vedotin alone and with rituximab was 2.4 mg/kg, which was well tolerated, with encouraging clinical activity in r/r NHL.

Introduction: Previously reported results from this ongoing study showed clinical activity for polatuzumab vedotin (PoV), an antibody drug conjugate (ADC) containing the anti‐mitotic MMAE targeting CD79b, at a dose of 2.4 mg/kg, in combination with rituximab (R) in relapsed/refractory (r/r) diffuse large B‐cell lymphoma (DLBCL) and follicular lymphoma (FL) when patients (pts) were treated until progression (Morschhauser ASH 2014, Advani ASCO 2015). Obinutuzumab (G) is a next generation antiCD20 mAb with enhanced antibody-dependent cellular cytotoxicity.and direct cell death. We report here the safety and efficacy results for this Phase Ib/II study of PoV at 1.8 mg/kg combined with G treated for up to 8 cycles in r/r DLBCL or FL (ClinicalTrials.gov NCT01691898). Methods: This multicenter, open-label Ph Ib/II study evaluated the combination of PoV with G in pts with r/r DLBCL or FL. Primary objective was to assess the anti-tumor activity of PoV + G; secondary endpoints included safety and tolerability and pharmacokinetics. Adult pts (≥18 years) were treated with PoV 1.8 mg/kg intravenously with G every 21 days for a total of 8 cycles. Assessments for anti-lymphoma activity were performed following 4 cycles of study treatment (tx) and at the end of study tx (EOT) by Lugano 2014 criteria. Patients: As of Apr 5, 2016, 70 (38 DLBCL, 32 FL) pts were evaluable for safety. The demographic and baseline characteristics are included in Table 1. Results: Of 70 safety-evaluable patients, 49 (70%) experienced a treatment-related adverse event (AE) of which most were mild (Gr 1/2; 34 of 49). The most common AEs in >20% of pts were Gr 1-3 fatigue (43%), Gr 1-2 diarrhea (34%), Gr 1-3 nausea (30%), Gr 1-2 constipation (21%) and Gr 1 headache (20%). Among the 70 pts who received at least 1 dose of tx, 46% (32/70) pts had Grade 3/4 AEs and 29% (13 DLBCL, 7 FL) had serious AEs (SAEs). The most common Grade 3/4 AE was neutropenia (13%) and the most common SAE occurring in >10% was infections (15%) including pneumonia (n=3), influenza, urinary tract infection (n=2 each), sepsis, sinusitis, and respiratory tract infection (n=1 each). Four pts (2 DLBCL, 2 FL) had Gr 2 peripheral neuropathy (PN, n=2) and peripheral sensory neuropathy (n=2). Onset of PN occurred after cycle 2 and cycle 3 in the DLBCL pts and after cycle 5 in both FL pts. All 4 pts required dose reduction of PoV (1.8 to 1.4 mg/kg). Five pts discontinued PoV due to AE (3 DLBCL, 2 FL), including Gr 3 GI perforation (n=1), Gr 3 malignant neoplasm (n=1), and Gr 3 delirium and urinary tract infection (n=1) in pts with DLBCL, and Gr 5 pneumonia and Gr 2 elevated bilirubin (n=1, each) in FL pts. Eight deaths (1 due to pneumonia in a pt with FL and 7 due to progressive disease in pts with DLBCL) were reported. The median number of treatment cycles received was 2 (0-8) and 5 (0-8) for the pts with DLBCL and FL, respectively. Best objective response rate by PET was available for 44 evaluable (21 DLBCL, 23 FL) pts. The regimen was active in heavily pre-treated pts with an ORR of 78% for FL and 52% for DLBCL (see Table 2). Updated safety and efficacy data will be presented. Conclusions: Early results from this ongoing study show that this novel combination of PoV, at 1.8 mg/kg with fixed duration (≤ 8 cycles), plus G has an acceptable safety profile with evidence of clinical activity in r/r DLBCL or FL pts who were heavily pretreated or refractory to the last prior regimen. Disclosures Brunvand: Millennium / Takeda: Speakers Bureau; Celgene: Speakers Bureau; Genentech: Speakers Bureau. Chen:Seattle Genetics: Consultancy. Press:Roche / Genentech: Consultancy, Research Funding. Chiappella:Pfizer: Speakers Bureau; Celgene: Speakers Bureau; Roche: Speakers Bureau; Janssen-Cilag: Speakers Bureau; Teva: Speakers Bureau; Amgen: Speakers Bureau. Diefenbach:Genentech: Honoraria, Research Funding. Jones:Genentech, Inc.: Employment. Hirata:Genentech: Employment. Flinn:Janssen: Research Funding; Gilead Sciences: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; ARIAD: Research Funding; RainTree Oncology Services: Equity Ownership.

The IL-2 gene is a T cell specific gene that is expressed early during the activation-specific T lymphocyte development program. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assays have defined DNA/protein interactions at the IL-2 promoter cis-elements in vitro. To determine if the trans-activators documented in T cell nuclear extracts actually bind the IL-2 promoter in vivo, ligation mediated PCR (LMPCR) genomic footprinting was performed on the IL-2 promoter in both activated and non-activated T cells and HL60 promyelocytes, which do not express the IL-2 gene. The in vivo footprints indicate that the IL-2 gene transcription start site and TATA sequence are protected in both activated and resting T cells, prior to the appearance of detectable IL-2 steady state message. The distal NF-AT and the NF kappa B sites are each footprinted and the Oct/OAP site contains hypersensitive residues in the unstimulated T lymphocytes. Additional residues are protected in each of these sites after T cell activation. The proximal NF-AT site (NF-IL-2B) and the AP-1 site at -150 are protected in activated Jurkat T lymphocytes, but these two sites are not protected in activated Jurkat lymphocytes stably transfected a gene construct containing multiple NFAT binding sites.

Introduction Treatment of patients (pts) with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) with the combination of venetoclax (VEN), an oral, selective Bcl-2 inhibitor, and rituximab yielded an ORR of 84% (Roberts et al. Haematologica 2015). Treatment of such pts with VEN in combination with obinutuzumab (Gazyva®, Gazyvaro™, G), a Type II, glycoengineered anti-CD20 antibody, may yield even better treatment outcomes. We present preliminary efficacy and updated safety data from an ongoing phase 1b study (NCT01685892) evaluating this combination in R/R or treatment-naïve (TN) pts with CLL in alternate treatment schedules. Methods Pts with CLL with an ECOG PS ≤1 and adequate organ function are enrolled in a study with a 3+3 design and cohorts ranging from 100 to 600 mg/day of VEN. Pts are assigned to one of two dosing schedules, starting treatment with either VEN (Schedule A) or G (Schedule B). Both schedules include tumor lysis syndrome (TLS) risk mitigation based on disease burden at screening, which includes a gradual VEN ramp-up to the assigned cohort dose. Six cycles of combination therapy will be given and then pts with R/R disease continue single-agent VEN until disease progression; TN pts will receive single-agent VEN for an additional 6 months. Dose-limiting toxicities (DLTs) are identified during the first 21 days of combination therapy in Schedule A or the first 35 days of combination therapy in Schedule B, and focus on TLS, infusion related reactions, and cytopenias. Based on a safety review of data from this trial, the 600 mg cohort will not be explored. Response is first assessed before Cycle 4 according to 2008 International Workshop on CLL guidelines. Results As of April 20, 2015, 32 pts (26 R/R and 6 TN) have been enrolled. Four R/R pts were unenrolled after a sponsor-initiated clinical hold secondary to TLS events in other VEN studies. Patient characteristics include a median age 62.5 (range, 45-80) years, and 62.5% male pts. TLS risk was assessed in 28 pts following protocol modifications adopted after a Sponsor-initiated clinical hold; 96.4% were at medium or high risk for TLS. The highest VEN dose administered in this study was 400 mg/day (administered to 11 R/R and 6 TN pts). Median time on study was 5.5 (range, 0.1-19.6) mo. for all pts and 2.8 (range, 0.9-2.8) mo. for TN pts. Among pts exposed to VEN, dose interruptions were observed in 17/27 (63%) pts. A summary of AEs is presented in Figure 1. Laboratory TLS was observed in 4/32 (12.5%) pts and all were able to continue study treatment after resolution of electrolyte changes; no cases of clinical TLS occurred. One pt with R/R disease in cohort 1 discontinued study participation following disease progression (the pt completed 6 cycles of combination treatment). A second pt with R/R disease in cohort 1 died secondary to acute respiratory failure; Richter's transformation also was suspected in this pt but not confirmed. Twenty pts with R/R disease and 6 TN pts remain on the study. At least 1 response evaluation has been performed in 17 pts with R/R disease. The overall response rate (ORR) by investigator assessment was 100%; 4/17 (23.5%) pts achieved complete response/complete response with incomplete bone marrow recovery (CR/CRi). Among the 13 (76.5%) pts with PRs after 3 cycles of therapy, 3 have improved to CR/CRi at assessments 28 days after completing C6D1. Full MRD data will be available in the near future but early analyses suggest some patients may achieve MRD negative status by Cycle 4. Conclusion These preliminary data suggest that VEN + G can be safely administered in pts with CLL with no difference in tolerability between R/R and TN subgroups. AEs appear to be manageable and no pt has discontinued study participation secondary to cytopenia, the most frequently observed AE. Data suggests that the TLS prophylaxis measures are effective even in patients with a higher disease burden. An expansion phase is planned using a 400 mg per day dose of VEN in R/R and TN pts following a review of safety data assessing potential differences between dosing schedules. The preliminary efficacy data suggest this regimen may be an important option in patients with CLL; a phase 3 study evaluating VEN+G is ongoing. Disclosures Flinn: Cephalon, Inc; Teva Pharmaceutical Industries Ltd; Genentech, inc; Gilead: Research Funding. Off Label Use: Venetoclax is an investigational drug that is not yet approved in this indication. Brunvand:Celgene: Speakers Bureau; Millenium: Speakers Bureau. Choi:Gilead: Consultancy, Other: Advisory Board, Speakers Bureau; AbbVie: Consultancy, Other: Advisory Board, Research Funding. Dyer:Roche Pharmaceuticals: Speakers Bureau; ONO Pharmaceuticals: Research Funding; Gilead: Research Funding. Gribben:Janssen: Honoraria; Celgene: Consultancy, Honoraria; Gilead: Honoraria; Roche/Genentech: Honoraria; Pharmacyclics: Honoraria. Hillmen:Janssen: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Roche Pharmaceuticals: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Pharmacyclics LLC, an AbbVie Company: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Celgene: Research Funding. Jones:Acerta Pharma BV: Research Funding. Li:Genentech, Inc.: Employment. Mobasher:Genentech, Inc.: Employment. Vosganian:Genentech, Inc.: Employment. Kipps:Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor; Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor.