ALBERT

Description: Introduction Maximizing proplatelet formation from cultured megakaryocytes will be a critical step in ex-vivo production of platelets for transfusion. However, mechanisms triggering proplatelet formation are not completely identified. Apoptosis factors are suggested to play an important role in initiating the process of platelet release. However, their role is controversial. Megakaryocytes might employ the apoptotic machinery to facilitate platelet production through the intrinsic pathway and caspase 3/9 activation, and pro-survival factors, such as Bcl-xL, are necessary to enable megakaryocyte survival during proplatelet formation. However, removing the pro-apoptotic intrinsic pathway factors Bak and Bax did not limit proplatelet formation. To shed some light on this controversy we performed experiments in which we increased megakaryocyte proplatelet formation by inhibiting two vital cytoskeletal proteins, actin and myosin-II. We found that such an inhibition of actin or myosin-II late in megakaryocytopoiesis correlated with activation of the apoptosis pathways. We analyzed the effect of actin or myosin-II inhibition on the extrinsic and intrinsic apoptosis pathways and whether the increase in proplatelet formation due to actin or myosin inhibition was dependent on apoptosis. Methods Human cord blood derived CD34+ cells were isolated and cultured with thrombopoietin (TPO) and stem cells factor (SCF) for 11 days. Megakaryocytes were isolated on day 8 and replated in culture medium containing only TPO. The day 8 megakaryocytes were treated with an actin inhibitor (AI, Latrunculin-A) 10µM or myosin-II inhibitor (MI, Blebbistatin) 20µM. On day 11 of culture, cell ploidy was analyzed by flow cytometry using propidium Iodide. Proplatelets were counted using inverted light microscopy and their structure characterized by fluorescence microscopy using a β-1 tubulin-specific antibody. Western blots were performed for Bcl-xL, Bak, and Bax. Mitochondrial outer membrane permeabilization (MOMP) and phosphatidylserine (PS) externalization (Annexin-V binding) were measured using flow cytometry. Caspases 3 and 7 were analyzed using a luminescence assay (CaspaseGlo3/7). In some experiments apoptosis was inhibited on day 8 with a pan-caspase inhibitor ZVAD-fmk (20µM). All results were compared to untreated control megakaryocytes. Results Treatment with either actin or myosin-II inhibitor increased the level of polyploidization in megakaryocytes (both p=0.03). There was no additive effect in final ploidy with combination of reagents. Proplatelet extension by megakaryocytes treated with AI or MI was higher than untreated control cells (p=0.02, p=0.05) and proplatelet structure was normal as assessed by fluorescence microscopy. Bcl-xL, Bak and Bax protein levels were not different from controls after AI or MI treatment. MOMP and PS externalization were increased in both AI (p=0.04) and MI (p=0.04) treated megakaryocytes compared to untreated controls. Caspase 3/7 activities were also increased in treated megakaryocytes. The addition of a pan-caspase inhibitor prevented the release of proplatelets in both AI and MI treated megakaryocytes. Discussion Inhibition of actin or myosin-II led to increased levels of polyploidization and proplatelet formation in cultured megakaryocytes. This increased proplatelet formation was simultaneous with increased final apoptosis activation. Apoptosis analysis showed no difference in protein levels of the intrinsic pathway pro-apoptotic (Bak and Bax) or pro-survival (Bcl-xL) factors. However, both flow cytometry and caspase 3/7 assays showed that the final apoptosis process was activated in cells treated with AI or MI. Thus the apoptosis activation was mediated through a pathway independent of the intrinsic pathway factors Bcl-xL, Bak and Bax. Proplatelet formation in response to AI or MI was inhibited by the pan-caspase inhibitor, suggesting that the effects of AI and MI on proplatelet formation are caspase dependent. These results suggest that inhibition of actin or myosin-II late in megakaryocytopoiesis increases proplatelet formation through activation of apoptosis. Our findings suggest that proplatelet formation is dependent on the activation of apoptosis but may not be mediated through the intrinsic pathway. Determining the pathway leading to final apoptosis and proplatelet formation deserves further investigation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1015 Introduction: Platelets are mediators of inflammation as well as thrombosis. Platelets are necessary for normal inflammation and play a primary role in the immune clearance of certain pathogens. Platelets secrete cytokines when they are activated, and can also synthesize proteins de novo when activated, such as IL-1b. Sickle Cell Disease (SCD) is characterized by an activated immune system, and it is now recognized that much of the pathology of SCD results from an inflammatory vasculopathy. Platelets may mediate some of the vascular injury through their inflammatory function, as indicated by: 1) elevated platelet counts in SCD, 2) platelets circulating in an activated state in SCD, as judged by activation dependent surface markers, and 3) elevated blood levels of platelet derived inflammatory proteins, such as soluble CD40 ligand. Hypothesis: A direct mechanism through which platelets could impact the immune system in SCD would be to produce and secrete cytokines. We hypothesized that the identity profile and quantity of cytokines produced and secreted by platelets would be altered in SCD compared to controls, and that those alterations would be associated with clinical status. We measured the expression levels of cytokine mRNA in resting and activated platelets in patients with SCD and compared those to controls. We also measured thrombin-induced secretion of cytokines from highly purified platelets in buffer and compared that to controls. Cytokines representing TH1, TH2, and TH17 immune phenotypes were measured. Methods: Whole blood was collected from adult patients with SCD undergoing exchange transfusions. Platelets were isolated by sequential centrifugation followed by passage through an affinity column for glycophorin-A and CD45. Platelets were activated with 0.125U/ml thrombin for 18 hours in M199 medium at 37C, and then the supernatant (the platelet releasate) was analyzed for cytokine content. Cytokines were measured using BD Cytometric Bead Array for Th1/Th2/Th17 profile: IL-1β (driver of inflammation, secreted from platelets), IL-2, IL-4, IL-6 (induction/maintenance of autoimmunity), IL-10, IL-17a (maintenance of autoimmunity), TNF-α, IFN-γ, and sCD40L (secreted from platelets). Total RNA was isolated from either fresh platelets or platelets after activation and analyzed by QRT-PCR. Relative mRNA quantity was measured using certified mRNA primer sets and SYBR green detector, using β-actin as an internal control. Results: Both IL-6 and TGFβ mRNA levels were significantly increased to more than nine- and six-fold over control levels, respectively. IL-1β and IL-10 mRNA levels were also increased over controls, but this did not reach significance. Platelet secretion of IL-6, IL-1β, and soluble CD40 ligand was increased approximately 300-, 21-, and 5-fold, respectively, in sickle cell platelets compared to controls. The alterations of cytokine mRNA and cytokine secretion were more pronounced in patients with alloantibodies than in those without. These preliminary findings support the idea of platelets as active modulators of SCD inflammation, and indicate a novel mechanism through which antiplatelet agents may be beneficial in SCD. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2289 Introduction: One of the goals of stem cell medicine is the production of platelets from stem cells for transfusion therapy. The process of platelet production from megakaryocytes is complex and depends on a wide spectrum of internal and external stimuli. The degree of cell polyploidization, amount of demarcation membrane system (DMS), and the cells capacity to form proplatelets are major determines of the quantity of platelets released by each megakaryocyte. Both polyploidization and proplatelet formation depend on the synchronous function of microtubules and actin/myosin. This complex machinery is regulated in part by the actions of the C-Myc and NF-E2 promoters. These promoters play a crucial role during polyploidization and proplatelet formation. We have examined the potential role of these promoters in the efficacy of various chemical and cell culture-based methods of driving hematopoietic stem cells to megakaryocyte differentiation and platelet production. Methods: Human cord blood derived CD34+ cells were isolated and 5×104cells were cultured with thrombopoietin (TPO) and stem cells factor (SCF) for 12 days. Megakaryocytes were cultured along with reagents that inhibit distinct mechanisms of the cytokinesis process: Rho-Rock inhibitor, Y27632 (RRI); Src-inhibitor, SU6656 (SI); Nicotinamide (NIC); Aurora-B inhibitor, ZM447439 (ABI); and Myosin Light Chain Kinase Inhibitor (MLCKI). Combinations of reagents were used in order to determine their interactions and to maximize megakaryocyte ploidy. The DMS was analyzed and quantified with Di-8 ANEPPS in flow cytometry and morphology was studied with Electron Microscopy (EM). On day 12 proplatelets were analyzed with an inverted microscope and platelets were counted with an Advia 120 cell counter. Total RNA was extracted and analyzed for C-Myc and NF-E2 mRNA by QRT-PCR. Results: All treatments increased megakaryocyte ploidy, except MLCKI. RRI reached the highest ploidy (p=0.0007), followed by NIC (p=0.003), SI (p=0.026) and ABI (p=0.018). Combinations all significantly increased polyploidization; however the only combination that equaled RRI alone was the combination of all of the other inhibitors (p

Description: Chimeric antigen receptor (CAR) T cell therapy has consistently shown significant results against acute lymphoblastic leukemia (ALL) in clinical trials1. However, results with other hematological or solid malignancies have been far more modest2. These disparate outcomes could be partially due to an inhibitory tumor microenvironment that suppresses CAR T cell function3. Thus, in order to expand the anti-tumor CAR T cell applications, a novel strategy in which these cells are capable of overcoming the hostile tumor microenvironment is needed. The cytokine interleukin-18 (IL-18) induces IFN-γ secretion, enhances the Th1 immune response and activates natural killer and cytotoxic T cells4. Early phase clinical trials that utilized systemic administration of recombinant IL-18 for the treatment of both solid and hematological malignancies have demonstrated the safety of this therapy5. We hypothesize that CAR T cells that constitutively secrete IL-18 could enhance CAR T cell survival and anti-tumor activity, and also activate cells from the endogenous immune system. To generate CAR T cells that constitutively secrete IL-18, we modified SFG-1928z and SFG-19m28mz CAR T cell constructs and engineered bicistronic human and murine vectors with a P2A element to actively secrete the IL-18 protein (1928z-P2A-hIL18 and 19m28mz-P2A-mIL18, respectively). Human and mouse T cells were transduced with these constructs and in vitro CAR T cell function was validated by coculturing the CAR T cells with CD19+ tumor cells and collecting supernatant for cytokine analysis. Both human and mouse CAR T cells secreted increased levels of IL-18, IFN-γ and IL-2. Proliferation and anti-tumor cytotoxic experiments were conducted with human T cells by coculturing CAR T cells with hCD19+ expressing tumor cells. 1928z-P2A-hIL18 CAR T cells had enhanced proliferation over 7 days and enhanced anti-tumor cytotoxicity over 72 hours when compared to 1928z CAR T cells (p=0.03 and 0.01, respectively) Next, the in vivo anti-tumor efficacy of the IL-18 secreting CAR T cell was tested in xenograft and syngeneic mouse models. Experiments were conducted without any prior lympho-depleting regimen. In the human CAR T cell experiments, Scid-Beige mice were injected with 1x106 NALM-6 tumor cells on day 0 and 5x106 CAR T cells on day 1. Survival curves showed a significant improvement in mouse survival with the 1928z-P2A-hIL18 CAR T cell treatment when compared to 1928z CAR T cell (p=0.006). Subsequently, to determine if IL-18 secreting CAR T cells could also improve anti-tumor efficacy in immunocompetent mice, we tested the murine 19m28mz-P2A-mIL18 CAR T cells in a syngeneic mouse model. The C57BL/6 hCD19+/- mCD19+/- mouse model was utilized and injected with 1x106 EL4 hCD19+ tumor cells on day 0 and 2.5 x106 CAR T cells on day 1. Mice treated with 19m28mz-P2A-mIL18 CAR T cells had 100% long-term survival, when compared to 19m28mz (p

Description: Abstract 4820 Introduction: The large-scale laboratory production of platelets for transfusion purposes using cord blood stem cells is one of goals of stem cell research. One step toward this goal will be to produce and expand megakaryocytes with high ploidy which are capable of releasing higher amounts of platelets than low ploidy cells. Megakaryocyte polyploidization requires a combination of distinct cellular mechanisms, including actin polymerization, myosin activation, microtubule formation, and increased DNA production. Normal bone marrow megakaryocytes reach a DNA content of 128N, with a modal ploidy of 16N. In contrast, cultured human megakaryocytes have typically reached a maximum ploidy of 16N with the majority of cells at 2N/4N. In this study we combined the inhibition of each of the principle mechanisms driving cytokinesis with the goal of driving polyploidization of cultured CD34+ umbilical cord blood cells. The effects of inhibiting each cytokinesis process was characterized by its effects on cell expansion and ploidy, and the additive or inhibitory effects of combining inhibition of multiple processes was determined. The results of this study provide a strategy to produce high ploidy megakaryocytes and provide new insight into the mechanisms underlying megakaryocyte polyploidization. Methods: Human cord blood derived megakaryocytes were cultured with thrombopoietin (TPO) and stem cell factor (SCF) along with reagents that inhibit different mechanisms of the cytokinesis process: Rho-Rock inhibitor, Y27632 (RRI); Src-inhibitor, SU6656 (SI); Nicotinamide (NIC); Aurora-B inhibitor, ZM447439 (ABI); and Myosin Light Chain Kinase Inhibitor (MLCKI). Reagents were added in one single day (day 8) or on three consecutive days (days 8–10) and ploidy was analyzed on day 11 of culture with PI/RNase. Combinations of reagents were used in order to determine their additive or inhibitory interactions and to maximize megakaryocyte ploidy. Results: Treatment with RRI resulted in the highest megakaryocyte ploidy. Up to 48% of megakaryocytes were high ploidy (≥ 8N), with highest ploidy ≥ 64N (p = 0.0007 vs. control). Treatment with NIC resulted in up to 24% of cells reaching high ploidy, with a maximum ploidy of ≥ 32N (p = 0.003 vs. control). Treatment with SI resulted in up to 24% megakaryocytes reaching high ploidy, with a maximum ploidy of ≥ 32N (p = 0.026 vs. control). Treatment with ABI resulted in up to 28% of megakaryocytes reaching high ploidy, with a maximum ploidy of ≥ 32N (p = 0.018 vs. control). The MLCKI had no effect in final ploidy. The combinations of reagents all significantly increased both percentage of cells reaching high ploidy and the highest ploidy compared to control. However, none of the combinations achieved a more robust effect in final ploidy than the RRI alone. However, the combination of MLCKI with SI, NIC and ABI increased the high ploidy cells up to 51.3% with a maximum ploidy ≥ 64N (p 〈 0.0001 vs. control) (Table 1). Conclusion: The RRI proved to be the most effective agent in driving umbilical cord-derived megakaryocyte polyploidization. All other reagents tested, except MLCKI, also moderately increased megakaryocyte ploidy. RRI and MLCKI, both reagents that inhibit the late stages of cytokinesis and furrow formation, resulted in opposite ploidy outcomes, suggesting that RRI acts on ploidy through pathways other than inhibition of myosin activation. While MLCK is active primarily in the late stages of cytokinesis, the Rho/Rock pathway overlaps with other signaling pathways involved in cytokinesis, including src activation in actin polymerization and Aurora kinase activity in microtubule formation. Although inhibition of myosin light chain activation by itself did not appear to drive polyploidization, the combination of MLCKI with other cytokinesis inhibitors increased polyploidization to the same extent as RRI. Our results indicate that induction of high ploidy in cord blood derived megakaryocytes involves a combination of distinct cytokinesis pathways. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Axi-cel is a US FDA-approved autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy for treatment of adult patients (pts) with relapsed or refractory large B cell lymphoma after ≥ 2 prior lines of therapy. In ZUMA-1, the pivotal study of pts with refractory large B cell lymphoma, the objective response rate (ORR) was 82%, including a 58% complete response (CR) rate (Neepalu and Locke, et al. N Engl J Med. 2017). Grade ≥ 3 cytokine release syndrome (CRS) and neurologic events were observed in 12% and 31% of pts, respectively, and were generally reversible. Checkpoint proteins, such as PD-1 and PD-L1, have been shown to be expressed on both CAR T cells and in the tumor microenvironment and subsequently upregulated after CAR T cell infusion (Vranic, et al. PLoS One. 2017; Cherkassky, et al. J Clin Invest. 2016; Galon, et al. ASCO 2017. #3025). This suggests that axi-cel activity could be augmented by incorporating PD-L1 blockade. This end of Phase 1 analysis of ZUMA-6 examines the safety and preliminary efficacy of axi-cel in combination with the anti-PD-L1 antibody atezolizumab (atezo) in pts with refractory diffuse large B cell lymphoma (DLBCL; NCT02926833). Methods: Eligible pts (≥ 18 years) with refractory DLBCL, defined as stable or progressive disease to last line of therapy or relapse within 12 months after autologous stem cell transplant, must have recieved prior CD20-targeting and anthracycline-containing regimen and had ECOG ≤ 1 and adequate bone marrow and organ function. Pts received low-dose conditioning with fludarabine 30 mg/m2/day and cyclophosphamide 500 mg/m2/day × 3 days followed by axi-cel infusion at a target dose of 2 × 106 cells/kg. Atezo was administered at 1200 mg every 21 days for 4 doses starting on Day 21, 14, and 1 post-axi-cel infusion for Cohorts 1, 2, and 3, respectively. This report describes Phase 1 results from all 3 cohorts. Incidence of dose-limiting toxicities (DLTs) was the primary endpoint. Secondary endpoints included the frequency of adverse events (AEs), disease response, pharmacokinetics, and biomarkers. Results: As of January 19, 2018, 12 pts have received axi-cel and at least 1 dose of atezo (3 in Cohort 1; 3 in Cohort 2, 6 in Cohort 3). Median age was 55 years (range, 30 - 66). Most pts (9/12, 75%) had received ≥ 3 prior therapies, and 4 pts (33%) had an International Prognostic Index score of 3 or 4. The median follow-up from axi-cel infusion was 4.4 months (range, 0.8 - 12.6), with 50% of pts having ≥ 6 months of follow-up. Eight pts (67%) have received all 4 doses of atezo, and 11/12 pts have received all scheduled doses of atezo. One pt in Cohort 3 experienced a DLT of Grade 4 thrombocytopenia and neutropenia lasting longer than 30 days. All pts experienced at least 1 AE (92% Grade ≥ 3), with no apparent exacerbation or recurrence of axi-cel-related toxicity following atezo infusion. Only 1 Grade ≥ 3 AE was attributed solely to atezo. Overall, the most common grade ≥ 3 AEs were anemia (9/12, 75%), encephalopathy (5/12, 42%), and neutropenia (5/12, 42%). Grade ≥ 3 CRS and neurologic events occurred in 3 (25%) and 6 (50%) pts, respectively. The ORR in evaluable pts was 9/10 (90%), with 6 pts (60%) in CR and 3 (30%) in partial response (PR); 2/6 pts (33%) had converted to CR at month 6 and month 9 after initially achieving a PR. CAR T cell expansion as measured by area under the curve in the first 28 days (AUC0-28) was over 2-fold higher in ZUMA-6 than the median observed in pts with DLBCL in ZUMA-1 (ZUMA-6: median, 823 cells/µL × days, range, 99 - 2301; ZUMA-1: median, 357 cells/µL × days, range, 5 - 11,507; Figure). Median CAR T cell levels remained higher than ZUMA-1 beyond 28 days. However, initial peak CAR T cell levels were similar (ZUMA-6: median, 68 cells/µL, range, 9 - 274; ZUMA-1: median, 32 cells/µL, range, 1 - 1513). Interferon-γ (IFNγ) levels peaked within the first week after axi-cel infusion and reached a median of 730.5 pg/mL (range, 212 - 1876). The median peak IFNγ level in pts from ZUMA-6 was 1.5-fold higher than that from pts enrolled in Cohort 1 of ZUMA-1 (493.8 pg/mL, range, 32.4 - 1876). Conclusions: PD-L1 blockade with atezo following axi-cel infusion has a manageable safety profile, with a low incidence of DLTs and no clinically significant evidence of increased incidence of AEs. Encouraging efficacy results support the opening of Phase 2 of ZUMA-6 in which 22 pts will be treated according to the Cohort 3 schedule. Pharmacokinetic data suggest the potential for enhanced CAR T cell expansion. Figure. Figure. Disclosures Locke: Kite Pharma: Other: Scientific Advisor; Novartis Pharmaceuticals: Other: Scientific Advisor; Cellular BioMedicine Group Inc.: Consultancy. Miklos:Kite - Gilead: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy, Research Funding; Pharmacyclics - Abbot: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Genentech: Research Funding; Janssen: Consultancy, Research Funding. Herrera:Merck, Inc.: Consultancy, Research Funding; Immune Design: Research Funding; Pharmacyclics: Consultancy, Research Funding; KiTE Pharma: Consultancy, Research Funding; Seattle Genetics: Research Funding; Gilead Sciences: Research Funding; AstraZeneca: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Genentech: Consultancy, Research Funding. Westin:Apotex: Membership on an entity's Board of Directors or advisory committees; Celgen: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Membership on an entity's Board of Directors or advisory committees. Lee:Kite Pharma, Caladrius Biosciences: Employment; Kite Pharma, Caladrius Biosciences: Equity Ownership; Kite Pharma: Other: TRAVEL, ACCOMMODATIONS, EXPENSES. Rossi:KITE: Employment. Zheng:Kite Pharma: Employment. Avanzi:Kite Pharma: Employment. Roberts:KITE: Employment. Sun:Kite, a Gilead Company: Employment.

Description: Background: ZUMA-5 (NCT03105336) is a multicenter Phase 2 study of axi-cel, an autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy, in patients with relapsed/refractory (R/R) indolent non-Hodgkin lymphoma (iNHL). In an interim analysis of ZUMA-5, axi-cel demonstrated high rates of durable responses (Jacobson CA, et al. ASCO 2020. #8008). Although most patients achieved a durable response, a few patients in ZUMA-5 relapsed. CAR T cell retreatment has resulted in limited responses for aggressive NHL (Locke FL, et al. ASCO 2020. #8012); however, the clinical efficacy of retreatment in iNHL is unknown. Here, we report outcomes of patients with R/R iNHL retreated with axi-cel in the primary analysis of ZUMA-5. Methods: In ZUMA-5, eligible adults (≥ 18 years) with iNHL, specifically follicular lymphoma (FL) or marginal zone lymphoma (MZL), had R/R disease after ≥ 2 lines of therapy. Patients were eligible for retreatment if they progressed after achieving a complete response (CR) or partial response (PR) at the month 3 post-infusion assessment, had no evidence of CD19 loss in progression biopsy, and had no Grade 4 cytokine release syndrome (CRS) or neurologic events (NEs) with first treatment. At both first treatment and retreatment, patients received conditioning chemotherapy followed by axi-cel at a target dose of 2 × 106 CAR T cells/kg. Results: As of March 12, 2020, 11 patients (9 with FL and 2 with MZL) were retreated with axi-cel. Before first treatment, 82% of patients had stage 3 - 4 disease, 91% had ≥ 3 FLIPI, and 91% had high tumor bulk (by GELF criteria). The median prior lines of therapy was 4 (range, 2 - 7); 60% of patients progressed 〈 2 years after initial anti-CD20 mAb-containing therapy (POD24), and 82% had refractory disease. Patients with iNHL who were retreated had significantly higher tumor burden before first treatment (median sum of product diameters) than those who were not retreated (3981 vs 2303 mm2; P = .014). After first treatment, 10 patients achieved a CR, and 1 patient achieved a PR. The first median DOR was 8.3 months (range, 1.9 - 11.8). CRS occurred in 7 patients (4 had Grade 1 and 3 had Grade 2) and NEs occurred in 4 patients (3 had Grade 1 and 1 had Grade 3). Among patients with FL, those who received retreatment (n = 9) had lower median peak CAR T cell levels at first treatment versus other patients with FL (n = 115) who did not receive retreatment (13.2 vs 41.9 cells/µL; P = .024); median peak CAR T cell levels were also lower when normalized by tumor burden (0.003 vs 0.023 cells/µL × mm2; P = .006). Similar trends in CAR T cell expansion were observed in patients with MZL. All 11 patients with iNHL also responded to axi-cel retreatment, with 10 patients achieving a CR and 1 achieving a PR. With a median follow-up of 2.3 months, the median DOR to retreatment was not reached (range, 〈 1 - 8.4 months). Responses were ongoing for 9 patients (82%) at data cutoff. Comparable instances of CRS and NEs were observed with retreatment as with first treatment; CRS occurred with retreatment in 8 patients (6 had Grade 1, 2 had Grade 2), and NEs occurred in 4 patients (3 had Grade 1, 1 had Grade 2). No patient experienced Grade ≥ 3 CRS or NEs with retreatment. Median peak levels of IL-6, IL-2, and IFN-γ, cytokines typically associated with severe CRS and NEs, were numerically similar at retreatment and first treatment (IL-6, 5.4 vs 5.5 pg/mL; IL-2, 1.8 vs 0.9 pg/mL; IFN-γ, 62.9 vs 64.2 pg/mL). At retreatment, median tumor burden by sum of product diameters was lower than that before first treatment (674 vs 3981 mm2; P = .004). In concordance, median levels of TNFα and IL-2Ra, cytokines typically associated with tumor burden, appeared lower before retreatment than before first treatment in patients with FL (TNFα, 1.4 vs 5.3 pg/mL; IL-2Ra, 1.6 vs 19.4 ng/mL). Median peak CAR T cell levels were similar for patients with FL at retreatment as at first treatment (9.0 vs 13.2 CAR-positive cells/µL blood) and remained similar when normalized to tumor burden (0.006 vs 0.003 cells/µL × mm2). Conclusions: Based on a limited sample, axi-cel retreatment exhibited high response rates in patients with R/R iNHL. Similar safety profiles and CAR T cell expansion were observed at retreatment and first treatment, and a lower tumor burden was observed before retreatment than before first treatment. Confirmatory analyses with more patients and longer follow-up are needed. Disclosures Chavez: Kite, a Gilead Company: Consultancy, Speakers Bureau; Novartis: Consultancy; Celgene: Consultancy; AstraZeneca: Speakers Bureau; Gilead: Consultancy; Epizyme: Speakers Bureau; Genentech: Speakers Bureau; AbbVie: Consultancy; Karyopharm: Consultancy; BeiGene: Speakers Bureau; Bayer: Consultancy; Verastem: Consultancy; Pfizer: Consultancy; Morphosys: Consultancy, Speakers Bureau; Merck: Research Funding. Sehgal:Juno Therapeutics: Research Funding; Prothena: Research Funding; Gilead Sciences: Research Funding; Bristol-Myers Squibb: Research Funding; TP Therapeutics: Research Funding; Merck: Research Funding. Neelapu:Incyte: Other: personal fees; Karus Therapeutics: Research Funding; Bristol-Myers Squibb: Other: personal fees, Research Funding; Merck: Other: personal fees, Research Funding; Kite, a Gilead Company: Other: personal fees, Research Funding; Novartis: Other: personal fees; N/A: Other; Cellectis: Research Funding; Poseida: Research Funding; Unum Therapeutics: Other, Research Funding; Calibr: Other; Takeda Pharmaceuticals: Patents & Royalties; Acerta: Research Funding; Legend Biotech: Other; Allogene Therapeutics: Other: personal fees, Research Funding; Pfizer: Other: personal fees; Precision Biosciences: Other: personal fees, Research Funding; Adicet Bio: Other; Cell Medica/Kuur: Other: personal fees; Celgene: Other: personal fees, Research Funding. Maloney:Bioline Rx: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; MorphoSys: Consultancy, Honoraria; Juno Therapeutics: Consultancy, Honoraria, Patents & Royalties: Patents are pending, but not issued, licensed, no royalties, no licensees., Research Funding; A2 Biotherapeutics: Consultancy, Current equity holder in publicly-traded company, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; Gilead Sciences: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Salles:Abbvie, Amgen, Celgene, Gilead, Janssen, Kite, Morphosys, Novartis, F. Hoffmann-La Roche, Takeda: Honoraria; Abbvie, Amgen, Celgene, Gilead, Janssen, Kite, Morphosys, Novartis, Roche, Takeda: Other: Participation to educational events; Abbvie, Autolus, BMS/Celgene, Debiopharm, Genmab, Kite/Gilead, Epizyme, Janssen, Karyopharm, Morphosys, Novartis, Roche, Takeda: Membership on an entity's Board of Directors or advisory committees; Abbvie, Autolus, BMS/Celgene, Debiopharm, Genmab, Kite/Gilead, Epizyme, Janssen, Karyopharm, Morphosys, Novartis, F. Hoffmann-La Roche, Takeda: Consultancy. William:Seattle Genetics: Research Funding; Dova: Research Funding; Guidepoint Global: Consultancy; Incyte: Research Funding; Kyowa Kirin: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Merck: Research Funding. Yang:Kite, a Gilead Company: Current Employment. Goyal:Kite, a Gilead Company: Current Employment. Chou:Gilead Sciences: Current Employment, Other: stock or other ownership; Five Prime Therapeutics: Other: stock or other ownership , Patents & Royalties; Kite, a Gilead Company: Other: travel support. Plaks:Gilead Sciences: Other: stock or other ownership ; Kite, a Gilead Company: Current Employment, Other: travel support. Avanzi:MSKCC: Patents & Royalties; Gilead Sciences: Other: stock or other ownership ; Kite, a Gilead Company: Current Employment, Other: travel support.

Description: 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.