Search

Chimeric antigen receptor (CAR)-modified T cells and bispecific T cell-engaging antibodies have demonstrated dramatic clinical responses in recent clinical trials. The hallmark of these novel highly active immunotherapies is nonphysiologic T cell activation, which has correlated not only with greatly increased efficacy but also with notable toxicity in some cases. We and others have observed a cytokine release syndrome (CRS), which correlates with both toxicity and efficacy in patients receiving T cell-engaging therapies. In addition to elevations in effector cytokines, such as interferon-γ, cytokines associated with hemophagocytic lymphohistiocytosis or macrophage activation syndrome, such as interleukin (IL)-10 and IL-6, may also be markedly elevated. Whereas corticosteroids may control some of these toxicities, their potential to block T cell activation and abrogate clinical benefit is a concern. Detailed studies of T cell proliferation and the resultant immune activation produced by these novel therapies have led to more targeted approaches that have the potential to provide superior toxicity control without compromising efficacy. One approach we have developed targets IL-6, a prominent cytokine in CRS, using the IL-6R antagonist tocilizumab. We will review the pathophysiology and management options for CRS associated with T cell-engaging therapies.

Advances in treatment regimens have improved outcomes for children with acute myeloid leukemia (AML) over the past several decades with 5-year mortality rates decreasing from over 80% in the mid-1970s to approximately 40–60% currently (1). These improvements are largely due to intensification of chemotherapy regimens, which carries the risk of increased treatment-related morbidity and mortality (2–5). Data suggest that children treated for AML are at high risk for sepsis, respiratory failure requiring mechanical ventilation, and circulatory shock requiring inotropic support (2, 6, 7). Initial reports of children with cancer receiving mechanical ventilation showed ICU mortality rates as high as 70–74% (8, 9). These outcomes led to wide variations in practice, presumably due to concerns regarding the appropriateness of offering intensive care to children with cancer (10–12). Although more recent studies have shown lower mortality rates in children with cancer receiving mechanical ventilation (25–65%) (13–22), these rates are substantially higher than the reported 7.2% mortality rate for a general population of mechanically ventilated children (23). Notably, among children with cancer who receive both mechanical ventilation and inotropic support, reported mortality rates have ranged from 54% to 69% (20, 24, 25). Despite the intensity of modern AML treatment regimens, there are few contemporary data documenting the frequency of ICU care, risk of specific organ failures, and mortality in admissions requiring ICU care within this patient population. A single-center study of hospital resource utilization in children with cancer demonstrated that approximately 50% of all charges were incurred by 12.7% of patients and these patients were more likely to have AML or to have received hematopoietic stem cell transplant (HSCT) or ICU care (26). These results support the hypothesis that patients with AML are at substantial risk of treatment-related complications requiring intensive supportive care services. However, to our knowledge, there are no data on outcomes relative to need for ICU resources in pediatric patients with AML. To address this gap in outcome data, we analyzed the Pediatric Health Information Systems (PHIS) database to study a large cohort of children treated for de novo AML prior to stem cell transplant at freestanding pediatric hospitals across the United States. The primary objective of this study was to describe the prevalence of ICU care, organ failure, sepsis, and hospital mortality rates in a homogenous pediatric AML population relative to nononcology pediatric patients. Based on our institutional experience, we hypothesized that a majority of children with AML requiring ICU care would survive but that children with AML would have higher mortality in admissions requiring ICU care than children without cancer. Furthermore, we hypothesized that among those requiring ICU care, the mortality rate would be substantially higher in children with AML who had sepsis than in the corresponding group of children without cancer.

Background: Following infusion of tisagenlecleucel, quantitative polymerase chain reaction (qPCR) can be used to measure in vivo kinetics of CAR transgene. The utility of qPCR to inform individual pt treatment decisions and measure functional persistence is an active area of research. Objective: To determine if CAR transgene detection by qPCR can be used to inform treatment decisions. Methods: Transgene levels in blood measured by qPCR from pivotal phase II studies in pts with relapsed/refractory pediatric and young adult acute lymphoblastic leukemia (pALL; ELIANA [NCT02435849, N=75] and ENSIGN [NCT02228096, N=29]) and adult diffuse large B-cell lymphoma (DLBCL; JULIET [NCT02445248, N=93]) were used to investigate the relationship between transgene persistence and clinical response. Results: In both pALL and DLBCL, there were detectable CAR transgene levels by qPCR in both responders and nonresponders. The geometric mean maximal expansion (Cmax) was similar between responding and nonresponding DLBCL pts, while a 1.7-fold difference was observed in pALL pts. For both DLBCL and pALL, high inter-individual variability in transgene levels was noted. Similarly, higher CAR-T cell expansion from flow cytometry data pooled from responding pALL and pediatric chronic lymphocytic leukemia pts was observed vs nonresponding pts (Mueller KT, et al. Blood 2017), while the levels in DLBCL pts were comparatively lower in blood, likely due to partitioning of functional CAR-T cells to target sites including lymph nodes. The median time to maximal transgene level (Tmax) ranged from 9-10 days in DLBCL responders and nonresponders and pALL responders, while nonresponding pALL pts showed delayed expansion with median Tmax of 20 days. The median time corresponding to last quantifiable transgene level (Tlast) and the half-life estimated from the terminal slope of the cellular kinetic profile, two indicators of persistence, were higher in responding vs nonresponding pts with DLBCL and pALL. Despite this general trend, in some cases, transgene levels were not detectable at later time points in pts with continued response. Although the majority of responding pts show persistent transgene levels, some pts maintained a favorable clinical response despite a decline in transgene levels to below the level of quantification. Conclusion: In both pALL and DLBCL, CAR transgene is initially detected at high levels with high variability in both responders and nonresponders. While the majority of responding pts tend to have persistent transgene levels, some pts maintain favorable clinical responses despite a lack of quantifiable transgene. These results indicate that transgene levels in peripheral blood are not definitive to guide clinical decisions in pALL and DLBCL. Further analysis is needed to understand how CAR transgene levels relate to disease burden and duration of response. Citation Format: Rakesh Awasthi, Karen Thudium Mueller, Gregory A. Yanik, Constantine S. Tam, Susana Rives, Joseph P. McGuirk, Michael A. Pulsipher, Michael W. Boyer, Ulrich Jaeger, Andre Baruchel, Gary D. Myers, Peter Borchmann, Stephen J. Schuster, Heather Stefanski, Michael Bishop, Edward R. Waldron, Ozlem Anak, Abhijit Chakraborty, Eric Bleickardt, Stephane Wong, Lida Bubuteishvili Pacaud, Edmund K. Waller, Shannon L. Maude. Evaluation of in vivo chimeric antigen receptor (CAR) transgene levels in patients (pts) treated with tisagenlecleucel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT237.

Background & Aim CTL019 (tisagenlecleucel) is an anti-CD19 genetically modified autologous T-cell therapy approved for treatment of relapsed/refractory pediatric and young adult B-cell acute lymphoblastic leukemia (ALL) and adult diffuse large B-cell lymphoma (DLBCL). For CTL019 manufactured at Penn, the product release criteria include a viability assessment. In clinical trials, viability specification for product release was ≥ 70% viable cells; whereas, for commercial tisagenlecleucel, viability specification for product release was ≥ 80%. We examined the relationship between CTL019 viability release testing and clinical outcomes. Methods, Results & Conclusion We analyzed CTL019 viability release testing in patients (pts) treated on prospective single institution clinical trials for non-Hodgkin lymphomas (NHL) (NCT02030834) and pediatric ALL clinical trials (NCT01626495, NCT02906371). The relationship of CTL019 product viability release testing and overall response, progression-free survival, overall survival, sensitivity/specificity of viability testing to predict complete response, and CAR T-cell expansion were examined. For 39 NHL pts, the median product viability was 89.8% (range: 73.7%-97.7%) and 3 pts had products with These results suggest that there is no dose-response relationship between CTL019 product viability (70-98%) and clinical outcomes.

TPS347 Background: Adoptive immunotherapy with Chimeric Antigen Receptor (CAR)-T cells has transformative potential for the treatment of cancer. However, a primary challenge to the success of these therapies in prostate cancer is the immunosuppressive microenvironment, including high levels of Transforming Growth Factor-beta (TGFβ), encountered by re-directed T cells upon tumor infiltration. Importantly, these immunosuppressive functions of TGFβ can be abrogated in T cells using a dominant negative TGFβ receptor (TGFβRdn), thereby enhancing antitumor immunity. In in vivo disseminated prostate cancer models, co-expression of TGFβRdn on PSMA-redirected CAR-T cells led to increased T cell proliferation, enhanced cytokine secretion, resistance to exhaustion, long-term persistence, and greater tumor eradication. Methods: We initiated a first-in-human phase 1 clinical trial to evaluate the safety and preliminary efficacy of lentivirally-transduced PSMA-directed/TGFβ-insensitive CAR-T cells (CART-PSMA-TGFβRdn) in men with metastatic CRPC. In preliminary dose-escalation cohorts, patients received a single dose of 1-3 x 107/m2 (Cohort 1) or 1-3 x 108/m2 (Cohort 2) CART-PSMA-TGFβRdn cells without lymphodepleting chemotherapy in a 3+3 design. In Cohort 3, patients will receive the MTD of CART-PSMA-TGFβRdn following a lymphodepleting regimen of cyclophosphamide and fludarabine. All patients provide newly obtained metastatic tumor biopsies at baseline, as well as on day +10 following the CAR-T cell infusion and at disease progression. CAR-T expansion and persistence in peripheral blood and trafficking to target tissues is evaluated via quantitative PCR of CART-PSMA-TGFβRdn DNA. Bioactivity of CART-PSMA-TGFβRdn cells is evaluated via multiplex immunoassays. Additional correlative studies include enumeration and phenotyping of circulating tumor cells and DNA. Cohorts 1 and 2 have been completed without observed DLT. Interestingly, a reversible cytokine release syndrome has been observed that is responsive to tocilizumab. Enrollment in Cohort 3 began in September 2018. Cohort expansions will examine serial CART-PSMA-TGFβRdn re-treatment strategies. Clinical trial information: NCT03089203.

Abstract CAR T cell therapy in relapsed B-ALL can result in complete response (CR) rates of 80-90%, but relapse-free survival declines to 60% within the first 12-months due to both CD19-positive and negative relapses. CD19-positive relapses that occur during this time are largely due to early CAR T cell loss. We hypothesize that inhibiting the PD-1:PD-L1 (programmed cell death 1) checkpoint axis may decrease T cell exhaustion, thereby improving CAR T cell function and persistence. We report our single institution experience of the use of PD-1 inhibitors in patients with relapsed or refractory B lymphoblastic malignancies treated with CD19-directed CAR T cell therapy. Methods: Patients treated with CD19-directed CAR T cell therapy (murine CTL019 or humanized CTL119) at the Children's Hospital of Philadelphia who demonstrated repeated early CAR T cell loss or partial/no response to CAR T cell therapy received a PD-1 inhibitor starting no sooner than 14 days after CAR T cell infusion and after resolution of cytokine release syndrome (CRS) symptoms, with the possibility of repeated doses up to every 3 weeks. Results: Fourteen patients, ages 4-17 years, with heavily pretreated, relapsed B-ALL (n=13) or B lymphoblastic lymphoma (n=1), were treated with CD19-directed CAR T cell therapy (CTL019, n=4; or CTL119, n=10) in combination with pembrolizumab (n=13) or nivolumab (n=1). Three of 6 patients treated with CD19 CAR T cells in combination with a PD-1 inhibitor for early B cell recovery re-established B cell aplasia (a reflection of CAR T cell function) for 5-15 months, 2 of whom have persistent B cell aplasia with ongoing pembrolizumab therapy. Four patients started pembrolizumab for bulky extramedullary disease unresponsive to or relapsed after CAR T cells, with 2 partial and 2 complete responses seen. In one patient, significant CAR T cell proliferation was measured within days of starting pembrolizumab and in temporal correlation to radiographic disease response. In 4 patients who failed to achieve disease remission with initial CAR T cell infusion, no CRs were achieved with the addition of pembrolizumab, although partial responses were seen, and one patient progressed with CD19-dim/negative disease. CRS symptoms and fever typical of CAR T cell proliferative responses were observed in 3/14 patients within 2 days of starting pembrolizumab. Other early and delayed adverse effects associated with PD-1 inhibition were tolerable or reversible upon discontinuation, and including 1 case each of acute pancreatitis, hypothyroidism, arthralgias, urticaria, as well as 4 patients with grade 3-4 cytopenias. No grade 5 toxicities or graft-versus-host disease flares occurred. Two patients discontinued pembrolizumab for delayed adverse effects after multiple doses; both patients relapsed/progressed with CD19+ disease a few weeks after discontinuation. Discussion: T cell exhaustion or activation induced CAR T death (AICD) has been suspected to contribute to poor persistence of CAR T cells. We hypothesized that the PD-1 checkpoint pathway may be involved in CAR T cell exhaustion in some cases, which may be overcome by checkpoint inhibition. Here, promising responses were specifically seen in those with early B-cell recovery and bulky extramedullary disease. In contrast, PD-1 inhibition had partial, but no durable, effect in the four B-ALL patients with poor initial marrow response to CAR T cell therapy alone, suggesting a different mechanism such as AICD may be responsible for poor initial responses. No unexpected or fatal toxicities were seen. This cohort shows initial evidence that checkpoint inhibitors can be used effectively and safely with CAR T cell therapy in children with relapsed B-ALL, and that this strategy may augment CAR T cell effect and persistence. Disclosures Teachey: Amgen: Consultancy; La Roche: Consultancy. Callahan:Novartis Pharmaceuticals Corporation: Consultancy. Porter:Genentech: Other: Spouse employment; Novartis: Other: Advisory board, Patents & Royalties, Research Funding; Kite Pharma: Other: Advisory board. Lacey:Novartis Pharmaceuticals Corporation: Patents & Royalties; Tmunity: Research Funding; Parker Foundation: Research Funding; Novartis Pharmaceuticals Corporation: Research Funding. June:Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Immune Design: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties. Maude:Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Introduction: CTL019 is an anti-CD19 genetically modified autologous T-cell immunotherapy developed at the University of Pennsylvania (Penn) that was recently approved for treatment of relapsed/refractory pediatric and young adult B-cell acute lymphoblastic leukemia (ALL) and adult relapsed/refractory diffuse large B-cell lymphoma (DLBCL) as tisagenlecleucel (Novartis). For ALL, the FDA-approved dose is 0.2 to 5.0 x 106 CAR-positive viable T cells per kg of body weight for patients ≤ 50 kg or 0.1 to 2.5 x 108 CAR-positive viable T cells for pts > 50 kg; for DLBCL, the FDA-approved dose is 0.6 to 6.0 x 108 CAR-positive viable T cells. For CTL019 manufactured at Penn, the dose is determined by flow cytometric staining of CAR-positive T cells, which are cryopreserved in product bags along with replicate aliquots of the final formulation in vials, simultaneously cryopreserved for release testing. The CTL019 product release criteria include a post thaw viability assessment using a vial of replicate aliquot of the final formulation for Trypan blue exclusion or dual fluorescence automated cell counting (Luna-FL, Logos Biosystems). There are no published data examining the relationship between CTL019 viability release testing and clinical outcomes. Methods: We analyzed CTL019 post thaw viability release testing in patients treated on one prospective single institution clinical trial of CD19-expressing non-Hodgkin lymphomas (NHL) (NCT02030834) and two single-institution prospective pediatric ALL clinical trials (NCT01626495 and NCT02906371). Patients were assessed for response to therapy and CAR T-cell expansion. Receiver operating characteristic (ROC) curves were constructed for prediction of complete responses based on sensitivity and specificity of CAR T-cell product post thaw viability release test results. Results: 39 pts with relapsed/refractory NHL (24 diffuse large B-cell lymphoma and 15 follicular lymphoma) were enrolled and received the protocol-specified dose of CTL019. Best response rate was 56% (22/39) complete responses (CR). 123 pts with relapsed/refractory pediatric ALL were enrolled and received the protocol-specified dose of CTL019. Best response rate was 96% (118/123) CR/complete remission with incomplete blood count recovery (CRi). For patients with NHL infused with CTL019, product % viability had a median of 89.8% viability (range: 73.7%-97.7%); product % viability quintiles were as follows: 20%-tile=81.7%, 40%-tile=88.3%, 60%-tile=91.1%, 80%-tile=94.8%). ROC area for NHL patients was 0.47 (95%CI: 0.28-0.65). For patients with ALL infused with CTL019, product % viability had a median of 89.3% viability (range: 56.0%-98.4%); product % viability quintiles were as follows: 20%-tile=82.3%, 40%-tile=87.5%, 60%-tile=90.9%, 80%-tile=94.4%). ROC area for ALL patients was 0.52 (95%CI: 0.32-0.71). For patients with NHL, progression-free survival (PFS) was not significantly influenced by product viability release test results by Cox proportional hazards (HR: 1.0, 95%CI: 0.94-1.09, p=0.7). For patients with NHL, peak CAR T-cell expansion was not significantly correlated with product viability release test results (r2=0.12, p=0.5). Data collection for Cox analysis to investigate the effect of release test viability on PFS and correlation of release test viability with peak CTL019 expansion in ALL is ongoing and will be presented. Conclusions: Our data suggest that, within the ranges obtained in these trials, there is no clear dose-response relationship between CTL019 product viability release test results and clinical response rates in pediatric and young adult ALL or DLBCL. Figure Figure. Disclosures Chong: Novartis: Consultancy. Levine:Cure Genetics: Consultancy; Brammer Bio: Consultancy; CRC Oncology: Consultancy; Incysus: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties; Jazz Pharmaceuticals: Consultancy. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Siegel:Novartis: Research Funding. Maude:Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees. Frey:Novartis: Consultancy; Servier Consultancy: Consultancy. Porter:Genentech: Other: Spouse employment; Novartis: Other: Advisory board, Patents & Royalties, Research Funding; Kite Pharma: Other: Advisory board. June:Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding. Schuster:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Consultancy, Honoraria; Merck: Consultancy, Honoraria, Research Funding; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nordic Nanovector: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees.

Background Quantitative polymerase chain reaction (qPCR) is an analytical method that has been used to investigate the in vivo kinetics of chimeric receptor antigen (CAR) transgene following the infusion of tisagenlecleucel. B cell aplasia, likely an "on-target toxicity" of tisagenlecleucel, has been considered a measure of functional persistence. (Maude SL et al. Blood 2015;125(26):4017-4023) Although the CAR transgene can be detected in peripheral blood of tisagenlecleucel treated patients, it is unclear whether CAR transgene detection by qPCR could be reliably used to inform treatment decision in an individual patient. Methods Transgene levels in blood measured by qPCR from pivotal phase II studies in relapsed/refractory (r/r) pediatric and young adult acute lymphoblastic leukemia (B-ALL) patients (pts) (ELIANA [NCT02435849, N=75]; ENSIGN [NCT02228096, N=29]) and adult diffuse large B cell lymphoma (DLBCL) pts (JULIET [NCT02445248, N=93]) were used to investigate the relationship between transgene persistence and clinical response. Results To determine whether CAR qPCR measurements are associated with or predictive of response, CAR transgene levels and timing of peak levels were examined. In both ALL and DLBCL pts, there were detectable CAR transgene levels by qPCR in both responders and non-responders. The geometric mean maximal expansion (geo mean Cmax) was similar between responding and non-responding adult DLBCL pts, while 1.7 fold differences were observed in pediatric ALL pts (geo mean Cmax in copies/µg: responders, 32700, n=79; non-responders, 19500, n=10; Table 1). For both DLBCL and ALL pts, high inter-individual variability in transgene levels was noted. Similarly, higher CAR-T cell expansion from flow cytometry data pooled from responding pediatric ALL and chronic lymphocytic leukemia (CLL) pts were observed relative to non-responding pts (Mueller KT et al. Blood 2017;130(21):2317-2325), while the levels in DLBCL pts were comparatively lower in blood, likely due to partitioning of functional CAR-T cells to the target sites including lymph nodes. The median time to maximal transgene level ranged from 9-10 days in DLBCL responders and non-responders and pediatric ALL responders, while non-responding pediatric ALL pts showed delayed expansion with median Tmax of 20 days. The median time corresponding to last quantifiable transgene level (Tlast), an indicator of persistence, was higher in responding pts compared to non-responding pts, indicating a trend for longer persistence in both DLBCL and ALL pts with continued response (Table 1). Similarly, the half-life estimated from the terminal slope of the cellular kinetic profile, an additional indicator of persistence, was higher in responding pts relative to non-responding pts for both DLBCL and ALL (Table 1). Despite this general trend, in some cases, transgene levels were not detectable at later time points in pts with continued response. The swimmer plot for representative responder ALL (Figure 1a) and DLBCL pts (Figure 1b) with responses and transgene levels demonstrate that although the majority of responding pts show persistent transgene levels, some pts maintained a favorable clinical response despite a decline in transgene levels to below the level of quantification of 50 copies/µg. Conclusion In both ALL and DLBCL, CAR transgene is initially detected at high levels with high variability in both responders and non-responders. While the majority of responding pts tend to have persistent transgene levels, some pts maintain favorable clinical responses despite a lack of quantifiable transgene. These results indicate that qPCR testing for CAR transgene in blood of tisagenlecleucel treated pts should not be used for making treatment decisions for individual pts. In addition, the qPCR measurements in peripheral blood do not reflect on the trafficking of CAR positive cells to sites outside peripheral blood. The assessment by flow cytometry remains an important assay to distinguish high expression in responding vs non-responding pts in ALL and CLL, and further evaluation of target tissue is needed in DLBCL to understand the utility of CAR expression as a means to distinguish responder and non-responders. Also, further data are needed to improve our understanding of how CAR transgene levels relate to disease burden and duration of response and whether this information is clinically useful. Disclosures Awasthi: Exelixis: Equity Ownership; Celgene: Equity Ownership; Novartis Institutes for Biomedical Research: Employment. Mueller:Novartis Institutes for Biomedical Research: Employment; Novartis Pharmaceuticals Corporation: Equity Ownership, Other: Patent pending. Tam:Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BeiGene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Research Funding. Rives:Amgen: Consultancy, Other: advisory board ; Novartis Pharmaceuticals Corporation: Consultancy, Other: Symposia, advisory boards ; Jazz Pharma: Consultancy, Other: Symposia, advisory boards ; Shire: Consultancy, Other: Symposia, advisory boards . McGuirk:Bellicum Pharmaceuticals: Research Funding; Fresenius Biotech: Research Funding; Novartis Pharmaceuticals Corporation: Honoraria, Other: speaker, Research Funding; Astellas Pharma: Research Funding; Gamida Cell: Research Funding; Kite Pharma: Honoraria, Other: travel accommodations, expenses, speaker ; Pluristem Ltd: Research Funding. Pulsipher:Adaptive Biotech: Consultancy, Research Funding; Amgen: Honoraria; CSL Behring: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau. Jaeger:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria; Takeda-Millenium: Membership on an entity's Board of Directors or advisory committees; Takeda-Millenium: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Infinity: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; MSD: Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Baruchel:Novartis: Membership on an entity's Board of Directors or advisory committees; Shire: Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel, accommodations or expenses; Amgen: Consultancy; Roche: Consultancy; Servier: Consultancy; Celgene: Consultancy. Myers:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding, Speakers Bureau. Balke-Want:Novartis Pharmaceuticals Corporation: Honoraria. Schuster:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Consultancy, Honoraria, Research Funding; Dava Oncology: Consultancy, Honoraria; Nordic Nanovector: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Research Funding. Stefanski:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Speakers Bureau. Bishop:Novartis Pharmaceuticals Corporation: Speakers Bureau; Juneau Therapeutics: Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; United Healthcare: Employment. Waldron:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Anak:Novartis Pharma AG: Employment. Chakraborty:Novartis Institutes for Biomedical Research: Employment. Bleickardt:Novartis Pharmaceuticals Corporation: Employment. Wong:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Bubuteishvili Pacaud:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Waller:Kalytera: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celldex: Research Funding; Pharmacyclics: Other: Travel Expenses, EHA, Research Funding; Cambium Medical Technologies: Consultancy, Equity Ownership. Maude:Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Background: Chimeric antigen receptor T-cells (CAR-T) have emerged as a promising treatment for children with relapsed/refractory acute lymphoblastic leukemia (ALL). While CAR-T outcomes have been published, little data exist on how to manage these patients for the weeks to months between T-cell collection and CAR-T infusion. Unlike stem cell transplant (SCT), where higher burden of disease is associated with poor outcomes and is often a contraindication, successful outcomes for children with high disease burden pre-CAR-T have been demonstrated. Thus, traditional high-intensity chemotherapy for relapsed ALL that aims to minimize disease burden but carries significant morbidity may not be the best option for pre-CAR-T populations. We thus compared our population-based experience in using high vs. low-intensity chemotherapy regimens to bridge patients in terms of toxicity, inpatient days, and success in reaching CAR-T infusion. Methods: The Hospital for Sick Children (Sickkids) is the provincial referral centre for cellular therapy for children in Ontario, Canada. All Ontario children referred to Sickkids between 2014-2018 for first T-cell collection with intent to proceed to CAR-T therapy were included and followed until CAR-T infusion, decision to pursue alternative therapy, or death. Bridging regimen details were collected and classified as low vs. high intensity based on whether such therapy was likely associated with >7 days of neutropenia. Disease and outcome variables were compared between high vs. low intensity regimens using Chi squared, Fisher's exact, or Wilcoxon tests. Results: The cohort included 32 patients with a median age of 9.7 years at the time of first T-cell collection [interquartile range (IQR) 6.0-12.3]. The median number of previous relapses was 2 (IQR 1-2), 14 (44.0%) had undergone prior SCT, and 4 (12.5%) had Down syndrome. The vast majority of patients (28, 87.5%) were successfully bridged to receive CAR-T therapy with a median time to infusion of 81 days (IQR 60-105). Two patients experienced manufacturing failure and pursued SCT instead, 1 died of toxicity, and 1 was still awaiting infusion at the end of the study period. Low-intensity bridging regimens were used following collection for 19 (59.4%) patients, most often based on low-dose intravenous methotrexate, 3-drug induction (vincristine/steroids/asparaginase), or maintenance therapy. The most common high-intensity regimens included cyclophosphamide/etoposide, high dose cytarabine, or 4-drug induction (vincristine/steroids/asparaginase/anthracycline). Patients receiving high-intensity therapy did not seem to have more aggressive disease prior to starting bridging treatment (as indicated by peripheral blasts, number of previous relapses, prior SCT) that would have justified choosing high-intensity treatments. Patients receiving initial high-intensity regimens were however more likely to have been collected in first half of the study period (Table 1). Patients receiving initial high-intensity regimens also developed more microbiologically documented infections and experienced a greater number of inpatient days (Table 1). Excluding patients experiencing manufacturing failure or still awaiting CAR-T at the end of the study period, the likelihood of receiving CAR-T also did not vary [high-intensity regimen - 11/12 (91.7%) vs. low-intensity regimen - 17/17 (100%); p=0.41]. Conclusions: We demonstrate in our population-based cohort of heavily pre-treated and high-risk patients that initial low-intensity chemotherapy had a very high likelihood of successfully bridging children to CAR-T infusion. Low-intensity bridging regimens were associated with lower rates of toxicity and higher quality of life as indicated by fewer inpatient days. Low-intensity regimens should be considered the first line option in this population. Disclosures Grupp: Jazz Pharmaceuticals: Consultancy; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding. Maude:Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees.

BACKGROUND Tisagenlecleucel is an FDA approved chimeric antigen receptor (CAR)-T cell therapy that reprograms T cells to eliminate CD19+ B cells. ELIANA (NCT02435849) is a phase 2 pivotal study of tisagenlecleucel in pediatric/young adult patients (pts) with CD19+ r/r B-cell acute lymphoblastic leukemia (ALL), the first global trial of a CAR-T cell therapy. The primary objective was met, with an overall remission rate (ORR) of 81% (complete remission [CR] + CR with incomplete blood count recovery [CRi]). Here we present an update of ELIANA, with additional pts and additional 11 mo follow-up from the previous report (Maude et al. N Engl J Med 2018). METHODS Eligible pts were aged ≥3 y at screening and ≤21 y at diagnosis and had ≥5% leukemic blasts in bone marrow. Tisagenlecleucel was centrally manufactured at 2 sites (Morris Plains, NJ, USA and Leipzig, Germany) by lentiviral transduction of autologous T cells with a vector encoding for a second generation 4-1BB anti-CD19 CAR and expanded ex vivo. Tisagenlecleucel was provided to pts at 25 study centers in 11 countries on 4 continents using cryopreserved apheresed mononuclear cells, central production facilities, and a global supply chain. The primary endpoint, ORR within 3 mo and maintained for ≥28 d among infused pts, was assessed by an independent review committee. Secondary endpoints included duration of remission (DOR), overall survival (OS), safety, and cellular kinetics. RESULTS As of April 13, 2018, 113 pts were screened and 97 enrolled. There were 8 manufacturing failures (8%) and 10 pts (10%) were not infused due to death or adverse events (AEs). Following lymphodepleting chemotherapy in most pts (76/79; fludarabine/cyclophosphamide [n=75]), 79 pts were infused with a single dose of tisagenlecleucel (median dose, 3.0×106 [range, 0.2-5.4×106] CAR-positive viable T cells/kg), and all had ≥3 mo of follow-up or discontinued earlier (median time from infusion to data cutoff, 24 mo [range, 4.5-35 mo]). Median age was 11 y (range, 3-24 y); 61% of pts had prior hematopoietic stem cell transplant (SCT). Among the 65 pts with CR/CRi, 64 (98%) were MRD- within 3 mo. Median DOR by K-M analysis was not reached (Figure): responses were ongoing in 29 pts (max DOR, 29 mo and ongoing); 19 pts relapsed before receiving additional anticancer therapy (13 died subsequently); 8 pts underwent SCT while in remission, 8 received additional anticancer therapy (non-SCT) and 1 discontinued while in remission. The probability of relapse-free survival at 18 mo was 66% (95% CI, 52%-77%). Median OS was not reached; OS probability at 18 mo was 70% (95% CI, 58%-79%). Cytokine release syndrome (CRS) occurred in 77% of pts (grade [G] 3/4; 48%; graded using the Penn scale); 39% of pts received tocilizumab for treatment of CRS with or without other anti-cytokine therapies; 48% of pts required ICU-level care for CRS, with a median ICU stay of 7 d. All cases of CRS were reversible. Most common G 3/4 nonhematologic AEs (>15%) other than CRS were neutropenia with a body temperature >38.3°C (62% within 8 wk of infusion), hypoxia (20%), and hypotension (20%). 13% of pts experienced G 3 neurological events, with no G 4 events or cerebral edema. Based on laboratory results, 43% and 54% of pts had G 3/4 thrombocytopenia and neutropenia not resolved by d 28; the majority of events resolved to G ≤2 by 3 mo. 25 post-infusion deaths were reported: 2 within 30 d (1 disease progression, 1 cerebral hemorrhage); 23 after 30 d of infusion (range, 53-859 d; 18 disease progression, 1 each due to encephalitis, systemic mycosis, VOD [hepatobiliary disorders related to allogeneic-SCT], bacterial lung infection, and an unknown reason after study withdrawal). Tisagenlecleucel expansion in vivo correlated with CRS severity, and persistence of tisagenlecleucel along with B-cell aplasia in peripheral blood was observed for ≥2.5 y in some responding pts. Analysis of B-cell recovery and correlation with relapse will be presented. CONCLUSIONS With longer follow-up, the ELIANA study continues to confirm the efficacy of a single infusion of tisagenlecleucel in pediatric and young adults with ALL without additional therapy. AEs were effectively and reproducibly managed globally by appropriately trained personnel at study sites. The achievement of high overall response rates and deep remissions, in combination with the median duration of response and overall survival not being reached, further corroborate previously reported results. Figure. Figure. Disclosures Grupp: Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties. Maude:Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees. Rives:Shire: Consultancy, Other: Symposia, advisory boards ; Jazz Pharma: Consultancy, Other: Symposia, advisory boards ; Novartis Pharmaceuticals Corporation: Consultancy, Other: Symposia, advisory boards ; Amgen: Consultancy, Other: advisory board . Baruchel:Celgene: Consultancy; Amgen: Consultancy; Roche: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel, accommodations or expenses; Novartis: Membership on an entity's Board of Directors or advisory committees; Shire: Research Funding; Servier: Consultancy. Bittencourt:Novartis Pharmaceuticals Corporation: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria. Bader:Riemser: Research Funding; Cellgene: Consultancy; Medac: Patents & Royalties, Research Funding; Neovii: Research Funding; Novartis: Consultancy, Speakers Bureau. Laetsch:Bayer: Consultancy; Pfizer: Equity Ownership; Eli Lilly: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy; Loxo Oncology: Consultancy. Stefanski:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Speakers Bureau. Myers:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding, Speakers Bureau. Qayed:Novartis: Consultancy. Pulsipher:CSL Behring: Consultancy; Amgen: Honoraria; Adaptive Biotech: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Speakers Bureau. Martin:Novartis Pharmaceuticals Corporation: Research Funding; Jazz Pharmaceuticals: Research Funding. Nemecek:Novartis Pharmaceuticals Corporation: Other: advisory boards. Boissel:Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees. Leung:Novartis Pharmaceuticals Corporation: Employment. Eldjerou:Novartis Pharmaceuticals Corporation: Employment. Yi:Novartis Pharmaceuticals Corporation: Employment. Mueller:Novartis Institutes for Biomedical Research: Employment; Novartis Pharmaceuticals Corporation: Equity Ownership, Other: Patent pending. Bleickardt:Novartis Pharmaceuticals Corporation: Employment.

The mechanisms underlying the maintenance of long-lasting humoral immunity are not well understood. Studies in mice indicate that plasma cells (PCs) can survive up to a lifetime, even in the absence of regeneration by B cells, implying the presence of long-lived PCs as a mechanism for long-lasting immunity. Evidence from humans treated with anti-CD20, which depletes circulating B cells, also suggests B-cell-independent long-term survival of some PCs. On the other hand, antibody responses may be sustained solely by short-lived PCs with repopulation from clonally related memory B cells. To explore PC longevity and humoral immunity in humans, we investigated the fate of PCs and their antibodies in adult and pediatric patients who received chimeric antigen receptor-based adoptive T-cell immunotherapy targeting CD19 to treat B-cell lineage malignancies (CTL019). Treatment with CTL019 is frequently associated with B-cell aplasia that can persist for years. Serum antibody titers to vaccine-related antigens were measured, and quantitative assessment of B cells and PCs in blood and bone marrow was performed at various time points before and after CTL019 therapy. While total serum immunoglobulin concentrations decline following CTL019-induced B-cell aplasia, several vaccine/pathogen-specific serum immunoglobulin G and A (IgG and IgA) titers remain relatively stable for at least 6 and 12 months posttreatment, respectively. Analysis of bone marrow biopsies after CTL019 revealed 8 patients with persistence of antibody-secreting PCs at least 25 months post-CTL019 infusion despite absence of CD19(+)CD20(+) B cells. These results provide strong evidence for the existence of memory B-cell-independent, long-lived PCs in humans that contribute to long-lasting humoral immunity.

SummaryGenomic profiling has identified a subtype of high-risk B-progenitor acute lymphoblastic leukemia (B-ALL) with alteration of IKZF1, a gene expression profile similar to BCR-ABL1-positive ALL and poor outcome (Ph-like ALL). The genetic alterations that activate kinase signaling in Ph-like ALL are poorly understood. We performed transcriptome and whole genome sequencing on 15 cases of Ph-like ALL and identified rearrangements involving ABL1, JAK2, PDGFRB, CRLF2, and EPOR, activating mutations of IL7R and FLT3, and deletion of SH2B3, which encodes the JAK2-negative regulator LNK. Importantly, several of these alterations induce transformation that is attenuated with tyrosine kinase inhibitors, suggesting the treatment outcome of these patients may be improved with targeted therapy.

The field of adoptive cell transfer includes chimeric antigen receptor (CAR) engineered T cells, constructs that emerged from basic research into principles of immunology and have transformed into clinically effective therapies for haematological malignancies. T cells engineered to express these artificial receptors hold great promise, but also carry significant risk. While permanent genetic modification of mature T cells appears safe, modulating their in vivo function is difficult, partly because the robust response can trigger other arms of the immune system. Suicide systems and toxicity management with cytokine blockade or signal transduction modulators have emerged as a new frontier in this field, a far cry from early problems getting CAR T cells to work at all. Currently, clinical trials in patients with relapsed or refractory B cell malignancies treated with CD19-specific CAR T cells have induced durable remissions in adults and children. Results from these trials indicate that more work needs to be done to understand biomarkers of efficacy, the role of T cell persistence and how to integrate this care into standard practice. Cell therapy will not be a 'one size fits all' class of medicine, and here we will discuss the development of this therapy and important questions for its future.

3051Background: Chimeric antigen receptor (CAR) T cells targeting CD19 have shown excellent responses in patients with relapsed/refractory B-ALL. However, a subset of patients either do not respond...

3044Background: Tisagenlecleucel, a chimeric antigen receptor (CAR) T-cell therapy, contains a murine single chain variable fragment (mCAR19) binding domain. Humoral immunity to anti-mCAR19 had no ...

Introduction. Anti-CD19 chimeric antigen receptor T cells (CART19 or CTL019) have shown impressive clinical activity in B-cell acute lymphoblastic leukemia (B-ALL) and are poised to receive FDA approval. However, some patients relapse after losing CD19 expression. Since CD22 remains highly expressed in relapsed/refractory (r/r) B-ALL even in these patients, anti-CD22 CART (CART22) have been developed. The National Cancer Institute (NCI) reported 4/9 complete remission (CR) in patients receiving CART22, with 100% CR at the highest T cell dose (NCT02315612)(S hah NN, ASH 2016 #650). Patients and Methods. We generated a second-generation CAR22 differing from that used by the NCI only by the use of a longer linker [4x(GGGGS); LL vs. 1x(GGGGS); SL] between the light and heavy chains of the scFv (Fig. 1 A). This construct was tested in two pilot clinical trials in adults (NCT02588456)and children with r/r-ALL (NCT02650414). CART22 cells were generated using lentiviral transduction as in our previous studies. The protocol-specified CART22 dose was 2x106-1x107 cells/kg for pediatric patients For the preclinical studies, we generated CART22LL and CART22SL and tested them in vivo using xenograft models. NOD-SCID gamma chain deficient (NSG) mice were engrafted with either a luciferase+ standard B-ALL cell line (NALM6) or primary B-ALL cells obtained from a patient relapsing after CART19 (CHP110R). We also used 2-photon imaging to study the in vivo behavior and immune synapse formation and flow cytometry to asses T cell activation. Results. CART22 cells were successfully manufactured for 10/12 patients. In the adult cohort 3/3 patients developed CRS (gr.1-3) and no neurotoxicity was observed; in the pediatric cohort out of 5 evaluable patients (1 discontinued for lineage switch to AML on pre-infusion marrow), 3/5 developed cytokine-release syndrome (CRS) (all grade 2) and 1 patient had encephalopathy (gr.1). CART22 cells expanded in the PB with median peak of 1977 (18-40314) copies/ug DNA at day 11-18. Interestingly, in an adult patient who had previously received CART19 a second CART19 re-expansion was observed following CART22 expansion (Fig 1 B). At day 28, in the adult cohort the patient who was infused in morphologic CR remained in CR, while the other 2 had no response (NR); in the pediatric cohort 2/5 patients were in CR, 1 in partial remission (PR) that then converted to CR with incomplete recovery at 2 months, and 2 NR. No CD22-negative leukemia progression was observed. Since our results with a long linker appeared inferior compared to the previously reported CART22 trial (short linker), we performed a direct comparison of the 2 different CAR22 constructs. In xenograft models, CART22SL significantly outperformed CART22LL (Fi 1 C) with improved overall survival. Moreover, CART22SL showed higher in vivo proliferation at day 17 (Fig 1 D). Mechanistically, intravital 2-photon imaging showed that CART22SL established more protracted T cell:leukemia interactions than did CART22LL, suggesting the establishment of productive synapses (Fig 1 E). Moreover, in vivo at 24 hrs higher T cell activation (CD69, PD-1) was observed in CART22SL from the BM of NALM-6-bearing mice. Conclusions. Here we report the results of two pilot clinical trials evaluating the safety and feasibility of CART22 therapy for r/r B-ALL. Although feasible and with manageable toxicity CART22LL led to modest clinical responses. Preclinical evaluation allowed us to conclude that shortening the linker by 15 amino acids significantly increases the anti-leukemia activity of CART22, possibly by leading to more effective interactions between T cells and their targets. Finally, with the caveats of cross-trial comparison, our data suggest that xenograft models can predict the clinical efficacy of CART products and validate the use of in vivo models for lead candidate selection Disclosures Ruella: Novartis: Patents & Royalties, Research Funding. Maude: Novartis Pharmaceuticals: Consultancy, Other: Medical Advisory Boards. Engels: Novartis: Employment. Frey: Novartis: Research Funding. Lacey: Novartis: Research Funding; Genentech: Honoraria. Melenhorst: Novartis: Research Funding. Brogdon: Novartis: Employment. Young: Novartis: Research Funding. Porter: Incyte: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Immunovative Therapies: Other: Member DSMB; Genentech/Roche: Employment, Other: Family member employment, stock ownship - family member; Servier: Honoraria, Other: Travel reimbursement. June: WIRB/Copernicus Group: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celldex: Honoraria, Membership on an entity's Board of Directors or advisory committees; Immune Design: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Grupp: Jazz Pharmaceuticals: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Other: grant; University of Pennsylvania: Patents & Royalties; Adaptimmune: Consultancy. Gill: Novartis: Patents & Royalties, Research Funding.

Relapsed and refractory acute lymphoblastic leukemia (ALL) remains difficult to treat, with minimal improvement in outcomes seen in more than 2 decades despite advances in upfront therapy and improved survival for de novo ALL. Adoptive transfer of T cells engineered to express a chimeric antigen receptor (CAR) has emerged as a powerful targeted immunotherapy, showing striking responses in highly refractory populations. Complete remission (CR) rates as high as 90% have been reported in children and adults with relapsed and refractory ALL treated with CAR-modified T cells targeting the B-cell–specific antigen CD19. Distinct CAR designs across several studies have produced similar promising CR rates, an encouraging finding. Even more encouraging are durable remissions observed in some patients without additional therapy. Duration of remission and CAR-modified T-cell persistence require further study and more mature follow-up, but emerging data suggest these factors may distinguish CAR designs. Supraphysiologic T-cell proliferation, a hallmark of this therapy, contributes to both efficacy and the most notable toxicity, cytokine release syndrome (CRS), posing a unique challenge for toxicity management. This review will discuss the current landscape of CD19 CAR clinical trials, CRS pathophysiology and management, and remaining challenges.

Genomic instability has long been considered a key factor in tumorigenesis. Recent evidence suggests that DNA damage may be widespread in early pre-neoplastic states, with deregulation of cyclin-dependent kinase (Cdk) activity a driving force. Increased Cdk activity may critically reduce licensing of origins of DNA replication, drive re-replication, or mediate overexpression of checkpoint proteins, inducing deleterious cell cycle delay. Conversely, inhibition of Cdk activity may compromise replication efficiency, expression of checkpoint proteins, or activation of DNA repair proteins. These vital functions point to the impact of Cdk activity on the stability of the genome. Insight into these pathways may improve our understanding of tumorigenesis and lead to more rational cancer therapies.

Cyclin-dependent kinases (Cdk) promote cell proliferation, are often deregulated in human cancers, and are targets of ongoing cancer chemotherapy trials. We show here that Cdk activity is also required in human cells to maintain function of the Chk1 pathway, a key component of the response to DNA damage or stalled replication. Chk1 expression was markedly reduced in primary fibroblasts and U2OS osteogenic sarcoma cells by treatment with small molecule Cdk inhibitors or induction of a dominant-negative mutant of Cdk2. The findings of decreased Chk1 activity and accumulation of Cdc25A, a protein targeted for degradation by Chk1, confirmed that Chk1 function was impaired. Furthermore, Cdk inhibition triggered a DNA damage response, characterized by the accumulation of activated forms of ATM and Chk2 as well as nuclear foci containing phosphorylated substrates of ATM/ATR, including histone H2AX (γH2AX). Time course experiments showed that the bulk of ATM activation followed Chk1 down-regulation. Chk1 RNA interference combined with partial inhibition of DNA replication was sufficient to evoke the DNA damage response. Conversely, ectopic expression of Chk1 blunted induction of γH2AX foci by Cdk inhibitors, indicating that Chk1 down-regulation was necessary to elicit the full phenotype. Finally, both Cdk and Chk1 inhibitors enhanced the cytotoxity of etoposide, a DNA-damaging agent. These results define a pathway through which Cdk inhibition can mediate DNA damage and potentially enhance the efficacy of extant cancer chemotherapies.

Targeted immunotherapy has become critical for the successful treatment of many forms of cancer, particularly therapeutic antibodies with cytotoxic abilities. No safe and effective immunotherapies have been developed for T-cell acute lymphoblastic leukemia (T-ALL). CD38 is a transmembrane glycoprotein found on the cell surface of activated T cells, terminally differentiated B cells, but relatively low levels on normal lymphoid and myeloid cells. Daratumumab (dara) (Darzalex, Janssen Biotech, Inc.) is a human IgG1κ monoclonal antibody that binds to a unique CD38 epitope and was recently FDA approved for the treatment of refractory multiple myeloma. In order to ensure CD38 is a relevant target in T-ALL and surface expression does not change with chemotherapy, we evaluated CD38 expression by flow cytometry (FACS) from 21 patients with T-ALL (10 early T-cell precursor (ETP) and 11 non-ETP) at diagnosis and after one month of induction chemotherapy. All of the samples had detectable CD38 expression which did not change significantly after induction (mean CD38 MFI at diagnosis vs end-induction: 3.27 log vs 3.19 (S.I.; p = 0.25). Therefore we hypothesized that targeting CD38 with dara would be effective against T-ALL. In order to test this hypothesis, we xenografted primary ALL blasts from 15 different patients, including 7 with ETP-ALL and 8 with non-ETP T-ALL. Mice were randomized to dara (200 μg /mouse intraperitoneally (IP) weekly) vs control (isotype control at 200 μg/mouse IP weekly; 5 mice per arm for each sample) after they developed >1% peripheral blood (pb) blasts by FACS. Disease burden was assessed by FACS enumeration of pb blasts weekly and splenic blasts at sacrifice. We demonstrate striking efficacy of dara monotherapy in 6 of 7 ETP-ALL samples with reduction of pb and splenic blasts. Mice treated with high disease burden from 5 of the 8 non-ETP samples were moribund immediately after dara injection, possibly from aggregates of antibody bound to tumor cells leading to pulmonary embolism or tumor lysis, and were therefore inevaluable. We have since repeated the experiments for 3 of the samples, treating the mice after injection but before detectable engraftment of peripheral blasts. Dara was effective in all 3 samples. Experiments are ongoing for the other 2 samples. Overall, we have found Dara was effective in 4 of 6 evaluable non-ETP T-ALL samples. In summary, we found dara is a highly effective novel monotherapy for T-ALL in preclinical models. Based on these results, we are developing an early phase trial for children and young adults with relapsed/refractory T-ALL. Note: This abstract was not presented at the meeting. Citation Format: Karen Lee Bride, Tiffaney Vincent, Soo-Yeon L. Im, Tori Fuller, Theresa Ryan, David M. Barrett, Shannon L. Maude, Mignon L. Loh, Michelle L. Hermiston, Stephan A. Grupp, Brent L. Wood, David T. Teachey. Preclinical efficacy of daratumumab in acute lymphoblastic leukemia [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 2642. doi:10.1158/1538-7445.AM2017-2642

10507 Background: Anti-CD19 CAR-T cell therapies have shown high efficacy in inducing durable marrow responses in patients with relapsed/refractory CD19+ ALL. We now report on outcome of 10 patients with extramedullary (EM) involvement of ALL treated with CAR-T, including 5 patients who had EM disease at time of infusion. Methods: We identified patients treated on pediatric phase 1/2a trials of murine (CTL019) or humanized (CTL119) anti-CD19 CAR-T cells for isolated EM or BM/EM relapse of ALL. EM relapse was defined as involvement of non-CNS site by imaging +/- pathology within 12 months (mos) of infusion. Post infusion, patients had diagnostic imaging done at 1, 3, 6, 9, and 12 mos. Results: Among 97 patients receiving CAR-T, ten (CTL019, n=6; CTL119, n=4) were identified who had EM involvement on average 2.3 mos (range 0-9 mos) prior to infusion; including 5/10 at time of infusion. Sites of EM relapses included testes, sinus, parotid, bone, uterus, kidney and skin, and 5 patients had multiple sites of EM involvement. Patients ranged from 2-4 relapses of their ALL pre-CAR-T. Two had isolated EM relapse (sites were parotid and multifocal bony lesions in one; testis and sinus in second). All 10 patients had undergone hematopoietic stem cell transplantation prior to EM relapse, 2 had received radiation directed to the EM site prior to CAR-T. Five patients evaluated by serial imaging had objective responses: 2 had resolution of EM disease by day 28; 2 had resolution by 3 mos; 1 had continued decrease in size of uterine mass at 3 and 6 mos and underwent hysterectomy at 8 mos with no evidence of disease on pathology. In the 4 patients with prior history of skin or testicular involvement, there was no evidence by exam at day 28. One patient had progressive EM disease within 2 weeks of CAR-T cell infusion and died at 6 weeks. Three relapsed with CD19+ disease [1 skin/medullary- died at 38 mos post CAR-T; 2 medullary (1 died at 17 mos, 1 alive at 28 mos)]. The remaining 6 are alive and well at median follow-up of 10 mos (range 3-16 mos) without recurrence of disease. Conclusions: Single agent CAR-T immunotherapy can induce potent and durable responses in patients with EM relapse of their ALL. Clinical trial information: NCT01626495, NCT02374333.

103 Background: CD19-targeted CAR T cells show CR rates of 70-95% in B-ALL. Yet a subset of patients do not respond or relapse due to poor CAR T cell expansion and persistence. We hypothesized that PD-1 checkpoint pathway inhibition may improve CAR T cell expansion, function and persistence. Methods: Four children with relapsed B-ALL treated with murine (CTL019) or humanized (CTL119) anti-CD19 CAR T cells received 1-3 doses of the PD-1 inhibitor pembrolizumab (PEM) for partial/no response or prior history of poor CAR T cell persistence starting 14d-2mo post CAR T cell infusion. Results: PEM increased and/or prolonged detection of circulating CAR T cells in all 4 children, with objective responses in 2/4. It was well tolerated, with fever in 2 pts and no autoimmune toxicity. Pts 1-3 received CTL119 for CD19+ relapse after prior murine CD19 CAR T cells. Pt 1 had 1.2% CD19+ residual disease despite expansion with detectable CTL119 by D28 and received PEM at 2mo for progressive disease with decreasing circulating CTL119. CTL119 became detectable at 0.2% of CD3+ cells by flow cytometry, but disease progressed. Pt 2 had no response after initial CTL119 expansion with a rapid disappearance by D28. After CTL119 reinfusion with PEM added 14d later, circulating CAR T cells remained detectable at 4.4% by D28, but disease progressed with decreased CD19 expression. In Pt 3, prior treatment with both CTL019 and CTL119 produced CR with poor CAR T cell persistence followed by CD19+ relapse. CTL119 reinfusion combined with PEM at D14 resulted in CR with prolonged CTL119 persistence (detectable at D50 compared to loss by D36 after 1st CTL119 infusion). Pt 4 received PEM for widespread extramedullary (EM) involvement at D28 post CTL019 infusion despite marrow remission. Initial CTL019 expansion peaked at 63% at D10 and fell to 20% at D28. Resurgence of CTL019 expansion, with a 2nd peak of 70% 11d after PEM, was associated with dramatic reduction in PET-avid disease by 3mo post CTL019. Conclusions: PEM was safely combined with CAR T cells and increased or prolonged CAR T cell detection, with objective responses seen. Immune checkpoint pathways may impact response to CAR T cell treatments and warrant further investigation. Clinical trial information: NCT02374333, NCT02906371.

10531 Background: Treatment with CAR T-cells targeting CD19 for pediatric leukemia has demonstrated significant efficacy with the principal toxicity being cytokine release syndrome (CRS). However, the cardiac effects related to CAR T-cell therapy have not been systematically evaluated. Methods: A retrospective chart review was performed on a large cohort who received CAR T-cell therapy from April 2012 through September 2016. Baseline oncologic and cardiac characteristics were recorded. Cardiomyopathy was defined as an ejection fraction < 55%, shortening fraction < 28%, or diastolic dysfunction. Primary outcome was cardiac event, defined as need for inotropic support or echocardiographic decline in function. Descriptive and univariate analyses were performed. Results: 93 patients were included [55% male, mean age 11.8 yrs (range 1.7-27.1)]; 98% had B cell ALL. Prior to treatment, 15% had cardiomyopathy and 1 patient had single ventricle Fontan palliation. Cardiac events occurred in 34 (36%) with mean onset 4.8 days (range 1-9) after CAR T-cell infusion, of whom 12 (35%) had abnormal systolic or diastolic function on echocardiogram and 6 (18%) required milrinone for cardiac support. 21 (22%) patients had CRS requiring tocilizumab and vasoactive infusions; there were no cardiac-related deaths. Factors associated with cardiac events included higher pre-treatment blast % on bone marrow biopsy [OR 10.45 (95% CI 3.87-28.22) for blast > 10%; p < 0.001] but not cardiomyopathy (p = 0.356), total body irradiation (p = 0.717) or anthracycline dose (p = 0.711). At discharge, 7 (7.5%) patients had worse cardiac function than at baseline, but only 2 (2%) had persistent cardiac dysfunction by 6 months post-infusion. Pre-treatment factors were not associated with persistent dysfunction. Conclusions: This is the first study to describe the cardiac effects of CAR T-cell therapy in children. CAR T-cell therapy appears to be safe, even in patients with cardiomyopathy. Although one-third experienced a cardiac event, persistent cardiac dysfunction is rare. Children with a pre-treatment blast > 10% were most at risk for cardiac events, a finding that may help identify patients who warrant close observation and early intervention with pressor support.

137 Background: The adoptive transfer of autologous T cells genetically modified to express a CD19-specific, 4-1BB/CD3ζ-signaling CAR (CTL019) has shown remarkable activity and induces long-term remissions in a subset of patients with relapsed/refractory chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). In ALL, CTL019 induces a complete response (CR) in over 90% of patients while in CLL 25% of patients obtain a CR. It is not fully understood why only certain patients respond to therapy. Methods: We employed next generation sequencing of RNA (RNAseq) to identify predictive indicators of response to CTL019. We performed RNAseq on leukapheresis and manufactured product T cells prior to re-infusion from 35 CLL and 7 pediatric ALL patients with heavily pre-treated and high-risk disease. To characterize potency, we performed RNAseq on the infusion product after stimulation with the CAR. Results: We find that durable remission in CLL is associated with gene expression signatures of early memory and T-effector cells, while T cells from non-responding patients are enriched in signatures of T-regulatory cells, terminal differentiation, and exhaustion. In following the results from CLL, we find that pediatric ALL manufactured T cells are significantly enriched for an early memory, naïve T cell state and all achieved a CR. In parallel in vitro experiments, stimulation of the infusion product further demonstrated that CTL019 cells from CRs have an increased capacity for activation upon stimulation. We tested if we could extend these observations to identify a phenotype of T cells that is predictive of response prior to CTL019 manufacturing and find that the signatures predictive of response at the pre-infusion stage are also observed at the earlier leukapheresis time point. Conclusions: These findings suggest that intrinsic T cell fitness dictates response to CAR T cells. These gene expression signatures, along with additional immunological biomarkers, may be used to identify which patients are most likely to respond to adoptive transfer strategies and suggest manufacturing modifications that might potentiate the generation of maximally efficacious infusion products. Clinical trial information: NCT01029366, NCT01747486, NCT01626495.

A single-center trial of CD19 directed, lentiviral transduced chimeric antigen receptor (CAR) T cells (CTL019) for relapsed and refractory (r/r) B-ALL pediatric patients showed rates of CR >90% with prolonged CAR T cell persistence/CR without further therapy in the majority of patients infused (Maude NEJM 2014). We report here the feasibility, safety and efficacy of the first multicenter global pivotal registration CAR T cell trial. Features of this trial include: i) the first trial in which industry-manufactured cells were provided to all patients; ii) enrollment across 25 centers in the US, EU, Canada, Australia, and Japan; iii) successful transfer and manufacturing of cells in a global supply chain; and iv) successful implementation of cytokine release syndrome (CRS) management across a global trial. All patients had CD19 positive B-ALL with morphologic marrow tumor involvement at registration (>5% blasts), and were either primary refractory; chemo-refractory after first relapse, relapsed after second line therapy; or ineligible for allogeneic SCT. CTL019 was manufactured from patient PBMC under GMP conditions in the US, at a centralized "sponsor-owned" manufacturing facility, and supplied to all sites. The primary endpoint of overall remission rate (CR+CRi) within 3 months and secondary endpoints (EFS, DOR, OS and safety) were assessed by an independent review committee. Based on preliminary data as of March 2016, 57 patients were enrolled. There were 3 manufacturing failures (5%), 5 patients were not infused due to death or adverse events (9%), and 15 patients were pending infusion at the data cut off. Following fludarabine/cyclophosphamide lymphodepleting chemotherapy in the majority of the patients, 34 patients (median age 11 [3-23], 50% with prior HSCT) were infused with a single dose of CTL019 at a median dose of 2.9 x106 transduced CTL019 cells/kg (0.2 to 4). Among 29 patients reaching D28 prior to the data cutoff, 83% (24/29) achieved CR or CRi by local investigator assessment, all of which were MRD-negative. Two early deaths occurred prior to initial disease assessment, one due to disease progression and one due to intracranial hemorrhage. Two patients did not respond. One patient was in CR by BM at D28, but CSF was not assessed, therefore this patient was classified as "incomplete" assessment. Safety was managed by a protocol-specified CRS algorithm with no cases of refractory CRS. Using the Penn CRS grading scale, 82% of patients experienced CRS, with 7 grade 3 (21%) and 8 grade 4 (24%) events. 44% patients with CRS required anti-cytokine therapy; all received tocilizumab with or without other anti-cytokine therapy, with complete resolution of CRS. Besides CRS, the most common grade 3 and 4 non-hematologic AEs were febrile neutropenia (29%), increased bilirubin (21%), increased AST (21%), and hypotension (21%). 21% of patients experienced grade 3 or 4 neuropsychiatric events including confusion, delirium, encephalopathy, agitation and seizure; no cerebral edema was reported. CTL019 in vivo cellular kinetics by qPCR demonstrated transgene persistence in blood in responding patients at and beyond 6 months. Overall exposure (AUC 0-28d) and maximal expansion (Cmax) of CTL019 DNA measured by qPCR was higher in responding compared with non-responding patients. In summary, this pivotal global study in pediatric and young adult patients with r/r B-ALL receiving CTL019, confirms a high level of efficacy and a similar safety profile to that shown in the prior single center experience. Safety was effectively and reproducibly managed by appropriately trained investigators. The study has completed accrual. At the meeting, updated data from a planned formal interim analysis including safety, efficacy (primary and selected secondary endpoints), cellular kinetics, and impact of anti-cytokine therapy will be presented for more than 50 patients infused at 25 global sites. Disclosures Grupp: Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy. Laetsch:Novartis: Consultancy; Loxo Oncology: Consultancy. Bittencourt:Seattle Genetics: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Educational Grant. Maude:Novartis: Consultancy. Myers:Novartis Pharmaceuticals: Consultancy. Rives:Novartis: Consultancy; Jazz Pharma: Consultancy. Nemecek:Medac, GmbH: Research Funding; Novartis: Consultancy; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees. Schlis:Novartis: Honoraria. Martin:Jazz Pharmaceuticals: Other: One time discussion panel; Novartis: Other: Support of clinical trials. Bader:Medac: Consultancy, Research Funding; Riemser: Research Funding; Neovii Biotech: Research Funding; Servier: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Peters:Novartis: Consultancy; Jazz: Speakers Bureau; Amgen: Consultancy; Pfizer: Consultancy; Medac: Consultancy. Biondi:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Cellgene: Other: Advisory Board; BMS: Membership on an entity's Board of Directors or advisory committees. Baruchel:Servier: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Jazz: Consultancy; Baxalta: Research Funding. June:University of Pennsylvania: Patents & Royalties; Johnson & Johnson: Research Funding; Celldex: Consultancy, Equity Ownership; Pfizer: Honoraria; Immune Design: Consultancy, Equity Ownership; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Tmunity: Equity Ownership, Other: Founder, stockholder . Sen:Novartis: Employment. Zhang:Novartis: Employment. Thudium:Novartis: Employment. Wood:Novartis Pharmaceuticals: Employment, Other: Stock. Taran:Novartis: Employment. Pulsipher:Chimerix: Consultancy; Jazz Pharmaceutical: Consultancy; Novartis: Consultancy, Other: Study Steering Committee; Medac: Other: Housing support for conference.

T cells bearing a second-generation anti-CD19 chimeric antigen receptor (CAR) induce complete remission in >90% of patients with acute lymphoblastic leukemia (ALL) at our institution. However, disease may recur and we recently identified two molecular mechanisms of relapse (PMID: 26516065). We here present a novel mechanism of antigen-negative relapse in a pediatric ALL patient. A 21 year-old male patient was in third relapse at the time of enrollment onto our CTL019 trial (ClinicalTrials.Gov #NCT01626495). The patient achieved an MRD-negative complete remission 1 month after CTL019 infusion but relapsed nine months later. Quantitative PCR analysis of the transgene and flow cytometry for CAR19 protein analysis showed the expected expansion of the CART cells followed by log-normal decay following disease eradication. At relapse, however, the transgene copy numbers had increased without a concomitant rise in CAR19 protein-expressing T cells. The CAR protein was found to be expressed by the now CD19-negative CD45dimCD10+CD3negCD22+ leukemia. Molecular analysis via next-generation immunoglobulin heavy chain sequencing (NGIS) of the apheresis product, used for CTL019 manufacturing, and relapse marrow at 9 months demonstrated clonal identity of the relapsed clone, which carried two rearranged IgH alleles. Sequencing of the CD19, CD21, CD81, and CD225 loci did not reveal any mutations. The analysis of lentiviral vector integration sites (LVIS) of the infusion product and post-infusion specimens showed the following: a) the infusion product carried over 15,000 unique integration sites; b) only 7 LVIS were shared between this sample and month 9 and 20 relapse specimens, none of which were near proto-oncogenes; c) the relapsed leukemia carried two LVIS, one on chromosome 10, >50 kb distal from neuropilin (NRP1) and the second in an intron of proprionyl coenzyme A carboxylase-A (PCCA). Flow cytometric and qRT-PCR analysis of leukemic cells in the apheresis and relapse showed that NRP1 levels were indistinguishable, suggesting that the lentiviral vector did not act as an enhancer for NRP1. Furthermore, qRT-PCR demonstrated that the lentiviral integration did not affect the gene expression levels of PCCA. Investigation into the origins of the leukemic CAR transduction event showed that the patient did not exhibit replication-competent lentivirus. However, NGIS analysis of infusion product revealed the leukemic clonotypes this sample, indicating that the gene transfer occurred during the manufacturing of the CTL019 cells. A retrospective analysis of 115 aphereses from ALL patients showed that the index patient had an unusually high disease burden in the apheresis product with 63% of all cells expressing CD19; at harvest, however, the CTL019 product consisted of 99.21% T cells, highlighting the purging effect of the CD19-specific T cells during manufacturing. NGIS analysis of infusion products of 17 additional ALL patients also identified the leukemic clonotype(s) in 6 more products. Only one additional patient demonstrated CAR19 protein expression on the leukemic cells, and this clone was not dominant at relapse (0.075% of all leukemic cells expressed the CAR). Our investigation into the biology of CAR19-expressing ALL cells showed the following: 1) the in vitro analysis of BBζ-signaling CAR19 showed no evidence of cytokine secretion; 2) the infusion of the baseline leukemia and CAR19-expressing leukemic cells from the same patient in mice did not demonstrate differential pharmacodynamics, even after restimulation with human CD19-expressing murine B cells in vivo; 3) the CD19 protein was detectable using flow cytometry and confocal microscopy, but only with an antibody recognizing an intracellular epitope; 4) importantly, the relapsed clone was indeed resistant to killing by CART19 cells in a xenograft model yet retained sensitivity to anti-CD22 CAR T cells. In conclusion, our data therefore show that a single leukemic cell accidentally transduced with CAR19 survived the 10-day manufacturing process and, upon reinfusion into the patient, was the sole clone at relapse 9 months later. This leukemic clone evaded CTL019 detection via downregulation of the target antigen in a cell-autonomous fashion. Disclosures Lacey: Novartis: Research Funding. Xu:Novartis: Research Funding. Ruella:novartis: Patents & Royalties: Novartis, Research Funding. Barrett:Novartis: Research Funding. Kulikovskaya:Novartis: Research Funding. Ambrose:Novartis: Research Funding. Patel:Novartis: Research Funding. Reich:Novartis: Research Funding. Scholler:Novartis: Patents & Royalties: Royalties, Research Funding. Nazimuddin:Novartis: Research Funding. Fraietta:Novartis: Patents & Royalties: Novartis, Research Funding. Maude:Novartis: Consultancy. Gill:Novartis: Patents & Royalties, Research Funding. Levine:Novartis: Patents & Royalties, Research Funding; GE Healthcare Bio-Sciences: Consultancy. Orlando:Novartis: Employment. Grupp:Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Research Funding. June:Tmunity: Equity Ownership, Other: Founder, stockholder ; Pfizer: Honoraria; Immune Design: Consultancy, Equity Ownership; Celldex: Consultancy, Equity Ownership; University of Pennsylvania: Patents & Royalties; Johnson & Johnson: Research Funding; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding. Melenhorst:Novartis: Patents & Royalties: Novartis, Research Funding.

Background: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and is most often of B-cell lineage (Roberts, et al. NEJM 2014). One subtype of B-cell ALL, Philadelphia chromosome-like ALL (Ph-like ALL), is BCR-ABL negative with a gene expression signature similar to that of BCR-ABL positive ALL and is prognostic for poor clinical outcomes (Roberts, et al. NEJM 2014). Ph-like ALL is often associated with rearrangements involving the cytokine receptor-like factor 2 (CRLF2) component of the thymic stromal lymphopoietin receptor (TSLPR) leading to its overexpression (Shochat, et al., JEM 2011). TSLPR is a heterodimer of CRLF2 and IL-7Rα that signals to promote the proliferation and differentiation of B-cell progenitors and acts to promote B-cell transformation in the context of Ph-like ALL (Maude, et al. Blood 2012). Glucocorticoid (GC) therapy plays a central role in the treatment of childhood ALL and resistance to GCs confers a poor prognosis (Piovan, et al. Cancer Cell, 2013). This study examined the role of TSLPR signaling in mediating primary GC resistance and the effects of downstream signal transduction inhibitors to confer GC sensitivity. Methods: Viably cryopreserved splenocytes were obtained from 19 patient-derived xenografts of Ph-like ALL banked in the Children's Oncology Group or Children's Hospital of Philadelphia leukemia biorepositories. Assays were also performed using the Mutz 5 Ph-like ALL cell line. Flow cytometry was used to assess the protein expression of TSLPR and GC receptor (GR). Levels of pERK and pAKT were measured by phosphoflow cytometry at baseline and following TSLP stimulation. Cells were cultured in the presence of 1µM dexamethasone (dex), a GC, with or without 1µM trametinib, a MEK1/2 inhibitor, or 1µM MK2206, a pan-AKT inhibitor, in the presence of TSLP and viability was assessed by Hoechst staining and flow cytometry at 48 hours. Results: Of the 19 Ph-like ALL samples in this study, 11 were CRLF2-rearranged (CRLF2R) and 8 were non-rearranged (CRLF2NR). CRLF2 rearrangements involved P2RY8 or IGH and 9 of 11 samples had concomitant activating mutations in JAK1 or JAK2. CRLF2NRsamples expressed a variety of other translocations involving genes such as JAK2, PDGFR, and ABL1. CRLF2R cells were shown to have significantly greater TSLPR protein expression relative to CRLF2NR cells (p = 0.03). In the presence of TSLP, CRLF2 rearrangement status predicted responsiveness to dex, with CRLF2Rsamples demonstrating significant resistance to dex relative to CRLF2NRsamples (p = 0.004). There was no significant reduction in cell viability following dex treatment in CRLF2R samples (p = 0.5), while dex effectively attenuated cell viability in CRLF2NRsamples (p = 0.008). Importantly, there was no difference in GR expression between these two groups (p = 0.6). CRLF2R samples demonstrated hyperresponsiveness to TSLP stimulation, with a significant induction of pERK and pAKT that exceeded the response of CRLF2NRsamples (p = 0.007 and p = 0.0005, respectively) despite no differences in basal phosphoprotein levels between the two sample groups. Inhibition of MAPK signaling with trametinib or of AKT signaling with MK2206 significantly sensitized CRLF2Rcells to dex in the presence of TSLP when used in combination relative to dex alone (p = 0.0003 and p < 0.0001, respectively) and resulted in a significant reduction in cell viability relative to untreated cells (p < 0.0001 and p < 0.0001, respectively). The Mutz 5 cell line, which expresses both a CRLF2 rearrangement and a JAK2 activating mutation, was used to assess the effect of simultaneous pathway inhibition. Mutz 5 cells were treated with dex alone or dex in combination with trametinib and/or MK2206. While dex alone had no significant effect on cell viability, the addition of trametinib or MK2206 resulted in a 43% and 36% reduction in cell viability, respectively. Furthermore, combined treatment with dex, trametinib, and MK2206 resulted in a 72% reduction in cell viability, demonstrating the efficacy of simultaneous MAPK and PI3K pathway inhibition to confer dex sensitivity. Conclusion: MAPK and PI3K pathway signaling play a role in mediating primary GC resistance in CRLF2R Ph-like ALL, making these pathways potential therapeutic targets for enhancing the efficacy of GC therapy in this patient group. Disclosures Maude: Novartis: Consultancy. Teachey:Novartis: Research Funding.

Chimeric antigen receptor (CAR)-modified T cells with CD19 specificity (CTL019) are a highly effective novel immune therapy for relapsed/refractory ALL. Cytokine release syndrome (CRS) is the most significant and in some cases a life-threatening toxicity. These patients are often neutropenic and immunosuppressed and at high risk for sepsis. In addition, there have been cases where concurrent infectious complications potentially fueled underlying CRS leading to hypotension and hypoxia refractory to anti-cytokine directed therapies (Frey et al. abstract 2296, ASH 2014). Discriminating between sepsis and CRS can be a significant clinical challenge in the critical time-window required to initiate effective therapy, especially given that the treatments for CRS, including anti-cytokine therapies and corticosteroids, may worsen severe infections. We recently published an extensive study of 43 cytokines and soluble cytokine receptors, and clinical biomarkers in a cohort of 63 CTL019-treated ALL patients. Peak levels of 24 cytokines in the first month after infusion were highly associated with severe CRS, and CRS predictive models were described (Teachey et al. Cancer Discovery, June 1, 2016 6; 664). The object of these current studies was to compare biomarker profiles in CRS and sepsis, and attempt to identify a profile that would discriminate between the two clinical syndromes, particularly at the time of ICU admission. We evaluated 50 cytokines, soluble receptors, and other serum biomarkers in 66 adult and pediatric patients treated with CTL019 and who developed CRS (ALL-CRS cohort), 15 patients not treated with CTL019 but who developed sepsis leading to ICU admission (sepsis cohort), and 10 normal healthy children (control cohort). Of the 66 patients treated with CTL019 and who developed CRS, 30 (45%) required ICU admission. The biomarkers tested included the ones described in our published work plus a panel of seven additional biomarkers (Angiopoietin 2, [ANG2] sCD163, Pentraxin 3 [PTX3], sCD14, PAI-1, P-selectin, ICAM-1) that have previously been reported in the literature to be associated with sepsis. After adjusting for multiple comparisons, the following biomarkers were significantly elevated in sepsis compared to normal subjects: ANG2, GCSF, IFNα, IL1RA, IL4, IL6, MIG, MIP1α, PTX3, TNFα, sCD163, sCD30, sIL-1RI, sIL-1RII, sIL-2Rα, sIL-4R, sRAGE, sTNFRI, sTNFRII, sVEGFR1, sVEGFR2, sVEGFR3, and VEGF, whereas IL13 and RANTES were significantly lower in sepsis. In subjects with ALL and CRS, within 72 hours of ICU admission following CTL019 therapy (N=29), 23 biomarkers were significantly different compared to sepsis after adjusting for multiple comparisons; 16 were elevated in CRS: GM-CSF, HGF, IFN-γ, IFN-α, IL-10, IL-15, IL-5, IL-6, IL-8, IP-10, MCP1, MIG, MIP-1β, sIL-2Rα, sTNFRI, and sTNFRII; whereas 7 were elevated in sepsis subjects: CD163, IL-1β, sCD30, sIL-4R, sRAGE, sVEGFR-1, and sVEGFR-2. Figure 1 shows representative box-plots for sCD163 (elevated in sepsis) and IP-10 (elevated in CRS). Detailed biomarker profiling and an algorithm for discriminating between sepsis and CRS in patients treated with CTL019 will be presented. These comprehensive profiling data provide novel insights into CRS biology and importantly, will help us discriminate between CRS and sepsis in patients who become critically ill after receiving CAR T cell therapy. These data have direct translational therapeutic relevance. Disclosures Lacey: Novartis: Research Funding. Shaw:Novartis: Research Funding; Vitality Institute: Research Funding. Teachey:Novartis: Research Funding. Weiss:NIGMS K23GM110496: Research Funding; ThermoFisher Scientific: Honoraria; Up-To-Date: Patents & Royalties: Up-To-Date. Chen:Novartis: Research Funding. Frey:Novartis: Research Funding; Amgen: Consultancy. Porter:Genentech: Employment; Novartis: Patents & Royalties, Research Funding. Maude:Novartis: Consultancy. Grupp:Novartis: Consultancy, Research Funding; Pfizer: Consultancy. Pequignot:Novartis: Research Funding. June:Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Johnson & Johnson: Research Funding; Celldex: Consultancy, Equity Ownership; Pfizer: Honoraria; Immune Design: Consultancy, Equity Ownership; Tmunity: Equity Ownership, Other: Founder, stockholder ; University of Pennsylvania: Patents & Royalties. Melenhorst:Novartis: Research Funding.

Introduction CTL019 is a novel, investigational, chimeric antigen receptor (CAR) immunotherapy whereby autologous T cells are genetically modified with a chimeric antigen receptor to target CD19 on the surface of malignant as well as healthy B cells. The cellular kinetics of CTL019 have been evaluated in several trials for patients with relapsed/refractory CD19+ leukemias, including pediatric acute lymphoblastic leukemia (pALL), adult ALL (aALL), and chronic lymphocytic leukemia (CLL) (Maude 2014, Porter 2015). Methods The cellular kinetic profile of CTL019 was determined in peripheral blood (PB) and bone marrow (BM) through serial measurements using flow cytometry and quantitative real-time polymerase-chain-reaction (qPCR) assay in 3 studies comprised of (i) 55 pALL patients (NCT01626495), (ii) 28 adult CLL patients from a dose selection study (NCT01747486), and (iii) 14 CLL and 6 adult ALL patients (NCT01029366). The flow cytometry assay used a CAR19-specific anti-idiotype antibody to enumerate CTL019 T cells as a % of CD3+ T cell (Porter 2015). Cellular kinetic parameters included: maximal extent of expansion as measured by peak copies of CTL019 DNA and peak % by flow cytometry (Cmax), area under the curve at day 28 (AUC0-28d) describing expansion and persistence in the first month, and time to reach Cmax (Tmax). Parameters were derived by non-compartmental methods. Where estimable, persistence was described by the half-life (T1/2) based on the slope of the terminal phase. Results Following infusion, CTL019, expansion and persistence was evident in the patients who responded to CTL019 as measured by both PK assays across all 3 studies. Table 1 summarizes (arithmetic mean (SD)) the CTL019 kinetic parameters. With complete remission (CR/CRi), CTL019 cells undergo rapid in vivo expansion beyond the original CTL019 dose with maximal expansion at a mean of 11 days in pALL and aALL and approximately 14-18 days in adult CLL as determined by qPCR and flow cytometry (Table 1). In CR/CRi patients the transgene level-profiles in PB reveal a kinetic profile with an initial rapid expansion followed by a slower decay function with some fluctuations of transgene over time resulting in higher AUC0-28d and Cmax, while non-responder (NR) patients tend to have a lower expansion and faster decay (shorter T1/2)of CAR positive T-cells resulting in lower AUC0-28d and Cmax by, leaving the mechanism to be further explored. In pALL, significantly higher AUC0-28d and Cmax were observed in CR/CRi patients compared to NR patients by flow cytometry; however, a wide range of mean AUC0-28d and Cmax was observed in NR patients (n=3) resulting from significant expansion in one NR patient as determined by qPCR. In CLL, the exposure metrics AUC0-28d and Cmax were approximately 12 times higher in CR/CRi patients compared with PRi/NR/PD in NCT01747486; a similar trend was observed in NCT01029366. Similar findings were captured by the flow cytometry based measurements as summarized in Table 1. In pALL and CLL, CR/CRi patients tend to maintain higher levels of CTL019 transgene over longer periods of time (>6 months) compared to NR patients as demonstrated by the longer T1/2 value. Cellular kinetic parameters were not summarized by response category for aALL due to the small sample size (n=5 CR/CRi; n=1 NR). CTL019 transgene levels ranged from below the limit of quantification (BLQ) to 178,000 copies/ug in aALL patients with CR/CRi and BLQ to 21,900 copies/ug in the NR. CTL019 positive cells were also shown to traffic to BM at 1 month in responders (CR/CRi), irrespective of the disease. Conclusions Overall, significantly higher levels of in vivo proliferation and persistence were observed in patients who successfully responded to CTL019 (i.e. CR/CRi/PR) compared to NRs in both CLL and (adult and pediatric) ALL patients, as captured by both analytical measures, indicating that the kinetics of CTL019 T cells and that proliferation and persistence of CTL019 reasonably predicts response to therapy. These are the first three studies to demonstrate that cellular kinetics may predict responses to CAR based cellular therapy. These results imply that measures to increase proliferation and persistence of CAR T cells may enhance responses in resistant patients. Figure. CTL019 concentration-time profiles for %CD3+/CTL019+ measured by flow cytometry and cellular kinetic parameters for qPCR and flow cytometry for p-ALL and adult CLL Figure. CTL019 concentration-time profiles for %CD3+/CTL019+ measured by flow cytometry and cellular kinetic parameters for qPCR and flow cytometry for p-ALL and adult CLL Disclosures Mueller: Novartis Pharmaceuticals: Employment. Chakraborty:Novartis Pharmaceuticals: Employment, Equity Ownership. Wood:Novartis Pharmaceuticals: Employment, Other: Stock. Awasthi:Novartis Pharmaceuticals: Employment. Quintas-Cardama:Novartis Pharmaceuticals: Employment, Equity Ownership. Han:Novartis Pharmaceuticals: Employment, Equity Ownership. Maude:Novartis: Consultancy. Grupp:Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding; Genentech: Employment. Frey:Novartis: Research Funding; Amgen: Consultancy. Marcucci:Novartis: Research Funding. Levine:GE Healthcare Bio-Sciences: Consultancy; Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Research Funding. June:Celldex: Consultancy, Equity Ownership; Immune Design: Consultancy, Equity Ownership; Pfizer: Honoraria; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; University of Pennsylvania: Patents & Royalties; Tmunity: Equity Ownership, Other: Founder, stockholder ; Johnson & Johnson: Research Funding. Lacey:Novartis: Research Funding.

Background: CTL019 is an investigational therapy derived from autologous T-cells expressing a CD19-specific chimeric antigen receptor (CAR). A single center, phase I/IIa trial of CTL019 showed complete and durable remissions in pediatric/young adult patients (pts) with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL) (Maude et al NEJM 2014); these results have yet to be reproduced in a multicenter setting. Here, we report results from a 6-month interim analysis of the first multicenter phase II trial of an engineered cell therapy in leukemia. Methods: 9 US sites participated in this single-arm phase II study in pediatric/young adult pts with R/R B-ALL. Leukapheresis products were shipped for centralized manufacturing according to the University of Pennsylvania (Penn) process in an academic-industry collaboration. T cells were transduced with a lentiviral vector encoding a CAR composed of anti-CD19 scFv, CD3ζ and 4-1BB domains. Following lymphodepletion with fludarabine and cyclophosphamide, a single dose of CTL019 cells was administered (target dose 2.0-5.0×106 cells/kg for ≤50 kg and 1.0-2.5×108 cells for >50 kg). The primary endpoint was overall remission rate (ORR = CR + CRi [CRi, complete remission with incomplete blood count recovery] maintained at 2 evaluations ≥28 days apart) as determined by an Independent Review Committee. Secondary objectives included minimal residual disease (MRD), relapse-free survival (RFS), overall survival (OS) and safety. All analyses were performed on infused patient set. Results: 29/35 pts enrolled (82.9%) were infused with CTL019; 6 withdrew prior to infusion (2 manufacturing failures [1 lack of growth, 1 contamination]; 4 deaths [median, 48 days from enrollment; 2 progressive disease, 1 multi-organ failure, 1 pneumonia]). Mean bone marrow involvement at enrollment was 68.2% (SD, 27.3%; Table 1). 2 pts did not receive lymphodepleting chemotherapy due to leukopenia. Collection, manufacturing and infusion were feasible in a multicenter setting with a median time from enrollment to infusion of 37 days. Target cell dose was met in 24/33 (72.7%) manufactured products. ORR in all infused pts was 69.0% (20/29 pts; 98.95% CI 43.6, 88.1). Of the 5 pts who received CTL019 below the target dose, 2 achieved CR/CRi. Of note, deep remission with no evidence of MRD ( Conclusions: In this first multicenter trial of CAR-modified T cell therapy, CTL019 therapy was feasible and efficacious, showing a high ORR with durable remissions in pediatric/young adult pts with R/R B-ALL. Despite the high rate of toxicity with CTL019 in the R/R setting, the rate of grade 3 or 4 CRS was comparable to the single center study, and standardized management of CRS was successful in a multicenter trial with no deaths attributable to CRS. In this highly refractory population, a vast majority of eligible pts can be successfully infused in a timely fashion and outcomes appear reproducible in a multicenter setting despite a more heterogeneous population than the single center study. The trial is continuing under Novartis manufacturing. Disclosures Maude: Novartis: Consultancy. Pulsipher:Medac: Other: Travel support for a study group; Chimerix: Consultancy, Other: Advisory Board ; Jazz Pharmaceutical: Consultancy, Other: Advisory Board; Novartis: Consultancy, Other: Advisory Board, Steering Committee for Phase II Study. Grupp:Pfizer: Consultancy; Novartis: Consultancy, Research Funding. Davies:Novartis: Honoraria. Verneris:Bimogen: Other: Advisory Board. Schlis:Novartis: Honoraria. Driscoll:Novartis: Consultancy. June:Immune Design: Consultancy, Equity Ownership; Pfizer: Honoraria; Celldex: Consultancy, Equity Ownership; Novartis: Honoraria, Patents & Royalties, Research Funding; Johnson & Johnson: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership. Levine:GE Healthcare Bio-Sciences: Consultancy; Novartis: Patents & Royalties, Research Funding. Wood:Novartis Pharmaceuticals: Employment, Other: Stock. Yi:Novartis: Employment.

Background: Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is an uncommon childhood leukemia that has been associated with very poor clinical outcomes in some studies. ETP-ALL cells arrest at a more immature differentiation stage than other T-lymphoblasts, and are hypothesized to retain multi-lineage differentiation potential, which may contribute to chemoresistance with standard lymphoid-directed therapy. Based on the recent clinical success of chimeric antigen receptor (CAR)-modified T-cells in children with B-ALL, we sought to identify potential surface protein targets on ETP lymphoblasts using differential gene expression analysis combined with a bioinformatic algorithm to predict surface expression. Methods: Cell-surface targets on ETP-ALL were predicted by identifying overexpressed transcripts based on gene expression and a bioinformatic algorithm to predict surface expression. Using several gene expression platforms and reference databases, (Oncogenomics website-Pediatric Oncology Branch, NCI, Gene Expression Omnibus, Gene Ontology, Human Protein References Database) ETP-ALL samples were compared to peripheral blood mononuclear cell (PBMC) controls on an individual transcript basis. A list of the top 25 transcripts was generated based on cell surface proteins, and the resultant list ordered by the degree of difference from PBMC controls. We next used human leukemia cells from six established ETP-ALL patient-derived xenograft (PDX) models using flow cytometry to evaluate for cell surface expression of proteins encoded by the overexpressed transcripts. Additionally, since CD7 and CD33 expression on ETP-ALL patient samples is universal with minimal normal tissue distribution, we developed two new second-generation anti-CD7 or anti-CD33 CAR constructs using a 41-BB/CD3ζ backbone. Results: Multiple gene transcripts encoding cell surface proteins potentially amenable to CAR T-cell targeting were overexpressed in ETP-ALL cells in comparison to PBMC controls. Many of these proteins are involved in cell signaling, cell adhesion, and metastasis, and thus potentially important for leukemic cell survival. TSPAN7 (also known as TALLA-1) was the strongest differentially expressed transcript. Despite identification of several transcripts, we did not detect increased surface expression of multiple antigens that were identified as top 25 transcripts, including TALLA-1, MCAM, EPHB6, or TSLPR. Interestingly, TALLA-1 was expressed on the more mature T-cell ALL lines, JURKAT and HPB-AU, suggesting that the surface expression of TALLA protein may be developmentally regulated. Although a new target could not be identified, given the universal expression of CD7 and CD33 on ETP-ALL, we proceeded with development of CARs targeting these antigens. CD33 CAR T-cells had excellent in vitro activity in human AML cell line MOLM-14 with minimal anti-leukemia activity in six tested ETP-ALL PDX models, perhaps due to their lower CD33 expression. We next tested T-cells transduced with a bicistronic CD7-redirected CAR with a truncated EGFR (EGFRt) to facilitate measurement of transduction efficiency and to provide a CAR deletion method. Despite high EGFRt surface expression in transduced T-cells, these CD7 CAR T-cells did not demonstrate in vitro activity against ETP-ALL or mature T-ALL samples despite high CD7 surface expression on all leukemia cell lines. We postulated that abnormal CAR distribution within the T-cell itself could be a potential factor in the observed lack of CD7 CAR T-cell activity. Using fluorescent-labeling to assess CAR surface membrane distribution, we detected high intracellular expression of the CD7 CAR, and noted that it did not traffic to the cell surface. Conclusions: We applied multimodal techniques to evaluate for cell surface expression on ETP-ALL that could serve as a target for immunotherapy. Although novel targets could not be identified, we were able to design an active anti-CD33 CAR. Further studies are in progress to evaluate what degree of antigen expression is needed to be amenable to targeted therapy. Additionally, ongoing studies are assessing whether optimization of CAR design can enhance cell surface trafficking and thereby potentially improve the anti-leukemia efficacy of CD7 CAR T-cells. Disclosures Orentas: Lentigen Technology, Inc.: Employment. Maude:Novartis: Consultancy. Teachey:Novartis: Research Funding.

Background Targeted immunotherapy with CTL019, CD19-specific chimeric antigen receptor (CAR)-modified T cells, can produce potent and sustained responses in children with relapsed/refractory acute lymphoblastic leukemia (ALL). However, a subset of patients has limited persistence, which can increase the risk of relapse. Most CAR single chain variable fragment (scFv) domains, including that of CTL019, are of murine origin; therefore, anti-mouse reactivity is one potential cause of immune-mediated rejection that may be overcome by fully human or humanized CAR designs. We developed a humanized anti-CD19 scFv domain and now report on treatment with humanized CD19-directed CAR T cells (CTL119). Design A pilot/phase 1 study of CAR-modified T cells containing a humanized anti-CD19 scFv domain (CTL119) enrolled children and young adults with relapsed/refractory B-ALL with or without prior exposure to a CAR T cell product. Patients previously treated with CD19-specific CAR-modified T cells were eligible if they met 1 of 3 criteria: 1) CD19+ relapse 2) no response to prior CAR T cell therapy or 3) early B cell recovery indicating poor persistence of CAR T cells. Patient-derived T cells were transduced ex vivo with a lentiviral vector encoding a CAR composed of CD3z, 4-1BB, and humanized anti-CD19 scFv domains and activated/expanded with anti-CD3/CD28 beads. The humanized scFv domain was developed by grafting the complementary determining regions of both the heavy and light chains onto human germline acceptor frameworks. Patients received lymphodepletion with cyclophosphamide and fludarabine 1 week prior to infusion with CTL119. Results Thirty children and young adults aged 29 mo-24 yr were infused with CTL119. Eighteen patients had received prior allogeneic stem cell transplant (SCT). Eleven patients who previously received murine-derived CD19-specific CAR-modified T cells (CTL019, n=7; other, n=4) were retreated for B cell recovery (n=5), CD19+ relapse (n=5), or no response to prior CAR T cells (n=1). CNS disease or other extramedullary disease was the indication for enrollment in 6 and 3 patients, respectively. At assessment 1 month after infusion, 26/30 patients (87%) achieved a complete response (CR), defined as morphologic remission with B cell aplasia. Of 11 patients previously treated with murine CD19-specific CAR-modified T cells, 7 (64%) achieved a CR at 1 month, 4 demonstrated no response. Multiparameter flow cytometry for minimal residual disease (MRD) was negative at a detection level of 0.01% in 5/7 responding patients. Two responding patients with positive MRD progressed to CD19+ relapse at 1.6 and 3 mo. In patients with no prior exposure to a CD19 CAR T cell product, MRD-negative CR was achieved in 19/19 patients (100%). One patient relapsed with CD19+ extramedullary disease at 2.8 mo. With a median follow-up of 4.2 mo (range, 1.0-14.1 mo) for all responding patients in both cohorts, 23/26 remain in remission with 1 proceeding to SCT in remission. B cell aplasia, a functional marker of CD19-targeted CAR T cell persistence, continued for 3 months or more in 11/18 patients with adequate follow-up: 1/6 retreatment, 10/12 CAR-naïve. Cytokine release syndrome (CRS) was observed in 28/30 patients and mild in most patients (grade 1, n=6; grade 2, n=18). Three patients experienced grade 3 CRS requiring supplemental oxygen or low-dose vasopressor support and 1 experienced grade 4 CRS requiring high-dose vasopressor and ventilatory support. Severe CRS was successfully managed with the IL6R antagonist tocilizumab in 3 patients. Neurologic toxicity included encephalopathy (n=5) and seizure (n=4) and was fully reversible. Conclusion In the first study of humanized anti-CD19 CAR T cells, CTL119 induced remissions in children and young adults with relapsed/refractory B-ALL, including 64% of patients previously treated with murine CD19-directed CAR T cells and 100% of CAR-naïve patients. Further investigation into CAR T cell persistence and anti-CAR responses will be vital to improve durable remission rates in this highly refractory population. Disclosures Maude: Novartis: Consultancy. Barrett:Novartis: Research Funding. Teachey:Novartis: Research Funding. Shaw:Novartis: Research Funding; Vitality Institute: Research Funding. Brogdon:Novartis: Employment. Scholler:Novartis: Patents & Royalties: Royalties, Research Funding. Marcucci:Novartis: Research Funding. Levine:GE Healthcare Bio-Sciences: Consultancy; Novartis: Patents & Royalties, Research Funding. Frey:Amgen: Consultancy; Novartis: Research Funding. Porter:Novartis: Patents & Royalties, Research Funding; Genentech: Employment. Lacey:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. June:Novartis: Honoraria, Patents & Royalties, Research Funding; Celldex: Consultancy, Equity Ownership; Pfizer: Honoraria; Immune Design: Consultancy, Equity Ownership; Johnson & Johnson: Honoraria; Novartis: Honoraria, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership. Grupp:Pfizer: Consultancy; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Research Funding.

Blinatumomab is a CD19/CD3-bispecific T-cell receptor-engaging (BiTE) antibody with efficacy in refractory B-precursor acute lymphoblastic leukemia. Some patients treated with blinatumomab and other T cell-activating therapies develop cytokine release syndrome (CRS). We hypothesized that patients with more severe toxicity may experience abnormal macrophage activation triggered by the release of cytokines by T-cell receptor–activated cytotoxic T cells engaged by BiTE antibodies and leading to hemophagocytic lymphohistiocytosis (HLH). We prospectively monitored a patient during blinatumomab treatment and observed that he developed HLH. He became ill 36 hours into the infusion with fever, respiratory failure, and circulatory collapse. He developed hyperferritinemia, cytopenias, hypofibrinogenemia, and a cytokine profile diagnostic for HLH. The HLH continued to progress after discontinuation of blinatumomab; however, he had rapid improvement after IL-6 receptor-directed therapy with tocilizumab. Patients treated with T cell-activating therapies, including blinatumomab, should be monitored for HLH, and cytokine-directed therapy may be considered in cases of life-threatening CRS. This trial was registered at www.clinicaltrials.gov as #NCT00103285.

3011Background: Targeted immunotherapy with chimeric antigen receptor (CAR)-modified T cells can produce potent anti-tumor responses. We previously reported complete remissions (CR) and prolonged persistence in children and adults with relapsed/refractory (r/r) acute lymphoblastic leukemia (ALL) treated with CD19-specific CART cells (CTL019). We now report on outcomes and longer follow-up of 59 children with r/r ALL. Methods: T cells collected from the patient were transduced with a lentiviral vector encoding a CAR composed of anti-CD19 scFv, CD3z, and 4-1BB domains, activated/expanded ex vivo with anti-CD3/CD28 beads, and then infused at a dose of 107 to 108cells/kg with a transduction efficiency of 2.3-45%. 54/59 patients received lymphodepleting chemotherapy the week prior to cell infusion. Results: Of 59 patients aged 20mo-24y with CD19+ ALL, 44 had detectable disease prior to CTL019 cell infusion, while 15 were MRD-. 39 were treated for relapse after prior stem cell transplant (SCT). 15 patients had ...

7002Background: Genetically modified T cells expressing an anti-CD19 CAR result in response rates of 90% in children with r/r CD19+ ALL. Cytokine release syndrome (CRS) is the most significant trea...

3007Background: Targeted immunotherapy with CTL019, CD19-specific CAR-modified T cells, can produce potent, sustained responses in children with relapsed/refractory acute lymphoblastic leukemia (AL...

CRLF2 rearrangements, JAK1/2 point mutations, and JAK2 fusion genes have been identified in Philadelphia chromosome (Ph)–like acute lymphoblastic leukemia (ALL), a recently described subtype of pediatric high-risk B-precursor ALL (B-ALL) which exhibits a gene expression profile similar to Ph-positive ALL and has a poor prognosis. Hyperactive JAK/STAT and PI3K/mammalian target of rapamycin (mTOR) signaling is common in this high-risk subset. We, therefore, investigated the efficacy of the JAK inhibitor ruxolitinib and the mTOR inhibitor rapamycin in xenograft models of 8 pediatric B-ALL cases with and without CRLF2 and JAK genomic lesions. Ruxolitinib treatment yielded significantly lower peripheral blast counts compared with vehicle (P < .05) in 6 of 8 human leukemia xenografts and lower splenic blast counts (P < .05) in 8 of 8 samples. Enhanced responses to ruxolitinib were observed in samples harboring JAK-activating lesions and higher levels of STAT5 phosphorylation. Rapamycin controlled leukemia burden in all 8 B-ALL samples. Survival analysis of 2 representative B-ALL xenografts demonstrated prolonged survival with rapamycin treatment compared with vehicle (P < .01). These data demonstrate preclinical in vivo efficacy of ruxolitinib and rapamycin in this high-risk B-ALL subtype, for which novel treatments are urgently needed, and highlight the therapeutic potential of targeted kinase inhibition in Ph-like ALL.

Background Targeted immunotherapy with T cells expressing a chimeric antigen receptor (CAR) can produce dramatic anti-tumor responses. We previously demonstrated complete remissions (CRs), prolonged persistence, and sustained responses in children and adults with relapsed/refractory (r/r) acute lymphoblastic leukemia (ALL) treated with CD19-specific CAR-modified T cells (CTL019). However, a subset (~25%) of patients has limited persistence of CTL019, which can increase the risk of relapse. We hypothesized that repeat infusion may prolong persistence in some patients but would be ineffective in patients with immune-mediated rejection. As most CAR single chain variable fragment (scFv) domains, including that of CTL019, are of murine origin, anti-mouse reactivity is one potential cause of immunogenicity that may be overcome by fully human or humanized CAR design. We now report on retreatment with murine or humanized CD19-directed CAR T cells. Aims We sought to 1) prolong persistence by reinfusion of murine CTL019 in children with r/r CD19+ ALL and evidence of poor persistence and 2) determine the safety and efficacy of CD19-directed CAR T cells with a humanized scFv domain in children with B cell recovery or CD19+ relapse after prior CAR T cell therapy. Method T cells collected from patients were transduced with a lentiviral vector encoding a CAR composed of murine or humanized anti-CD19 scFv, CD3z, and 4-1BB domains, activated/expanded ex vivo with anti-CD3/anti-CD28 beads, cryopreserved, and then infused. The humanized scFv was developed by grafting the complementary determining regions of both the heavy and light chains onto human germline acceptor frameworks. Results Of 50 patients in CR at 1 month after CTL019 infusion (50/53 CR), 14 patients received a repeat infusion of murine CTL019 at 3 and/or 6 months after initial infusion and 11 are evaluable for response. Indications were B cell recovery (n=4), CD19+ minimal residual disease (MRD, n=2), or undetectable CTL019 in peripheral blood by flow cytometry (≤0.1% CTL019+ of CD3+, n=5). Minimal toxicities were observed and included fever and fatigue. CTL019 reinfusion induced B cell aplasia for a second time in 1 of 4 children treated for B cell recovery. Two of these patients later relapsed, 1 with CD19+ and 1 with CD19- disease. Of 2 patients with CD19+ MRD, 1 had no response and subsequently experienced a CD19+ relapse and the second became MRD-negative but had B cell recovery. All 5 children reinfused for poor CTL019 persistence demonstrated continued B cell aplasia 3-15 months after repeat infusion with detectable CTL019 in peripheral blood by flow cytometry (0.2-2.8% CTL019+ of CD3+) in 4 patients. Of this group, 4 patients remain in remission 12-21 months after initial infusion, and 1 experienced a CD19- relapse. To overcome the potential for anti-murine immune-mediated rejection, patients previously treated with CAR-modified T cells were eligible for a phase 1 study of humanized CD19-directed CAR T cells (CTL119). Five children were infused for B cell recovery (n=3) or CD19+ relapse (n=2). Cytokine release syndrome (CRS), seen in 2 patients, was limited to fever, headache, and nausea and did not require vasopressor or respiratory support. Three patients showed no response and progressed (n=1) or relapsed with CD19+ (n=1) or CD19- (n=1) ALL. Responses were demonstrated in 2 patients: 1) MRD-negative CR in a patient treated for CD19+ relapse and 2) reduction in MRD and return of B cell aplasia in a patient treated for CD19+ MRD and B cell recovery, with subsequent progression to CD19+ relapse. Both responding patients were previously resistant to murine CTL019 reinfusion, suggesting a possible immune-mediated rejection mechanism. Ongoing studies are investigating anti-CAR responses. Conclusion Repeat infusion of murine CTL019 may prolong B cell aplasia in patients with early evidence of poor persistence and in a fraction of patients with B cell recovery. In the first demonstration of humanized anti-CD19 CAR efficacy, retreatment with CTL119 expressing a humanized scFv induced remission of ALL refractory to prior CD19-directed murine CAR T cell therapy, suggesting that immune-mediated rejection may be an important mechanism of resistance in the subset of patients with rapid CAR cell loss and loss of B cell aplasia. Further investigation into anti-CAR responses will be vital to improve durable remission rates in this highly refractory population. Disclosures Maude: Novartis: Consultancy, Research Funding. Off Label Use: CTL019 and CTL119 for relapsed/refractory ALL. Rheingold:Endo: Other: Husband's employer, has equity interest; Novartis: Consultancy. Aplenc:Sigma Tau: Consultancy. Teachey:Novartis: Research Funding. Shaw:Novartis: Research Funding. Brogdon:Novartis: Employment. Loew:Novartis: Employment. Zheng:Novartis: Patents & Royalties. Levine:Novartis: Patents & Royalties, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding. Lacey:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. June:Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight. Grupp:Novartis: Consultancy, Research Funding.

Background. Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is associated with genomic alterations that activate JAK/STAT and PI3K/Akt/mTOR signal transduction and with poor clinical outcomes. Therapeutic disruption of PI3K pathway signaling in Ph-like ALL has been minimally investigated to date, however. We hypothesized that PI3K isoform-selective or dual PI3K pathway protein inhibition would robustly inhibit Ph-like ALL proliferation in vivoand abrogate aberrant signaling. Methods. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were engrafted with primary CRLF2/JAK-mutant or ABL/PDGFR-mutant Ph-like ALL specimens (Table 1) and treated with inhibitors of PI3K? (BYL719), PI3K? (idelalisib), PI3K/mTOR (gedatolisib), TORC1/TORC2 (AZD2014) or with vehicle. Treated patient-derived xenograft (PDX) models were assessed for pharmacodynamic inhibition of signal transduction phosphoproteins at 72 hours by phosphoflow cytometry and for residual ALL in murine spleens after 3-4 weeks of inhibitor or vehicle treatment by quantitative flow cytometry. Subsequent studies tested the efficacy of gedatolisib with the JAK1/2 inhibitor ruxolitinib (CRLF2/JAK-mutant models) or gedatolisib with the SRC/ABL inhibitor dasatinib (ABL/PDGFR-mutant models). Table 1. Genomic characteristics of Ph-like ALL specimens utilized for PDX studies. USI Disease status CRLF2/JAK alterations ABL/PDGFR alterations PALTWS D IGH@-CRLF2* PAMDKS D IGH@-CRLF2, JAK2R683G PAMDRM D IGH@-CRLF2,JAK2GPinsR683 ALL121 R IGH@-CRLF2, JAK2R683G ALL4364 R P2RY8-CRLF2, JAK2R683G PAKMVD D JAK1 S646F PAKYEP D BCR-JAK2 PAKKCA D EBF1-PDGFRB PAKVKK D NUP214-ABL1 PANSFD D ETV6-ABL1 USI = unique specimen identifier. D = de novo, R = relapse. * non-Ph-like by prediction analysis of microarrays. Results. All tested PDX models demonstrated inhibition of leukemia proliferation and abrogation of activated signaling with PI3K pathway inhibition. Gedatolisib treatment resulted in near-eradication of leukemia in CRLF2/JAK-mutant models (n=7) with mean 92.2% (range 86.0-99.4%) leukemia reduction vs vehicle treatment (p Conclusions. PI3K pathway inhibition is a biochemically relevant therapeutic approach for Ph-like ALL. Dual PI3K/mTOR inhibition with gedatolisib monotherapy potently inhibited leukemia proliferation and demonstrated additive or synergistic activity in combination with JAK or SRC/ABL inhibition in JAK-mutant or ABL/PDGFR-mutant Ph-like ALL, respectively. These data provide compelling rationale for testing combinations of signal transduction inhibitors without or with cytotoxic chemotherapy in children and adults with Ph-like ALL. Disclosures Off Label Use:preclinical testing of signal transaction inhibitors in Ph-like ALL models. Teachey:Novartis:Research Funding. Maude:Novartis:Consultancy, Research Funding. Perl:Actinium Pharmaceuticals:Consultancy; Asana Biosciences:Consultancy; Arog Pharmaceuticals:Consultancy; Ambit/Daichi Sankyo:Consultancy; Astellas US Pharma Inc.:Consultancy. Hunger:Sigma Tau:Consultancy; Jazz Pharmaceuticals:Consultancy; Spectrum Pharmaceuticals:Consultancy; Merck:Equity Ownership. Grupp:Novartis:Consultancy, Research Funding.

[Graphic][1] CAR T cells with anti-CD19 specificity have demonstrated considerable promise against highly refractory hematologic malignancies. Dramatic responses with complete remission rates as high as 90% have been reported in patients (pts) with relapsed/refractory ALL treated with CTL019 (Maude et al., NEJM 2014). Marked in vivo CAR T cell proliferation (100 to 100,000x) leads to improved efficacy but can be associated with adverse events, including cytokine release syndrome (CRS). To better understand manifestations of CRS, we studied clinical, laboratory, and biomarker data of 39 children and 12 adults with relapsed/refractory ALL treated with anti-CD19 CAR T cells. T cells were lentivirally transduced with a CAR composed of anti-CD19 single chain variable fragment/4-1BB/CD3 (Porter, NEJM 2011). 43 cytokines, chemokines, and soluble receptors (collectively termed cytokines hereafter) were serially measured, using Luminex bead array. Other biomarkers were tested in a CLIA/CAP certified lab. 48 of 51 pts developed grade 1-5 CRS (CRS1-5) (see Table). Most pts developed mild (grade 1-2) to moderate (grade 3) CRS (34/51). 14 pts developed severe (grade 4-5) CRS (12 grade 4, 2 adults with grade 5). 21 pts were treated with the IL-6 inhibitor tocilizumab, and most had rapid marked clinical improvement in CRS evidenced by quick resolution of fever and weaning of vasoactive medications. We found peak levels of 24 cytokines, including IFNg, IL6, IL8, sIL2Ra, sgp130, sIL6R, MCP1, MIP1a, and GM-CSF during the first month after CTL019 infusion were highly associated with CRS4-5 compared to CRS0-3, significant by the Holm-Bonferroni adjusted p-value. Analyzing cytokines from the first 3 days after infusion, sent before patients developed severe CRS, only 2 cytokines, sgp130 and IFNg, were strongly associated with later development of severe CRS (p10,000ng/ml), splenomegaly, and hypofibrinogenemia. Of the tested cytokines, 18 have been previously studied in children with HLH. We found a near identical pattern of cytokines differentially elevated in HLH also elevated in pts with CRS4-5 compared with CRS0-3. IL6, sIL6R, and sgp130 were markedly elevated in pts with CRS4-5; this IL6 cytokine pattern, along with the pronounced response to tocilizumab, establishes that IL6 trans-signaling is clinically relevant. These data represent the largest and most comprehensive profiling of the clinical and laboratory manifestations of CAR T cell related CRS and provide novel insights into CRS biology. They represent the first data that can accurately predict which pts treated with CAR T cells have a high probability of becoming critically ill. These data have direct therapeutic relevance and may guide future cytokine directed therapy. The first 4 authors contributed equally. | Gr1 | Supportive care only | | ---- | ------------------------------------------------------------------------------------------------------ | | Gr2 | IV therapies +/- hospitalization. | | Gr3 | Hypotension requiring IV fluids or low-dose vasoactives or hypoxemia requiring oxygen, CPAP, or BIPAP. | | Gr4 | Hypotension requiring high-dose vasoactives or hypoxemia requiring mechanical ventilation. | | Gr 5 | Death | Table 1. CRS grading Disclosures Teachey: Novartis: Research Funding. Off Label Use: tocilizumab. Lacey: Novartis: Research Funding. Shaw: Novartis: Research Funding. Melenhorst: Novartis: Research Funding. Frey: Novartis: Research Funding. Maude: Novartis: Consultancy, Research Funding. Aplenc: Sigma Tau: Consultancy. Chen: Novartis: Research Funding. Gonzalez: Novartis: Research Funding. Pequignot: Novartis: Research Funding. Rheingold: Endo: Other: Husband's employer, has equity interest; Novartis: Consultancy. June: Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight. Porter: Novartis: Patents & Royalties, Research Funding; Genentech: Other: Spouse Employment. Grupp: Novartis: Research Funding. [1]: /embed/inline-graphic-2.gif

BACKGROUND CARs combine a targeting antibody (scFv) domain with intracellular signaling domains. We have previously reported on CTL019 cells expressing an anti-CD19 CAR, which have resulted in up to 100,000x in vivo proliferation, durable anti-tumor activity, and prolonged persistence in pts with B cell tumors, including sustained CRs in adults and children with ALL (Grupp et al., NEJM 2013, Maude et al., NEJM 2014). We now report on outcomes and longer follow up of the first 53 pts with relapsed/refractory (r/r) ALL treated on our pilot trial in pediatric ALL. METHODS T cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3ζ, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into children with r/r CD19+ ALL. 48/53 pts received lymphodepleting chemotherapy the week prior to CTL019 infusion. The targeted T cell dose range was 107 to 108 cells/kg with a transduction efficiency of 3.6-45%. T cells for manufacturing were collected from the pt regardless of prior SCT status, and not their allo donors. RESULTS We treated 53 children and young adults with CD19+ ALL, median age 11y, (4-24y). To assess disease burden after lymphodepleting chemotherapy, pts had BM aspirations performed 1D prior to 1st CTL019 infusion: 41/53 pts had detectable ALL while 12 were MRD(-). A median of 4.3x106 CTL019 cells/kg (1-17.4x106/kg) were infused over 1-2D (1 pt got cells over 3D). There were no infusional toxicities >gr2, although pts who developed fevers within 24h of infusion did not receive a planned 2nd infusion of CTL019 cells. 50 pts (94%) achieved a CR, including a patient with CD19+ T ALL, 3 did not respond. MRD measured by clinical flow cytometry was All but 5 (90%) of pts developed grade 1-4 cytokine release syndrome (CRS) at peak T cell expansion. Detailed cytokine analysis showed marked increases of IL6 and IFNγ (both up to 1000x), and IL2R. Treatment for CRS was required for hemodynamic or respiratory instability in 28% of patients and was reversed in all cases with the IL6-receptor antagonist tocilizumab, together with short courses of corticosteroids in 9 pts. Although T cells collected from the 35 pts who had relapsed after allo SCT were median 100% donor origin, no GVHD has been seen. Grade 4 CRS was associated with high disease burden prior to infusion and with elevations in IL-6, ferritin (suggesting macrophage activation syndrome) and C reactive protein after infusion. Persistence of CTL019 cells can be detected by flow cytometry and/or QPCR, and results in the pharmacodynamic marker of CTL019 function, B cell aplasia, which continued for 3-39 months after infusion in pts with ongoing responses. B cell aplasia has been treated with IVIg without significant infectious complications. CONCLUSIONS: CTL019 cells can undergo robust in vivo expansion and can persist for 3 years or longer in children and young adults with r/r ALL, allowing for the possibility of long-term disease control without subsequent therapy such as SCT. This approach also has promise as salvage therapy for patients who relapse after allo SCT with a low risk of GVHD. CTL019 therapy is associated with a significant CRS that responds rapidly to IL-6-targeted anti-cytokine treatment. CTL019 cells can induce potent and durable responses for patients with r/r ALL; however, recurrence with cells that have lost CD19 is an important mechanism of CTL019 resistance. Rapid loss of CTL019 cells (prior to 3 months) is associated with a high risk of CD19+ relapse. CTL019 therapy has received Breakthrough Therapy designation from the FDA in pediatric and adult ALL, and phase 2 multicenter registration trials are well underway. Disclosures Grupp: Novartis: Consultancy, Research Funding. Maude:Novartis: Consultancy, Research Funding. Shaw:Novartis: Research Funding. Aplenc:Sigma Tau: Consultancy. Lacey:Novartis: Research Funding. Levine:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Research Funding. Rheingold:Novartis: Consultancy; Endo: Other: Husband's employer, has equity interest. Teachey:Novartis: Research Funding. Wood:Novartis Pharmaceuticals Corporation: Employment. Porter:Novartis: Other: IP interest, Research Funding; Genentech: Other: Spouse employment. June:University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight; Novartis: Research Funding.

CD19 is expressed on most B-cell neoplasms, including acute lymphoblastic leukemias (B-ALL). Chimeric antigen receptor (CAR)-armed T cells targeting CD19 (CART-19) yield complete remission rate of 70-90% in B-ALL patients; the bispecific anti-CD19/CD3 antibody blinatumomab also shows clinical efficacy. Yet relapses due to the apparent CD19 loss have been observed following both therapies. Here we investigated the molecular mechanisms of epitope loss using five B-ALL samples obtained after CD19-directed therapy. These samples were matched, when available, with CD19-positive pre-treatment leukemias. Although the CD19 epitope recognized by CART-19 was undetectable in relapsed specimens, in all cases the CD19 genomic sequence was retained and the mRNA levels were only slightly reduced, suggesting regulation at the level of transcript processing. Indeed, using RT-PCR and RNA-Seq we detected several alternatively spliced CD19 mRNA species lacking exons 5-6 and/or exon 2 (Δex2), which encodes the cognate CART-19 epitope. In the sets of matched samples, the relative abundance of the Δex2/5-6 mRNA isoforms was increased post-treatment, and accordingly truncated CD19 protein variants were detectable using antibodies recognizing alternative epitopes. In one relapsed leukemia with a nonsense mutation in exon 2 of CD19, Δex2 was the only expressed isoform. To determine the contribution of alternative splicing to immune escape, we introduced CD19-expressing retroviruses into murine neoplastic B-cells, which had lost endogenous CD19 expression following silencing of its main transcriptional regulator Pax5. While skipping of exon 2 made the resultant protein invisible to CART-19, it partly rescued the defects in cell proliferation associated with CD19 loss. Collectively, these data suggest the existence of a novel mechanism of resistance to immunotherapy, based not on mutations in the coding sequence but rather on selection for alternatively spliced target protein isoforms (e.g., CD19 Δex2). Our findings could lead to the development of additional CD19-targeting CARTs for treating relapsed patients. Citation Format: Elena Sotillo, David Barrett, Aseb Bagashev, Kathryn Black, Caludia Lanauze, Derek Oldridge, Robyn Sussman, Colleen Harrington, Elaine Y. Chung, Ted J. Hofmann, Shannon L. Maude, Nicole M. Martinez, Pichai Raman, Marco Ruella, David Allman, Elad Jacoby, Terry Fry, Yoseph Barash, Kristen W. Lynch, Crystal Mackall, John Maris, Stephen A. Grupp, Andrei Thomas-Tikhonenko. Alternative splicing of CD19 mRNA in leukemias escaping CART-19 immunotherapy eliminates the cognate epitope and contributes to treatment failure. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3143. doi:10.1158/1538-7445.AM2015-3143

CTLO19 cells are CAR-modified T cells which recognize CD19 and produce high durable remission rates for pts with relapsed or refractory acute lymphoblastic leukemia (ALL). Cytokine Release Syndrome (CRS) has emerged as the major treatment related effect from CTL019, with symptoms that include high fevers and malaise but can progress to capillary leak, hypoxia and hypotension. CRS occurs hours to days after CTL019 infusion and correlates with rapid in vivo CTL019 expansion and marked elevation of serum IL6. In most cases, CRS is self-limited or rapidly reversed with anti-cytokine directed therapies. Here we report 3 cases of refractory CRS in adult pts with ALL. Our experience offers insight into clinical and investigational parameters describing this syndrome; highlights the variance of CRS across disease types and illustrates complexities of CRS management during concurrent infectious illness. As of 7/1/14, 97 pts (30 pediatric ALL, 12 adult ALL, 41 CLL, 14 NHL) have been treated with CTLO19. To capture clinical manifestations of CRS across protocols, we developed a novel CRS grading scale which will be described. Severe CRS (Gr 3-5) occurred in 27 (64%) of ALL pts and only 16 (29%) of CLL/NHL pts (p=0.001). 12 adults with ALL received CTL019; 8/9 evaluable pts achieved CR (MRD negative) at 1 month and 1 pt with extramedullary disease had marked reduction of PET avid disease which is maintained at 1 yr. Severe CRS occurred in 11 of 12 adult ALL pts. CRS was self-limited in 2 pts, rapidly reversed with anti-IL6 directed therapy in 6 pts and was refractory to therapy, contributing to death in 3 pts who were not evaluable for disease response. No baseline attributes differentiate these 3 pts from the 9 adult ALL pts with manageable Gr1-4 CRS. We have shown however that ALL disease burden correlates with CRS severity (in press) and all 3 pts had significant disease burden at baseline. All received lymphodepleting chemotherapy with cyclophosphamide 300 mg/m2 q12h x 6 followed by infusion of CTLO19 cells. These 3 pts each received 6.50E+06, 6.70E+06 and 8.45E+06 CTLO19 cells/kg compared to median CTL019 dose of 3.62E+06 in the 9 adult ALL pts with manageable CRS. Pt 21413-03 developed CRS 12 hrs after infusion and tested positive for influenza B on D3. Despite broad spectrum antimicrobials (including oseltamivir) and anticytokine directed therapy with tocilizumab (4mg/kg x 2) and steroids, he died with refractory hypotension on D5. Pt 21413-06 had extensive disease after 2 prior allogeneic SCTs and developed CRS within 12 hrs of infusion. In addition to broad spectrum antibiotics, she received tocilizumab 8mg/kg (D 3, 6 and 12); intermittent high dose steroids (D 4-15) and etanercept (D14). She died D15 with hypotension, hypoxic respiratory failure and concurrent MDR pseudomonas sepsis and pneumonia. Pt 21413-11 developed CRS within 24 hrs of infusion. He received tocilizumab 8mg/kg (D3&4); siltuximab (D5&15) and intermittent high dose steroids (D 4-15). After an initial response, he developed recurrent fever, pulmonary infiltrates and blood cultures positive for stenotrophomonas. He died D15 with refractory hypoxia and hypotension. All 3 pts’ clinical CRS correlated with marked in vivo CTL019 expansion and progressive serum cytokine elevations (data to be shown). CONCLUSIONS: CRS is the major toxicity of CTL019 therapy and its clinical course varies depending on disease type (more frequent and severe in ALL) and disease burden (in ALL). The 3 refractory CRS cases described here (of 97 total pts treated) have all occurred in adult ALL pts with significant disease burden who received relatively high doses of CTL019 cells. In addition, all 3 had significant infectious complications which potentially fueled underlying CRS and/or were made more virulent due to impairment of immunity with administration of anti-cytokine directed therapies. Future protocol modifications will be made goal of limiting severity of CRS while maintaining high durable remission rates. Further exploration is planned to better correlate timing and choice of anticytokine directed therapy in relation to clinical and investigation parameters of CRS. Disclosures Frey: Novartis: Research Funding. Off Label Use: USe of CART19 cells to treat CLL. Levine:Novartis: Patents & Royalties, Research Funding. Lacey:Novartis: Research Funding. Grupp:Novartis: Consultancy, Research Funding. Schuster:Novartis: Research Funding. Hwang:NVS: Research Funding. Leung:Novartis: Employment. Shen:Novartis: Employment. Ericson:Novartis: Employment. Melenhorst:Novartis: Research Funding. June:Novartis: Patents & Royalties, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding.

BACKGROUND CARs combine a single chain variable fragment (scFv) of an antibody with intracellular signaling domains. We have previously reported on CTL019 cells expressing an anti-CD19 CAR. Infusion of these cells results in 100 to 100,000x in vivo proliferation, durable anti-tumor activity, and prolonged persistence in pts with B cell tumors, including sustained CRs in adults and children with ALL (Grupp et al., NEJM 2013, Maude et al., NEJM 2014). We now report on outcomes and longer follow up of the first 30 pts with relapsed, refractory ALL treated on our pilot trial in pediatric ALL. METHODS T cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3ζ, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into children with relapsed or refractory CD19+ ALL. 26/30 pts received lymphodepleting chemotherapy the week prior to CTL019 infusion. The targeted T cell dose range was 107 to 108 cells/kg with a transduction efficiency of 11-45%. T cells for manufacturing were collected from the pt regardless of prior SCT status, not allo donors. RESULTS 30 children median age 10y (5-22y) with CD19+ ALL were treated. 25/30 pts had detectable disease on the day before CTL019 cell infusion, while 5 were MRD(-). A median of 3.6x106 CTL019 cells/kg (1.1-18x106/kg) were infused over 1-3 days. There were no infusional toxicities >grade 2, although 9 pts developed fevers within 24 hrs of infusion and did not receive a planned 2nd infusion of CTL019 cells. 27 pts (90%) achieved a CR, including a patient with T cell ALL aberrantly expressing CD19+. 3 did not respond. MRD measured by clinical flow cytometry was negative in 23 responding pts and positive at 0.1% (negative at 3 mo), 0.09%, 0.22%, and 1.1% in 4 pts. With median follow up 8 mo (1-26 mo), 16 pts have ongoing CR, with only 3 patients in the cohort receiving subsequent treatment such as donor lymphocyte infusion or SCT, 6-month EFS measured from infusion is 63% (95% CI, 47-84%), and OS is 78% (95% CI, 63-95%). CTL019 cells were detected in the CSF of 17/19 pts and 2 pts with CNS2a disease experienced a CR in CSF. 10 pts with a CR at 1 mo have subsequently relapsed, half with CD19(-) blasts. 2/5 pts who relapsed with CD19(-) disease had previously been refractory to CD19-directed blinatumomab and subsequently went into CR with CTL019. Figure 1 Figure 1. All responding pts developed grade 1-4 cytokine release syndrome (CRS) at peak T cell expansion. Detailed cytokine analysis showed marked increases of IL6 and IFNγ (both up to 1000x), and IL2R. Treatment for CRS was required for hemodynamic or respiratory instability in 37% of patients and was rapidly reversed in all cases with the IL6-receptor antagonist tocilizumab, together with corticosteroids in 5 pts. Although T cells collected from the 21 pts who had relapsed after allo SCT were median 100% donor origin, no GVHD has been seen. Grade 4 CRS was strongly associated with high disease burden prior to infusion and with elevations in IL-6, ferritin (suggesting macrophage activation syndrome) and C reactive protein after infusion. Persistence of CTL019 cells detected by flow cytometry and/or QPCR, and accompanied by B cell aplasia, continued for 1-26 months after infusion in pts with ongoing responses. QPCR showed very high levels of CTL019 proliferation, with all patients achieving peak levels >5000 copies/ug gDNA and 26 patients with peak levels >15,000 copies/ug gDNA. B cell aplasia has been treated with IVIg without significant infectious complications. Probability of 6-mo CTL019 persistence by flow was68% (95% CI, 50-92%) andrelapse-free B cell aplasia was 73% (95% CI, 57-94%). CONCLUSIONS: CTL019 cells can undergo robust in-vivo expansion and can persist for 2 years or longer in pts with relapsed ALL, allowing for the possibility of long-term disease response without subsequent therapy such as SCT. This approach also has promise as a salvage therapy for patients who relapse after allo-SCT with a low risk of GVHD. CTL019 therapy is associated with a significant CRS that responds rapidly to IL-6-targeted anti-cytokine treatment. CTL019 cells can induce potent and durable responses for patients with relapsed/refractory ALL; however, recurrence with cells that have lost CD19 is an important mechanism of CLT019 resistance. CTL019 therapy has received Breakthrough Therapy designation from the FDA in both pediatric and adult ALL, and phase II multicenter trials have been initiated. Disclosures Grupp: Novartis: Consultancy, Research Funding. Barrett:Novartis: Research Funding. Chew:Novartis: Research Funding. Lacey:Novartis: Research Funding. Levine:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Research Funding. Rheingold:Novartis: Consultancy. Shen:Novartis: Employment. Wood:Novartis Pharma: Employment. Porter:Novartis: managed according to U Penn Policy Patents & Royalties, Research Funding. June:Novartis: Research Funding, Royalty income Patents & Royalties.