William Y. Go

The Osmoprotective Function of the NFAT5 Transcription Factor in T Cell Development and Activation

The NFAT5/TonEBP transcription factor, a recently identified rel/NF-κB family member, activates transcription of osmocompensatory genes in response to extracellular hyperosmotic stress. However, the function of NFAT5 under isosmotic conditions present in vivo remains unknown. Here we demonstrate that NFAT5 is necessary for optimal T cell development in vivo and allows for optimal cell growth ex vivo under conditions associated with osmotic stress. Transgenic mice expressing an inhibitory form of NFAT5 in developing and mature T cells exhibited a 30% reduction in thymic cellularity evenly distributed among thymic subsets, consistent with the uniform expression and nuclear localization of NFAT5 in each subset. This was associated with a 25% reduction in peripheral CD4+ T cells and a 50% reduction in CD8+ T cells. While transgenic T cells exhibited no impairment in cell growth or cytokine production under normal culture conditions, impaired cell growth was observed under both hyperosmotic conditions and isosmotic conditions associated with osmotic stress. Transgenic thymocytes also demonstrated increased sensitivity to osmotic stress. Consistent with this, the system A amino acid transporter gene ATA2 exhibited NFAT5 dependence under hypertonic conditions but not in response to amino acid deprivation. Expression of the TNF-α gene, a putative NFAT5 target, was not altered in transgenic T cells. These results not only demonstrate an osmoprotective function for NFAT5 in primary cells but also show that NFAT5 is necessary for optimal thymic development in vivo, suggesting that developing thymocytes within the thymic microenvironment are subject to an osmotic stress that is effectively countered by NFAT5-dependent responses.

Outcomes in refractory diffuse large B-cell lymphoma: Results from the international SCHOLAR-1 study

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. Although 5-year survival rates in the first-line setting range from 60% to 70%, up to 50% of patients become refractory to or relapse after treatment. Published analyses of large-scale outcome data from patients with refractory DLBCL are limited. SCHOLAR-1, an international, multicohort retrospective non-Hodgkin lymphoma research study, retrospectively evaluated outcomes in patients with refractory DLBCL which, for this study, was defined as progressive disease or stable disease as best response at any point during chemotherapy (>4 cycles of first-line or 2 cycles of later-line therapy) or relapsed at ≤12 months from autologous stem cell transplantation. SCHOLAR-1 pooled data from 2 phase 3 clinical trials (Lymphoma Academic Research Organization-CORAL and Canadian Cancer Trials Group LY.12) and 2 observational cohorts (MD Anderson Cancer Center and University of Iowa/Mayo Clinic Lymphoma Specialized Program of Research Excellence). Response rates and overall survival were estimated from the time of initiation of salvage therapy for refractory disease. Among 861 patients, 636 were included on the basis of refractory disease inclusion criteria. For patients with refractory DLBCL, the objective response rate was 26% (complete response rate, 7%) to the next line of therapy, and the median overall survival was 6.3 months. Twenty percent of patients were alive at 2 years. Outcomes were consistently poor across patient subgroups and study cohorts. SCHOLAR-1 is the largest patient-level pooled retrospective analysis to characterize response rates and survival for a population of patients with refractory DLBCL.

TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress: organic osmolyte-dependent and -independent pathways

TonEBP (tonicity-responsive enhancer binding protein) is a transcription factor that promotes cellular accumulation of organic osmolytes in the hypertonic renal medulla by stimulating expression of its target genes. Genetically modified animals with deficient TonEBP activity in the kidney suffer from severe medullary atrophy in association with cell death, demonstrating that TonEBP is essential for the survival of the renal medullary cells. Using both TonEBP knockout cells and RNA interference of TonEBP, we found that TonEBP promoted cellular adaptation to hypertonic stress. Microarray analyses revealed that the genetic response to hypertonicity was dominated by TonEBP in that expression of totally different sets of genes was increased by hypertonicity in those cells with TonEBP vs. those without TonEBP activity. Of over 100 potentially new TonEBP-regulated genes, we selected seven for further analyses and found that their expressions were all dependent on TonEBP. RNA interference experiments showed that some of these genes, asporin, insulin-like growth factor-binding protein-5 and -7, and an extracellular lysophospholipase D, plus heat shock protein 70, a known TonEBP target gene, contributed to the adaptation to hypertonicity without promoting organic osmolyte accumulation. We conclude that TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress in addition to organic osmolyte accumulation.

Optimization and direct comparison of the dimerizer and reverse tet transcriptional control systems.

Exogenously controlled gene expression systems are essential for both the in vivo analysis of gene function and the regulated delivery of therapeutic gene products. However, differences in experimental methods used to characterize the various systems prohibit informative comparisons. The purpose of this study was to identify an optimal system for regulated gene expression studies through a rigorous direct comparison of the dimerizer and the reverse tet transactivator (rtTA) transcriptional switch systems.

Identification of a domain in the carboxy terminus of CCK receptor that affects its intracellular trafficking

The carboxy-terminal region of many guanine nucleotide-binding protein (G protein)-coupled receptors contains important regulatory sequences such as an NP(x)2-3Y motif, a site of fatty acid acylation, and serine- and threonine-rich domains. The type A CCK receptor contains all of these, yet their significance has not been examined. We have, therefore, constructed a series of receptor site mutants and truncations that interfere with each of these motifs and expressed each in Chinese hamster ovary cells where they were studied for radioligand binding, cell signaling, receptor internalization, and intracellular trafficking. Each construct was synthesized and transported appropriately to the cell surface, where CCK bound with high affinity, elicited an inositol 1,4, 5-trisphosphate response, and resulted in internalization and normal trafficking. Thus modification or elimination of each of these established sequence motifs had no substantial effect on any of these parameters of receptor and cellular function. However, an additional construct that truncated the carboxy terminus, eliminating an additional 15-amino-acid segment devoid of any currently recognized sequence motifs, resulted in a marked change in receptor trafficking, with all other parameters of receptor function normal. This mutant receptor construct was delayed at the stage of early endosomes, delaying its progress to the lysosome-enriched perinuclear compartment from the rapid time course followed by wild-type receptor and all of the other constructs. It is proposed that this region of the CCK receptor tail contains a new motif important for intracellular receptor trafficking.The carboxy-terminal region of many guanine nucleotide-binding protein (G protein)-coupled receptors contains important regulatory sequences such as an NP(x)2-3Y motif, a site of fatty acid acylation, and serine- and threonine-rich domains. The type A CCK receptor contains all of these, yet their significance has not been examined. We have, therefore, constructed a series of receptor site mutants and truncations that interfere with each of these motifs and expressed each in Chinese hamster ovary cells where they were studied for radioligand binding, cell signaling, receptor internalization, and intracellular trafficking. Each construct was synthesized and transported appropriately to the cell surface, where CCK bound with high affinity, elicited an inositol 1,4,5-trisphosphate response, and resulted in internalization and normal trafficking. Thus modification or elimination of each of these established sequence motifs had no substantial effect on any of these parameters of receptor and cellular function. However, an additional construct that truncated the carboxy terminus, eliminating an additional 15-amino-acid segment devoid of any currently recognized sequence motifs, resulted in a marked change in receptor trafficking, with all other parameters of receptor function normal. This mutant receptor construct was delayed at the stage of early endosomes, delaying its progress to the lysosome-enriched perinuclear compartment from the rapid time course followed by wild-type receptor and all of the other constructs. It is proposed that this region of the CCK receptor tail contains a new motif important for intracellular receptor trafficking.

Abstract CT019: Primary results from ZUMA-1: a pivotal trial of axicabtagene ciloleucel (axicel; KTE-C19) in patients with refractory aggressive non-Hodgkin lymphoma (NHL)

Introduction: Outcomes for pts with refractory aggressive NHL are poor with current therapies (Crump, ASCO 2016). Results from the interim analysis of (n=62) of ZUMA-1, the 1st multicenter trial of an anti-CD19 chimeric antigen receptor (CAR) T cell, axi-cel, in refractory aggressive NHL, showed an objective response rate (ORR) of 79% (complete response [CR] 52%; Blood 2016;128:LBA-6). Here we present results from the primary analysis of ZUMA-1. Methods: Pts received a target dose of 2 × 106 anti-CD19 CAR T cells/kg after lowdose conditioning with cy/flu. Eligible pts (≥18 y) had diffuse large B cell lymphoma (DLBCL), primary mediastinal B cell lymphoma (PMBCL) or transformed follicular lymphoma (TFL); an ECOG performance status (PS) 0-1; and refractory disease (progressive or stable disease as best response to last prior therapy, or relapsed ≤12 m of autologous stem cell transplant [ASCT]). The primary endpoint for this analysis was ORR in the combined DLBCL, PMBCL, and TFL population. Key secondary endpoints were duration of response (DOR), overall survival (OS), and frequency of adverse events (AEs). The primary analysis was triggered when 92 pts had at least 6 m of follow-up. Results: As of January 27, 2017, 111 pts from 22 institutions were enrolled; 101 pts (91%) received axi-cel. Median age was 58 y (range, 23-76), 67% male, 85% stage IIIIV, 47% IPI 3-4, 77% refractory to ≥2nd line of therapy, and 21% relapsed ≤12 m of ASCT. Axi-cel was successfully manufactured in 110/111 (99%) pts with an average turnaround time from apheresis to the clinical site of 17 d. With an ORR of 82% (n = 92; P Conclusions: Axi-cel significantly improved ORR in patients with refractory aggressive NHL. The CR rate was 7-fold higher compared to historical controls (Crump, ASCO 2016) and nearly half the patients have an ongoing response. Axi-cel demonstrated significant clinical benefit with a manageable safety profile in pts lacking curative treatment options.[F.L.L. and S.S.N. contributed equally to this study.] Funding source: Kite Pharma and in part by funding from the Leukemia & Lymphoma Society (LLS) Therapy Acceleration Program treatment options. Citation Format: Frederick L. Locke, Sattva S. Neelapu, Nancy L. Bartlett, Lazaros J. Lekakis, David Miklos, Caron A. Jacobson, Ira Braunschweig, Olalekan Oluwole, Tanya Siddiqi, Yi Lin, John Timmerman, Jonathan W. Friedberg, Adrian Bot, John Rossi, Lynn Navale, Yizhou Jiang, Jeff Aycock, Meg Elias, Jeff Wiezorek, William Y. Go. Primary results from ZUMA-1: a pivotal trial of axicabtagene ciloleucel (axicel; KTE-C19) in patients with refractory aggressive non-Hodgkin lymphoma (NHL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT019. doi:10.1158/1538-7445.AM2017-CT019

Abstract CT020: Immune signatures of cytokine release syndrome and neurologic events in a multicenter registrational trial (ZUMA-1) in subjects with refractory diffuse large B cell lymphoma treated with axicabtagene ciloleucel (KTE-C19)

Introduction: Axicabtagene ciloleucel (axi-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy. ZUMA-1 is a multicenter registrational trial of axi-cel in patients with refractory, aggressive B-cell non-Hodgkin lymphoma. In a pre-specified interim analysis, ZUMA-1 met its primary endpoint with a 76% objective response rate and 47% complete response. The incidence of Grade 3 and higher (Gr 3+) cytokine release syndrome (CRS) and neurologic events (NE) was 13% and 29%, respectively, in 93 patients with 1 month follow-up (Neelapu ASH 2016). We present novel immune signatures of Gr 3+ CRS and NE by analyzing the axi-cel-related biomarker profile in association with clinical outcomes. Methods: In this interim analysis of 62 patients, 44 serum analytes pre- and post-axi-cel treatment were measured via ELISA at multiple timepoints during the first month. The number of CAR+ cells in blood was determined by qPCR. Kinetics and association of these markers with CRS and NE were analyzed. The treatment-related profile was defined by analytes with at least double an increase over baseline in at least 50% of patients. These analytes were selected and evaluated for association with Gr 3+ CRS or NE. Immune signatures were determined based on p-values applied to peak or cumulative levels of analytes and adjusted for multiplicity (stepdown method). Results: Out of 44 analytes measured, 12 demonstrated an increase by double over baseline in at least 50% of patients. This axi-cel-related biomarker profile includes immune proliferative/modulating cytokines, pro-inflammatory cytokines, markers of myeloid activation, and chemokines. Markers with the highest median-fold increase were IFNγ (44x), IL-10 (31x), IL-6 (26x), IL-15 (20x), and GRZB (17x). These peaked within 7-14 days and generally returned to baseline within 1 month post-treatment. Within this panel of 12 analytes elevated post-axi-cel treatment, markers associated with Gr 3+ CRS (p Citation Format: Frederick L. Locke, John Rossi, Xiaodong Xue, Sattva S. Neelapu, Daniel H. Ryan, Armin Ghobadi, Lazaros J. Lekakis, David Miklos, Caron A. Jacobson, Ira Braunschweig, Olalekan Oluwole, Tanya Siddiqi, Yi Lin, John Timmerman, Patrick M. Reagan, Marika Sherman, Janice Nagatani, Xiao Zhang, Lynn Navale, William Y. Go, Jeff Wiezorek, Adrian Bot. Immune signatures of cytokine release syndrome and neurologic events in a multicenter registrational trial (ZUMA-1) in subjects with refractory diffuse large B cell lymphoma treated with axicabtagene ciloleucel (KTE-C19) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT020. doi:10.1158/1538-7445.AM2017-CT020

Clinical Response in ZUMA-1, the Pivotal Study of Axicabtagene Ciloleucel (Axi-Cel) in Patients with Refractory Large B Cell Lymphoma, May Be Influenced by Characteristics of the Pretreatment Tumor Microenvironment (TME)

untreated high-grade diffuse large B-cell lymphoma. Patients: 40 untreated DLBCL patients from 4 centers were enrolled in a prospective study between August 2013 Apr 2018; stage II-IV; ECOG 0-3; median age 60 years (27-78); age 60y/<60y 57%/ 43%; age 55y/<55y 50%/50%; M/F 50%/50%; IPI: 67% highintermediate and 33% high risk; 22% with bone marrow involvement. All patients underwent 4-8 courses (2-4 cycles) of chemotherapy: RDA-EPOCH, R-HMA. For patients older than 60 years (or 55y with comorbidities) dose of R-HMA was reduced. In 4 cases of DLBCL with bone marrow involvement LEAM conditioning and autologous stem cell transplantation were applied. Results: The median follow-up is 41,5 months (6,7-52,4). There was no mortality associated with toxicity. The main non-hematological toxicities of R-HMA were infections (mucositis, pneumonia, sepsis, enteropathy) grades 1-2 and 3-4 in 90% and 10%, respectively. Hematological toxicity grade 4 for less than 4 days we observed only after courses R-HMA. Complete remission (CR) was achieved in 36 (90%) patients. With a median follow-up of 41 months, overall and disease-free survival of 40 patients constituted 76,3% and 69,7%, respectively. Age older than 55y, IPI, LDH and bulky disease were factors of poor prognosis for OS and DFS by Long Rank test. In a group of patients older than 55 years results of therapy seemed to be worse than in young patients: OS were 70,5% vs 78,6% (p1⁄40,005), DFS were 63,6% vs 80% (p1⁄40,013), respectively. But mortality in older patients didn’t associate with toxicity or disease relapse, unlike in young patients. Conclusions: The R-DA-EPOCH/R-HMA protocol demonstrated acceptable toxicity and high efficacy in patients with high-grade DLBCL. This protocol has shown optimistic results in the elderly patients.

745. Updated Phase 1 Results from ZUMA-1: A Phase 1-2 Multi-Center Study Evaluating the Safety and Efficacy of KTE-C19 (Anti-CD19 CAR T Cells) in Subjects with Refractory Aggressive Non-Hodgkin Lymphoma (NHL)

Introduction: Anti-CD19 CAR T cells with CD3-zeta and CD28 signaling domains showed durable remissions in subjects with relapsed/refractory advanced B cell malignancies at the NCI (Kochenderfer et al. Blood 2012, JCO 2014, ASH 2014). KTE-C19 utilizes the same anti-CD19 CAR construct as investigated in the NCI study in a 6-8 day manufacturing process (Better et al. ASCO 2014). ZUMA-1 is a phase 1-2 multicenter, open-label study evaluating the safety and efficacy of KTE-C19 in subjects with refractory aggressive B-cell NHL. Methods: Subjects received KTE-C19 at a target dose of 2 × 106 (minimum 1 × 106) anti-CD19 CAR T cells/kg after a fixed dose conditioning chemotherapy regimen of cyclophosphamide 500 mg/m2/day and fludarabine 30 mg/m2/day for 3 days. The primary objective of phase 1 was to evaluate the safety of KTE-C19 as determined by the incidence of dose-limiting toxicities (DLT). Key secondary objectives were overall response rate, duration of response, levels of CAR T cells in the blood, and levels of serum cytokines. Key inclusion criteria were ≥ 18 years old, ECOG 0-1, and chemotherapy-refractory disease defined as progressive disease or stable disease as best response to last line of therapy, or disease progression ≤ 12 months after autologous stem cell transplant (ASCT). Results: As of 20 Nov 2015, 7 subjects were dosed in the phase 1 portion of the study. All subjects were evaluable for safety and 6 were evaluable for efficacy with a median follow up time of 13 weeks post KTE-C19 infusion. One subject experienced a DLT of grade (gr) 4 encephalopathy and gr 4 cytokine release syndrome (CRS) and died due to an intracranial hemorrhage deemed unrelated to KTE-C19 per the investigator. Key safety and efficacy findings are summarized in the table. CRS and neurotoxicity were managed with supportive care, tocilizumab and systemic steroids. 5 of 7 subjects (71%) had an objective response including 4 complete remissions (57%). Three subjects have ongoing complete remission at 3 months. CAR T cells peaked within two weeks post infusion were detectable 1-3+ months post infusion. Updated clinical and biomarker results will be presented. Conclusions: The KTE-C19 regimen evaluated was safe for further study. The predominant toxicities include CRS and neurotoxicity which are generally reversible. Complete and partial responses have been observed in subjects with refractory disease. KTE-C19 can be centrally manufactured and administered in a multicenter trial. The potentially pivotal phase 2 portion of the study is ongoing. Clinical trial: NCT02348216.SubjectSex/Age/ECOGDisease TypeTreatment HistoryGr 3 or Higher KTE-C19-Related Adverse EventsBest Response1M/59/0DLBCLRelapse ≤ 12 mo after ASCTGr 3 encephalopathy (resolved)Partial Response2M/69/1DLBCLRefractory to 2nd line or later line chemotherapyGr 3 tremor (resolved) Gr 3 delirium (resolved) Gr 3 agitation (resolved) Gr 3 restlessness (resolved) Gr 3 somnolence (resolved)Complete Remission**3M/69/0DLBCLRefractory to 2nd line or later line chemotherapyGr 3 encephalopathy (resolved)Stable Disease4M/67/1DLBCLRelapse ≤ 12 mo after ASCTNoneComplete Remission Ongoing 3 mo+5F/34/0DLBCLRelapse ≤ 12 mo after ASCTGr 3 hypoxia (resolved)Complete Remission Ongoing 3 mo+6M/40/0DLBCLRelapse ≤ 12 mo after ASCTNoneComplete Remission Ongoing 3 mo+7F/29/1DLBCLRefractory to 2nd line or later line chemotherapyGr 4 CRS Gr 4 encephalopathyNE View Table in HTML mo – months, M – male, F – female, NE – not evaluable,*relapsed and retreated with an ongoing PR

Abstract CT135: Updated phase I results from ZUMA-1: a phase I-II multicenter study evaluating the safety and efficacy of KTE-C19 (anti-CD19 CAR T cells) in subjects with refractory aggressive non-Hodgkin lymphoma (NHL)

Introduction: Anti-CD19 CAR T cells with CD3 zeta and CD28 signaling domains showed durable remissions in subjects with relapsed/refractory advanced B cell malignancies at the NCI (Kochenderfer, JCO 2014). KTE-C19 utilizes the same CAR construct as investigated in the NCI study in a 6-8 day manufacturing process (Better, ASCO 2014). Updated results from the phase 1 portion of ZUMA-1 are presented here. Methods: Subjects received KTE-C19 at a target dose of 2 × 10⁁6 anti-CD19 CAR T cells/kg after a fixed dose conditioning chemo regimen of cyclophosphamide and fludarabine. The primary objective of phase 1 was to evaluate the safety of KTE-C19 as determined by the incidence of dose-limiting toxicities (DLT). Key secondary objectives were ORR, duration of response, and biomarkers. Key inclusion criteria were ? 18 years old, ECOG 0-1, and chemo-refractory disease defined as progressive disease or stable disease as best response to last line of therapy, or disease progression ? 12 months after ASCT. Results: As of 20 Nov 2015, 7 subjects were dosed. All subjects were evaluable for safety and 6 were evaluable for efficacy with a median follow up time of 13 weeks post KTE-C19 infusion. 1 subject experienced a DLT of grade (gr) 4 encephalopathy and gr 4 cytokine release syndrome (CRS) and died due to an intracranial hemorrhage deemed unrelated to KTE-C19 per the investigator. CRS and neurotoxicity were managed with supportive care, α-IL6R and steroids. 5 of 7 (71%) subjects responded including 4 CRs (57%). CAR T cells peaked within 2 weeks and were detectable 1-3+ months post infusion. Conclusions: The KTE-C19 regimen evaluated was safe for further study. The predominant toxicities include CRS and neurotoxicity which are generally reversible. CRs and PRs have been observed in subjects with refractory disease. The potentially pivotal phase 2 portion of the study is ongoing (NCT02348216). This study is supported in part by funding from The Leukemia & Lymphoma Society (LLS) Therapy Acceleration Program® Citation Format: Armin Ghobadi, Frederick L. Locke, Sattva S. Neelapu, Tanya Siddiqi, Julio C. Chavez, Chitra M. Hosing, Nancy L. Bartlett, Lihua E. Budde, Adrian Bot, John M. Rossi, Marika Sherman, Lynn Navale, Meg Elias, Jeff Wiezorek, William Y. Go. Updated phase I results from ZUMA-1: a phase I-II multicenter study evaluating the safety and efficacy of KTE-C19 (anti-CD19 CAR T cells) in subjects with refractory aggressive non-Hodgkin lymphoma (NHL). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT135.